Prevalence And Rapid Diagnosis Of Acute Bacterial Meningitis In Childhood In Bangladesh

1. INTRODUCTION AND REVIEW OF LITERATURE

1.1 INTRODUCTIONMeningitis is an inflammation of the membranes and cerebrospinal fluid (CSF) that encases and bathes the brain and spinal cord. Meningitis is a serious disease that includes several types. These include bacterial meningitis, acute bacterial meningitis, viral meningitis, aseptic meningitis and chronic meningitis.Meningitis is a serious disease that can be life-threatening and result in permanent complications, such as coma, shock, and death. Meningitis is a serious infection of one of the membranes that surrounds the brain. Acute meningitis caused by bacteria is called acute bacterial meningitis and develops very quickly in a matter of hours or days. Acute bacterial meningitis is generally the most serious type of meningitis. The pathogens that can cause many forms of meningitis are carried by humans in the nose and throat and are spread into the air by coughing and/or sneezing. Once pathogens are airborne, they can be picked up by anyone who breathes them into their respiratory tract. The pathogens then spread from the respiratory tract to the blood stream and to the nervous system.Symptoms of meningitis include a high fever and stiff neck. Serious complications can occur, especially with acute bacterial meningitis. In some cases death can happen in a matter of daysMaking a rapid and accurate diagnosis of meningitis begins with taking a thorough personal and family medical history, including symptoms, and completing a physical examination. Diagnostic tests include a lumbar puncture, also called a spinal tap, which involves withdrawing a small sample of cerebrospinal fluid (CSF) from the spine with a needle. The sample of CSF is tested for white blood cells and other indications of meningitis.Additional tests may be performed in order to rule out or confirm other diseases that may accompany meningitis or cause similar symptoms, such as high fever, headache, and neck stiffness. These may include a throat culture, CT, or X-rays.It is possible that a diagnosis of meningitis can be missed or delayed because some symptoms, such as fever, headache, and nausea and vomiting, are similar to symptoms of other diseases. In infants, signs of acute bacterial meningitis can include excessive crying, excessive sleepiness, difficulty with feeding, and a bulging of the soft spot on the top of the head. Serious complications of acute bacterial meningitis include kidney failure and permanent neurological damage, such as blindness, hearing loss, brain damage, and paralysis. Causes of bacterial meningitis :Meningococcus Pneumococcus Hib Tuberculosis E. coli Group B Streptococcus other causes of bacterial meningitis mainly in newborns:Proteus Klebsiella Citrobacter Salmonella Pseudomonas (type of Nosocomial infections) Serratia Achromobacter

Acute bacterial meningitis is one of the common causes of morbidity and mortality in children under 5 years of age in developing countries (Saha, 1997). It is well known that H. influenzae and S. pneumoniae and N. meningitidis are responsible for 80% of meningitis cases (Chowdhury et al., 1992).

1.2 MENINGITIS CAUSES: RISK FACTORS The following conditions have been cited in various sources as potentially causal risk factors related to Meningitis:Age under 1 year Age under 5 years Age group 15-24 Contacts of confirmed meningococcal cases Immune compromise Overseas travel - especially to Africa or parts of Asia where meningococcus is more common. Institutions - have been associated with meningococcal disease outbreaks More risk factors.Data on the aetiology of acute bacterial meningitis in Bangladesh are few. This study has been designed to shed light on the incidence, rapid diagnosis and aetiological trends in bacterial meningitis in childhood less than twelve years of age in Bangladesh.

1.3 BACTERIAL MENINGITIS The subarachnoid space and its CSF are relatively defenseless in stopping invasion by bacterial pathogens because of the CSF's paucity of phagocytic cells and low concentrations of complement and immunoglobulin. Unchecked invasion and multiplication of bacteria in the CSF result in meningitis. The pathophysiology of bacterial meningitis has been studied experimentally and is reasonably well understood (McGee et al., 1990, Saez-Llorens et al., 1991 and Tunkel et al., 1990). Inflammation of the meninges is initiated by the presence of bacterial lipopolysaccharide, teichoic acid, and/or other bacterial cell wall components in the subarachnoid space. The bacterial antigens stimulate monocytes to produce the cytokine interleukin-1 and stimulate macrophages, astrocytes, microglial cells, ependymal cells, and endothelial cells in the central nervous system to produce the cytokine tumor necrosis factor (cachectin). Tumor necrosis factor and interleukin-1 probably act synergistically to elicit inflammatory responses which manifest clinically as meningitis. A logical temporal sequence of such responses is as follows: chemotaxis and adherence of polymorphonuclear leukocytes to cerebral capillaries; damage to capillary endothelial cells; structural changes in the bloodbrain barrier; cytotoxic parenchymal edema; increased intracranial pressure; decreased intracranial perfusion; cerebral infarction; and focal or diffuse brain damage.Acute bacterial meningitis is a severe childhood illness. In developing countries like Bangladesh, it is a leading cause of bacterial meningitis (Salisbury, 1998), responsible for over 200,000 cases and more than 40,000 deaths annually (Mulholland et al., 1997; Salisbury, 1998). Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae remain important pathogens (Schlech et al., 1985, Schuchat et al., 1997). The prevalence of these organisms varies from place to place, by age and by season (Schlech et al., 1985), but N. meningitidis is more often the commonest cause of meningeal infection (Bell & Silber, 1971) with S. pneumoniae (Spink & Su, 1960) and H. influenzae (McGowan, 1974) being second and third respectively. However, the order is reverse in several studies in Bangladesh (Saha et al., 1997). Recurring epidemics of meningococcal disease (Booy & Kroll, 1998), increased antibiotic resistance among pneumococci (Goldstein & Acar, 1996), and failure to introduce conjugate Hib vaccines into many developing countries means that bacterial meningitis remains a serious global health problem.Children are most likely to get meningitis during their first year (Schlech et al., 1985; Panjarathinam & Shah, 1993). Children who were infected as neonates had more health and developmental problems than those who had meningitis when they were older than one month (Bedford et al., 2001). The rates of severe or moderate disability differ widely between children infected with different organisms (Bedford et al., 2001). Infection with

S. pneumoniae has been reported to be associated with a higher rate of disability than infection with H. influenzae and N. meningitides (Bedford et al., 2001; Baraff et al., 1993).Even with the advances in the development of many powerful antimicrobial agents, bacterial meningitis still remains a serious cause of morbidity and mortality in childhood. Although it is widely acknowledged that the consequences of meningitis in infancy may be severe, there are few studies that focus on the etiologic diagnosis of bacterial meningitis (Rahman et al., 1990; Saha et al., 1997; Saha 2003; Saha et al., 2005; Hoque et al., 2006), and no reliable data from large prospective studies that focus on the outcome of infection in infancy or fatality in Bangladesh. Since meningitis is potentially hazardous disease of childhood, diagnostic tests that are readily available, easy to interpret and simple to perform are of paramount importance. Lumbar puncture is frequently performed in primary care (Whistle et al., 1995; Visser & Hall, 1980). Commonly performed tests on cerebrospinal fluid (CSF) include protein and glucose levels, cell counts and differential, microscopic examination, and culture (Seehusen et al., 2003). Culture is the “gold standard” for determining the causative organism in meningitis (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. The present study aimed to etiologic diagnosis bacterial meningitis in a national paediatric hospital in Bangladesh using CSF culture method, and also to evaluate the usefulness of various laboratory procedures in diagnosing bacterial meningitis.Bacterial meningitis remains a very important disease worldwide. From its original recognition in 1805 until the early 1900, bacterial meningitis was virtually 100% fatal (Quagliarello VJ et al., 1997). According to a World Health Organization estimate, approximately 171,000 people worldwide die from bacterial meningitis each year. Even with antimicrobial treatment, fatality rates are as high as 5-10% in the developed world. The incidence and mortality rates are much higher in third- world countries (World Health Organization, 2004.). Between 10% and 20% of those who do survive bacterial meningitis suffer permanent damage such as mental retardation, deafness, or epilepsy. Addition to the tragedy is the fact that these deaths could have been avoided; either through vaccination or by accurate diagnosis and rapid intervention (Babiker et al., 1984).Acute bacterial meningitis is one of the most severe infectious diseases in the childhood. The global burden of the disease is high. Apart from epidemic, at least 1.2 million cases of meningitis are estimated to occur every year with 135,000 deaths (Tunkel et al., 1995). In developing countries like Bangladesh, it is a leading cause of bacterial meningitis (Salisbury, 1998), responsible for over 200,000 cases and more than 40,000 deaths annually (Salisbury, 1998 and Mulholland K, et al.1997). The disease is seen more in children than adults (Babiker et al., 1984). It is caused by a variety of organisms but the most important ones are Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in children and adults (Salisbury DM. and Dagbjartsson A et al., 1997). Other bacteria that have been less frequently implicated as pathogens include Streptococci; Gram-negative enteric or related organisms like Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa had been isolated infrequently from meningitis cases in Bangladesh (Hoque MM et al., 2006). The prevalence of these organisms varies from place to place, by age and season (Durand et al.1993). The specific pathogen causing bacterial meningitis varies around the world (Sigurdardottir et al., 1997, Tunkel et al., 2004 & Hussein et al., 2000). However, there is predominance of Gram- negative organisms as the etiological agents of bacterial meningitis (Van de Beek et al., 2004). To reduce death or predominant neurological sequelae as much as possible, a fast and correct diagnosis is of the utmost importance. The current standard for the diagnosis of bacterial meningitis is microscopic examination and subsequent culture of cerebrospinal fluid (CSF) (Schuurman et al., 2004). Since meningitis is potentially hazardous disease of childhood, diagnostic tests are readily available, easy to interpret and simple to perform are of paramount importance. Lumber puncture is frequently performed in primary care (Wiswell et al., 1995; Shattuck et al., 1992 and Visser et al., 1980). Commonly performed laboratory tests on CSF include protein and glucose levels, cell count and differential, microscopic examination, and culture (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. Culture is the gold standard for determining the causative organism in meningitis (Seehusen et al., 2003). The present study aimed to diagnosis bacterial meningitis using CSF culture method, to assess the prevalence and pattern of antimicrobial resistance of the isolated aetiological agents for proper selection of antibiotic therapy, and also to evaluate the usefulness of various laboratory procedures in diagnosing bacterial meningitis.

Bacterial meningitis is the most common and notable infection of the central nervous system, can progress rapidly, and can result in death or permanent debilitation. Not surprisingly, this infection justifiably elicits strong emotional responses and, hopefully, immediate medical intervention. The advent and widespread use of antibacterial agents in the treatment of meningitis have drastically reduced the mortality caused by this disease. The majority of patients with bacterial meningitis survive, but neurological sequelae occur in as many as one-third of all survivors (especially newborns and children) (Saez-Llorens et al., 1990 & Saez-Llorens et al., 1991) Bacterial meningitis is much more common in developing countries than in the United States.Meningitis is inflammation of the meninges that results in the occurrence of meningeal symptoms (e.g., headache, nuchal rigidity, and photophobia) and an increased number of white blood cells in the cerebrospinal fluid (CSF), i.e., pleocytosis (Razonable et al., 2005). Numerous infectious and non-infectious causes of meningitis are existed1. Acute bacterial meningitis denotes a bacterial cause of this syndrome. Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae are the most common causes of bacterial meningitis in children (Schlech et al., 1985). The prevalence of these organisms varies from place to place, by age and by season (Schlech et al., 1985), but N. meningitidis is more often the commonest cause of meningeal infection (Bell et al., 1971) with S. pneumoniae (Spink et al., 1960 ) and H. influenzae (McGowan, 1974.) being second and third respectively. However, the order is reverse in a study in Bangladesh (Saha et al., 1997).Children are most likely to get meningitis during their first year (Schlech et al., 1985; Health PT, et al. 2003 & Panjarathinam et al. 1993). Children who were infected as neonates had more health and developmental problems than those who had meningitis when they were older than one month (Bedford H, et al. 2001). The rates of severe or moderate disability differ widely between children infected with different organisms (Bedford et al. 2001). Infection with S. pneumoniae has been reported to be associated with a higher rate of disability than infection with H. influenzae and N. meningitides (Bedford et al., 2001; Baraff et al., 1993, McIntyre et al 1993 & Grimwood et al., 1995). Bacterial meningitis has high mortality rate. Prior to the introduction of antibiotics in the 1940S, case fatality rates for epidemic and endemic bacterial meningitis exceeded 70% (Rao et al., 1998). Since then, antibiotic use has reduced case fatality rates for meningitis caused by most bacteria to 25% or less, but no further reduction has been documented in the past two or three decades (Panjarathinam et al., 1993 & Rao et al., 1998). Despite advances in vaccine development and chemoprophylaxis, bacterial meningitis remains a major cause of death and long term neurological disabilities, such as mental retardation, convulsions and hydrocephalus. These are best prevented by early diagnosis and appropriate treatment of the disease. Although lower mortality rates have been reported in industrialized countries such as the United States of America (2.6%) (Pomeroy et al., 1990), higher rates have been reported in some developing countries and countries in the Middle East, such as Turkey (38%) (Gurses N.1997), Saudi Arabia (14.7%) (Srair et al., 1992), Sudan (28.6%) (Ahmed et al., 1996) and India (21.8%) (Deivananyagam et al., 1993). Of the developing countries, the case fatality rate of 13.0% in the Libyan Arab Jamahiriya is not the highest among the world reports (Rao et al., 1998).Although it is widely acknowledged that the consequences of meningitis in infancy may be severe, there are few studies that focus on the aetiologic diagnosis of bacterial meningitis (Saha et al., 1997, Rahman et al., 1990, Saha et al., 2003, Saha et al., 2005 and Hoque et al., 2006), and no reliable data from large prospective studies that focus on the outcome of infection in infancy or fatality in Bangladesh. Since meningitis is potentially hazardous disease of childhood, diagnostic tests that are readily available, easy to interpret and simple to perform are of paramount importance. Lumbar puncture is frequently performed in primary care (Wiswell et al., 1995, Visser et al., 1980 and Shattuck et al., 1992). Commonly performed tests on cerebrospinal fluid (CSF) include protein and glucose levels, cell counts and differential, microscopic examination, and culture (Razonable et al., 2005 & Seehusen et al., 2003). Culture is the “gold standard” for determining the causative organism in meningitis (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. The present study aimed to identify the aetiologic agents of bacterial meningitis in a pediatric hospital in Bangladesh using CSF Culture method, and also to evaluate the usefulness various laboratory procedures in diagnosing bacterial meningitis.Acute bacterial meningitis is an infection of the nervous system that results in inflammation of the meninges, the membranes that surround the brain and spinal cord. It occurs in the US with an annual incidence of

approximately three cases per 100,000 persons (Attia et al., 1999). Overall mortality attributable to bacterial meningitis in various case series has ranged from 15 % to 21 % (Sigurdardottir et al., 1997). Hence, it is extremely important that clinicians be knowledgeable of this condition for a diagnosis to be made and for timely therapy to be instituted.Bacterial meningitis is a serious disease with high morbidity and mortality. To reduce death or permanent neurological sequelae as much as possible, a fast and correct diagnosis is of the most importance. The current standard for the diagnosis of bacterial meningitis is microscopic examination and subsequent culture of CSF. However, this approach might have some disadvantages with regard to the desired rapidity and sensitivity. Results of culture may only be available after 24 to 48 h and sometimes for instance, when the number of viable organisms in the CSF is low it may take even longer. Moreover the sensitivity of microscopic examination and culture of CSF can be debated.First, bacterial concentration in the CSF has a profound effect on the results of microscopy. Regardless of the type of organisms in the CSF, the percentage of positive microscopic results is only 25% with <103 cfu/ml and 60% in the range of 103 to 105 cfu/ml.Second, in an extensive study over a period of 27 years, it appeared that culture might miss the diagnosis of bacterial meningitis in at least 13% of cases. Acknowledged reasons for this lack in the sensitivity are CSF obtained after the start of antibiotic treatment and meningitis due to the fastidious or slow growing microorganisms (Schuurman et al., 1998) Bacterial meningitis continues to be one of the most serious infectious diseases experienced during childhood and despite the availability of newer antibiotics and a greater understanding of the pathogenesis of the disease, a considerable mortality and morbidity may still be experienced. Knowledge of the causative organisms and their antibiotics sensitivity in a region may be of importance.First, when considering empirical therapy when a causative organism cannot be identified. Secondly, in view of the increasing availability of vaccines offering protection against this potentially devastating disease (Donald et al., 1996)Bacterial meningitis is relatively common, can progress rapidly, and can result in death or permanent debilitation. This infection justifiably elicits strong emotional reactions and, hopefully, immediate medical intervention. This review is a brief presentation of the pathogenesis of bacterial meningitis and a review of current knowledge, literature, and recommendations on the subject of laboratory diagnosis of bacterial meningitis. Those who work in clinical microbiology laboratories should be familiar with the tests used in detecting bacteria and bacterial antigens in cerebrospinal fluid (CSF) and should always have the utmost appreciation for the fact that results of such tests must always be reported immediately. Academic and practical aspects of the laboratory diagnosis of bacterial meningitis presented in this review include the following: anatomy of the meninges; pathogenesis; changes in the composition of CSF; etiological agents; processing CSF; microscopic examination of CSF; culturing CSF; methods of detecting bacterial antigens and bacterial components in CSF (counter-immunoelectrophoresis, coagglutination, latex agglutination, enzyme-linked immunosorbent assay, Limulus amebocyte lysate assay, and gas-liquid chromatography); use of the polymerase chain reaction; and practical considerations for testing CSF for bacterial antigens (Gray et al.,1992)In acute bacterial meningitis, the classic symptoms and signs of meningeal irritation are common, but these signs may be present in other diseases like acute viral meningitis, tuberculous meningitis, subarachnoid hemorrhage, etc. Most of the patients of acute meningitis usually receive broad spectrum antimicrobial therapy before any diagnostic approach taken. The CSF should be examined in every patient in whom the clinical findings are consistent with the possibility of meningitis. Alternative methods of CSF study have been developed which may be useful in patients commenced with antibiotic therapy before lumber puncture.Where culture is usually negative, detection of soluble bacterial antigens can help to reach a diagnosis. The Latex Particle Agglutination Test (LPAT) has been introduced for this purpose because it can detect comparatively very small quantity of antigens present.Although specificity of these tests is good, sensitivity is not better than a Gram stain. Therefore, negative results for a specific bacterial antigen do not rule out bacterial meningitis (Phillips et al., 1991).

The main limitation of LPAT is that it is positive only in the presence of specific polysaccharide surface antigens for H.influenzae type b (Hib), S. pneumoniae, E. coli, group B Streptococcus and N. meningitidis A, C, Y, W-135 antigens, while any other bacteria remain undetected. The Gram's staining method does not have this limitation. To optimize cost benefit ratio, the test should not be indiscriminately used. Thus, the LAPT should be done routinely when positive Gram stain and abnormal CSF values (WBC counts, glucose, protein concentrations and other markers) indicated bacterial meningitis.The antigens of common meningeal pathogens e.g. H. influenzae type b (Hib), S. pneumoniae, E. coli, group B Streptococcus and N. meningitidis are detected by the LPAT. The diagnosis of bacterial meningitis caused by specific pathogens is established by positive LPAT, but a negative test does not rule out other bacterial meningitis cases. A broad-spectrum antibiotic coverage is usually recommended as an initial treatment for suspected bacterial meningitis, which is very costly. The LPAT can diagnose these specific bacterial pathogens and specific antibiotic therapy can be given to reduce the emergence of bacterial resistance (Begum N, et al., 2007).Bacterial meningitis in infants and children is a serious clinical entity with signs and symptoms that commonly do not allow distinguishing the diagnosis and the causative agents. Acute meningitis is a common infection, predominantly aseptic (82– 90%), but when of bacterial origin (10-20%), it is infrequently associated with severe neurologically sequelae, especially when the diagnosis and treatment are late (Nigrovic et al., 2002 & Tatara et al., 2000). As it is difficult to distinguish between bacterial and aseptic meningitis in the initial state, most authors have recommended rapid initiation of antibiotics in children with acute meningitis, with conventional therapy until cerebrospinal(CSF)culture results become available, 48-72 hours later (Saez-Uorens et al., 2003 & El Bashir et al., 2003).The pattern of bacterial meningitis and its treatment during neonatal periods may over lap, especially in the first one to three months old in whom group B streptococcus, Haemophilus influenza-type b , meningococcus and pneumococcus may all produce meningitis (Tunkel et al., 2004 & Feigin et al., 1992). In children more than 3 months of age H. influenzae, Streptococcus pneumoniae, Neiseria meningitiditis are the commonest causative organism of bacterial meningitis. The aim of this study is to study the microbiological profile of CSF in childhood meningitis, over a period of one year (Saez-Uorens et al., 1990).In healthy children, the three most common organisms causing acute bacterial meningitis are S.pneumoniae, N meningitidis, and H. influenzae type b (Hib) (Freedom et al., 2001). Although Hib is the commonest causative agent), with the availability of Hib conjugate vaccine, the current likely hood of Hib meningitis in a child who has received at least two doses of vaccine was extremely rare. Lumbar puncture is the gold standard for the diagnosis and should be done in all suspected cases of meningitis unless contraindicated (Freedom et al., 2001). It helps to distinguish the microbial etiology of meningitis and encephalitis, and to rule out non-infectious causes of disease (Kneen et al., 2002). The myth about lumbar puncture complications among parents has to be resolved by the physician in order to get the consent to do the procedure Development of bacterial meningitis progress through the following steps: Bacterial colonization of the nasopharynx Mucosal inflammation and penetration into the blood stain Intravascular multiplication and entrance through the blood brain barer. Generation of inflammation within the subarachnoid space Neuronal cell injury and auditory nerve damage. Children with bacterial meningitis present in one of the following pattern: The most common and insidious form with non specific symptoms that progress over 2 to 5 days before meningitis is diagnosed. A more common rapid form, in which symptoms and signs of meningitis progress over one or two days. A fulminant course, with rapid deterioration and shock early in the course of illness. On physical examination, the fontanel of an infant may be bulging, presumably indicating increased intra cranial pressure; this sign is neither highly sensitive nor specific for meningitis but always requires evaluation. Most specific physical findings of meningitis are Kernig’s and Brundenzki sign and neck stiffness. Papilledema

is uncommon in a child with a uncomplicated meningitis and if present, suggest another cause such as subdural effusion, brain abscesses etc (Saez-Uorens et al., 1990). Petechial or purpuric rash and shock are classically associated with meningococcal meningitis but also can be occasionally caused by H. influenzae or S. pneumoniae (Oastenbrink et al., 2004 & Bingen et al., 2005).

Table 1: Laboratory values of components of CSF from healthy persons and from patients with meningitisa

TraitsCSF laboratory value

Protein Glucose Leukocytes Predominant cell (mg/dl) (mg/dl)b (perl )

Healthy personsNewborns 15 - 170 34 - 119 0 - 30Adults 15 - 50 40 - 80 0 - 10 Lymphocytes

(63-99)Monocytes (3-37)PMN (0-15)Adult patients

with:

Bacterial meningitis

Fungal meningitis

100Increased<100

40<30Normalc

1,000Increased<500

PMN(>50)LymphocytesPMN(early) and lymphocytes (late)

a. Data are commonly observed values. Notable exceptions to these values and overlap of values elicited by different etiological agents are not uncommon. PMN, polymorphonuclear leukocytes. b. The CSF glucose/serum glucose ratio usually is 0.6 (adults) or 0.74 to 0.96 (neonates and preterm babies). In-patients with bacterial meningitis, the ratios usually are <0.5 (adults) and <0.6 (neonates and preterm babies). c. Lower than normal glucose concentrations have been observed during some noninfectious disease processes and in some patients with viral meningoencephalitis due to herpesviruses, varicella-zoster virus, mumps virus, lymphocytic choriome

1.4 ETIOLOGICAL AGENTS OF BACTERIAL MENINGITISThe results of national surveillance studies have shown that both the etiological agents and mortality rates (0 to 54%) of bacterial meningitis depend on the season of the year and the age, sex, ethnic background, and geographic location of the patient (McGee et al., 1990, Schlech et al., 1985 and Wenger et al., 1990). multistate surveillance study of the etiological agents of bacterial meningitis (Wenger et al., 1990). H. influenzae was the most frequent cause of bacterial meningitis (2.9 cases per 100,000 populations) and, paradoxically, was associated with the lowest fatality rate (3%) of the five most frequent bacterial agents. Table 3: Shows the results of a 1986a

Bacterium No. (%)of cases

Incidence(cases/100,000)

Case fatalityRate (%)

Haemophilus influenzae 964(45) 2.9 3Streptococcus pneumoniae 379(18) 1.1 19Neisseria meningitidis 293(14) 0.9 13Streptococcus group B 122(5) 0.4 12Listeria monocytogenes 69(3) 0.2 22Otherb 331(15) 1.0 18

aData were obtained from a surveillance study by Wenger et al.,1990 and are used with permission of the publisher.

bOther bacteria include Streptococcus spp. other than group B, S. aureus, E. coli, S. epidermidis, Klebsiella spp., Enterobacter spp., Serratia spp., and Acinetobacter spp. On the other hand, Listeria monocytogenes was reported relatively infrequently (0.2 case per 100,000 population) but had the highest fatality rate (22%). Table 4 contains additional data from the aforementioned 1986 study and shows the distribution of etiological agents of bacterial meningitis in five commonly defined age groups. Streptococcus group B (Streptococcus agalactiae), H. influenzae, N. meningitidis, and Streptococcus pneumoniae were the leading causes of bacterial meningitis in neonates, young children, young adults, and adults and senior adults, respectively. Certain elements of a patient's history (e.g. predisposing factors, medical condition, epidemiology, occupation, and immune status) can suggest specific bacterial agents of meningitis (Isenberg et al., 1991; Kaufman et al., 1990 and McGee et al., 1990). Unusual and rare bacteria that have been reported to cause meningitis include Bacteroides fragilis (Odugbemi et al., 1985).

TABLE 4: Etiological agents of bacterial meningitis in five age groups (1986)a% of casesb caused by:Age Streptococ

-cus groupB

L.monocytogenes

H.influenzae

S.pneumoniae

N.meningitidis

Otherc

0-1 mo 49 9 5 3 1 332mo-4 yr

2 70 10 13 5

5-29 yr 2 2 8 17 42 2930-59 yr 4 6 5 37 10 38≥60 yr 3 14 4 48 3 28

aData were obtained from a surveillance study by Wenger et al., 1990 and are used with permission of the publisher. bThe percentages were extrapolated by us from the data in reference (Wenger et al., 1990 ).cOther bacteria include Streptococcus spp. other than group B, S. aureus,E. coli, S. epidermidis, Klebsiella spp., Enterobacter spp., Serratia spp., and Acinetobacter spp

1.5 STREPTOCOCCUS PNEUMONIAE1.5.1 Introduction Streptococcus pneumoniae is one of the leading causes of morbidity and mortality throughout the world including Bangladesh. It is most common cause of community acquired bacterial pneumonia and the second most common cause of bacterial meningitis in children less than five years of age in Bangladesh and across the developing world. It also causes abdominal infection, bactermia, otitis media and various diseases. Most children experience some form of pneumococcal infections and some develops sepsis or meningitis. Despite over a century of research, many aspects of pneumococcal disease remain obscure. The continued frequency and severity of pneumococcal disease, coupled with the knowledge that use of antimicrobial agents does not invariably present or death and the recent emergence of strains of S. pneumoniae resistant to most antimicrobial agents, all severe to underscore the need for better understanding of pneumococcal infections. Particular attention is presently concentrated on efforts to prevent these in infections by development of appropriate vaccine (Feigin and Cherry et al., 2000).Meningitis occupies only a small portion of the spectrum of diseases caused by pneumococcus. The organism has positioned itself as the leading cause of mortality inunder-five children in developing countries, mainly by causing pneumonia, a silent killer of third world children.World Health Organization (WHO) calculates that the organism, as a whole, by causing pneumonia, meningitis, sepsis and other diseases, kills more than 800,000 under-five children worldwide (WHO. Pneumococcal vaccines.Wkly Epidemiol Record 2003, Williams,et al., Lancet ID2002) and 90% of them are in the developing part of the world (httt://www.preventpneumo.org/disease_vaccines).

Approximately 20% to 40% of global total of four million deaths of children bellow five years of age occurred from pneumonia globally per year (Saha et al., 1999). Such a fetal causing agent (pneumonia) of human morbidity and mortality required special study and research in locally and globally but not a perfect study has been done in Bangladesh in this field. By considering the situation our study on pneumococci has special importance. Various antibiotic (penicillin) resistance or decreased susceptibility to antibiotics among the clinical isolates of S. pneumoniae became a threatening problem, which increase, dramatically in the last decade (Felminham et al., 1999). Bangladesh is not devoid of this situation due to the worldwide migration of the antibiotic resistant clones of S. pneumoniae along with the migration of the pneumococcal carrier or pneumococcal infected individuals. How fast the antibiotic resistant pneumococcus has been spread and grown up, could be understand by the following examples.In England and Wales, for example, erythromycin resistant rate increased from 2.8% to 8.6% in the year between 1990 to 1995 (Goldsmith et al., 1997). In a study in central Italy, erythromycin resistant rate increased from 7.1% to 32.8% in the year between 1993 to 1997 (Oster et al. 1999). In France, resistance to erythromycin was first described in 1976 and increased to 20% in 1984 then to 29% in 1990 (Geslin et al., 1992). On the other hand, the first case of clinically significant penicillin resistant isolates was reported in Australia in 1967 (Hansman et al., 1967) an abrupt increase of penicillin resistance among pneumococcal was reported in last decade. For instance, the frequency of penicillin resistance in Italy increases from 5.5% to 7.7% in the year between 1993 to 1996 (Marchese et al., 1998). The preliminary report from Bangladesh showed that 10% of S. pneumoniae strains were resistant to penicillin (Saha et al., 1992). If the above rate of antibiotic resistance is continued, all the antibiotics designed for S. pneumoniae will be infective against pneumococcal infection and that would be a threatening condition for the human health. But studies on the prevalence, serotypes and detection of antibiotics resistant clones and their distribution are few in numbers in Bangladesh. To understand and contain this crucial problem first we have to know the prevalent serotypes, patterns of antibiotics resistance and the clonal distribution of pneumococcal clinical isolates from the individuals in Bangladesh. Prospective neurodevelopmental assessments of pneumococcal meningitis patients in Bangladesh, 3-4 months and 12-24 months after discharge from the hospital, revealed that about 65% and 49% of cases survived with one or more impairments and permanent disabilities respectively, including deafness, vision loss, mental shortfall, and psychomotor deficits. Other studies in India (Gupta V, 1993), Pakistan (Qazi SA, et al., 1997), Sudan (Salih MA, et al., 1991), and Vanuatu (Carrol KJ, et al., 1994) have shown similar results.This high prevalence of disability from pyogenic meningitis cases can be attributedto poor care-seeking behavior, and delay in diagnosis and treatment of meningitis (Salih MA, et al., 1991 and Richardson MP, et al., 1997).

1.5.2 Bacteriology Streptococcus pneumoniae along with many other species of Streptococci, e.g., S. pyogenes; S. agalactiae, etc. belongs to the pyogenic category of Streptococcus genus. Table 1.6 shows the comparison between the pyogenic Streptococcal species (Bergy’s manual of systemic bacteriology 1998).

1.5.3 Morphology Streptococcus pneumoniae is encapsulated Gram-positive diplococci, oval or spherical in shape, and 0.5 to 1.25 m in diameter. It occurs singly in pairs and short chains occasionally as individual cocci (Willett et al., 1992). Continued laboratory cultivation, especially on unfavorable media or in the presence of type specific antibody, leads to the formation of larger chains or may be found in short chains and particularly with a low Mg++ concentration. Gram-positive reaction of young cells may be lost as culture ages and subsequently strains Gram-negative.

1.5.4 Biochemical properties Surface-active agents, such as bile salts or sodium deoxy-cholate stimulate autolysis. The test for “bile solubility” is useful in identification of pneumococci. They are inulin fermenters. Deoxy-cholate activates an

amidase that splits the tetra peptide from muramic acid in peptidoglycan. This reaction is properly regulated; normally function in wall morphogenesis and cell division.

1.5.5 Cultural characteristics The Streptococcus pneumoniae has complex nutritional requirements. It can be grown on chemically defined synthetic media but for primary isolation and routine culture, enriched infusion agar and broth such as trypticase soy brain heart infusion enriched with 5% defibrinated sheep or goat or horse blood, is recommended (Willett, 1992). The optimum PH and temperature for growth is 7.4 to7.8 and 370C, respectively. All pneumococcal strains require an increased CO2, concentration for primary isolation on solid agar media. A candle extinction jar or CO2 incubator should be used for this purpose. Colonies on blood agar plates are small, smooth and transparent. Low convex while tiny, they become flattened or depressed centrally, showing the “draughtsman form.” As they grow older. Some strain e,g. of type 3, which form large capsule, tent to remain convex (Mackie & Mc. Cartney 1996). Uncapsulated strains produce small rough colony. A partial clearance of blood and a greenish discoloration (α-hemolysis) is produced underneath. A narrow zone around the colonies formed when they are incubated aerobically. Aerobic incubation results in β-hemolysis due to pneumolysin ‘O’ activity. Unlike other Streptococcus, S. pneumoniae requires cholone for growth in defined media. Ethanolamine replaces choline but not on a molar basis. Ethanolamine substitution during growth leads to a number of physiological defects including:Resistance to autolysis, Aberrant cell division, Incompetence in transformation, and Phage resistance.

1.5.6 Physiology Reducing agents are essential for S. pneumoniae. Most strains require at least 4 of the B vitamins for growth as well as adenine, guanine, and uracil and 7 to 10 amino acids. S. pneumoniae is a facultative aerobic and its energy-yielding metabolism is fermentative, yielding primary low level of lactic acid. Under aerobic condition a significant amount of hydrogen peroxide (H2O2) accumulates, with some degree of acetic and formic acids. S. pneumoniae does not produce catalase or peroxidase and the accumulation of hydrogen peroxide kill the organism unless catalase is provided by the addition of RBC (Red blood cell) to the culture media (Willett, 1992). The organism tents to die fairly quickly in cultures e.g. in course of a day or two, particularly in aerobic cultures media without blood. The dead organism tents to undergo autolysis (Mackie et al., 1996).

1.5.7 Diagnosis of pneumococcal infections Demonstrating the presence of pneumococcal in a specimen of sputum, lung aspirate, CSF, urine or blood directly by Gram-stain and culture, and then identifying the culture in an optochin sensitivity test generally do diagnosis of S. pneumoniae. The approach is likely to be successful when a heavily infected specimen is collected early in the ilness and before the start of antibiotic therapy. Now a day, because of the emerging problem of antibiotic resistant pneumococci, definitive diagnosis is absolutely necessary. There are four ways of detection of pneumococci:Microbiological identificationSerological identificationMolecular biological testAnimal pathogenecity test

1.6 MICROBIOLOGICAL IDENTIFICATION 1.6.1 Gram-StainUnder microscopic examination, pneumococci appear as Gram-positive diplococci (Bergy’s manual of systemic bacteriology 1998, vol.II).

1.6.2 Optochin sensitivity testCommercially available optochin disks are applied to a quarter of a blood agar plate that has been streaked with a few colonies of the organism to be tested. After overnight incubation at 350C in either candle-extinction jar or a CO2 incubator, inhibition zones are measured. Zone of >14 mm with a 6 mm disc or >16mm with an mm disk indicates inhibition and identify the isolates as S. pneumoniae. Isolates laying smaller zone of inhibition should be subjected to an additional test for bile solubility to confirm their identities (Willett et al., 1992)

1.6.3 Bile solubility testPneumococci are soluble in bile but viridans and other Streptococci are not (Mackie and Mc. Cartney, 1996). This test is based on the presence of an autolytic amidase in pneumococci, which cleaves the bond between alanine and muramic acid in the peptidoglycan. Surface-active agents such as bile or bile salt, resulting in lysis of the organism, activate this amidase. For testing a neutral pH, 10% deoxycholate and viable young organisms are required (Willett et al., 1992).

1.6.4 Quellung ReactionThis is the most useful and rapid method for identification of S. pneumoniae. The test not only identifies an organism as a S.pneumoniae but also specifies its type (Willett et al., 1992). The Quellung or “capsular swelling” reaction is actually a reaction between the bacterial capsular polysaccharide and its homologous antiserum.

1.7 SEROLOGICAL IDENTIFICATION 1.7.1 Counter current immunoelectrophoresis (CIE)The method described by Dulake (1979), is an especially sensitive method for detecting capsular antigen and may give a positive result when the coagglutination test is negative (Mackie & Mc. Cartney, 1996). The methodology of CIE is based on the fact that most soluble bacterial antigens are negatively charged in slightly alkaline media (pH 8.2- 8.6). Under the same condition antibody (usually rabbit Ig) is neutralized or slightly changed. When an electric field is applied the resulting antigen-antibody complex forms a precipitation line, in between two walls. By varying antibody, the bacterial antigen can be determined.

1.8 MOLECULAR BIOLOGICAL METHOD 1.8.1 Polymerase chain reaction (PCR)The polymerase chain reaction (PCR) is a technique that can be used to find very low quantities of an infectious agent present in clinical sample by increasing the quantity of a specific nucleotide sequence continued within the organism by a process of detecting in-vitro DNA synthesis. With the advent of PCR technique (Mullis & Falona, 1987) several workers attempted to utilize this technique to detect S. pneumoniae from clinical samples. The pioneer in these cases is Rudolph et al., (1993). They developed a nested PCR protocol, and used blood sample from patient’s culture proven bacteremia. The next work for the detection of S. pneumoniae DNA in blood cultures by PCR was done by Hassan-King et al., 1994. The objective of his study was to develop a PCR assay that would improve the frequency of detection of S. pneumoniae in patients with septicemia. In August 1994 Zhang et al published his work on detection of S. pneumoniae penicillin binding protein 2B gene. In this study QIA Amp Kit was used for the processing of whole blood. Salo et al. From Finland in 1994 developed a PCR diagnosis method of S. pneumoniae by amplification of pneumolysin gene fragment in serum. They used a nested PCR strategy for the detection assay. Their study identified, besides all culture positive samples, 6 culture-negative samples for S. pneumoniae in 100 healthy patients by PCR. In February 1996, Hassan-King et al. published a multiplex PCR assay for the simultaneous detection of S. pneumoniae and H. influenzae type b. They used the primers from the autolysin’s gene and used blood as sample. A semi-nested PCR strategy for the detection of penicillin resistant S. pneumoniae from CSF was developed by Mignon et al. (1997) from South Africa. Very recently, an article from Israel has shown a prospective study for the detection of pneumococcal DNA in sera of children by single PCR (Dagon et al., 1998).

1.8.2 Identification by DNA probes hybridizationNucleic acid hybridization tests often reduce the time necessary to identify microorganism. They also allow laboratories to increase the number of types of pathogens that can be easily detected and identified. Nucleic acid probes are segments of DNA or RNA that have been labeled with enzymes, or radioisotopes and can bind with high specificity to complementary sequences of nucleic acid. Oligonucleotide probes can be chemically synthesized and purified with relative ease. DNA probe from lyt A gene was constructed and is used for S. pneumoniae detection. Dot blot hybridization is used for the assay. For the process 0.5N NaOH denatured the probe for lyt a gene. Denatured DNA (0.1 ml) was applied to 96 wells Bio-Dot apparatus containing the sample (Pozzi et al., 1989).

1.8.3 Drug resistance in Streptococcus pneumoniaeFollowing the introduction of penicillin in 1940, pneumococci wre regarded as uniformly sensitive to this antibiotic. This brief was so well establish that the use of in vitro susceptibility testing was not considered necessary (Zighelboin et al., 1981, Hussein et al., 1989). This idea persisted until 1967 when the first strain showing increased resistance to penicillin was isolated (Hansman and Bullen, 1967).Various patterns of S. pneumoniae resistant to drug other than penicillin had been reported. Strains resistant to penicillin, tetracycline, erythromycin, and chloramphenical in various combinations have been reported from different parts of the world (Hansman et al, 1974 & Cates et al, 1978). In the present study, resistance to tetracycline, Co-trimoxazole, chloramphenical and erythromycin was 70%, 43%, 12% and 4% respectively. Tetracycline resistant pneumococci strains averaged 13% in 1975 in England (Repoted of an Adhoe study Group on antibiotic resistance, 1977), 58% in 1983 in Hong Kong (Long et al., 1983), 67% in spain in 1982 (Casal, 1982), 67% in 1987 in Saudi Arabia (Mahgoub and Hussein, 1987) and 70% in the present study. Most studies indicate that approximately 10% of pneumococci are resistant to co-trimoxazole (Malatovic et al., 1981, Michel et al., 1983 and Henderson et al., 1988).In Bangladesh, the pneumococcus has been identified as the predominant cause of meningitis and pneumonia in children (Khan et al., 1989, Saha et al., 1988) and there are no reports of penicillin resistance of this organism. In a study all strains were sensitive to penicillin (Saha et al., 1988). And in other (Khan et al., 1989) no sensitivity pattern was shown in the first study, however, the total number of strains was only eight and disc diffusion method was used, with penicillin disc (10 micro gram) to detect resistance to the drug. The validity of this test is questionable since it does not detect penicillin resistant pneumococcus strains. Since 1989 Dr. S.K. Saha and his colleagues screened 51strains of pneumococci isolated from cerebrospinal fluids (CSF) (39) and blood (12) for resistance to penicillin. The minimum inhibitory concentrations (MIC) were measured on Muller-Hinton agar containing 5% sheep blood and various concentrations of antibiotic (Saha et al., 1998). Of these 51 strains 2 (3.9%) were fully resistant (MIC>1.0 micro gram/ml) 4 (7.8&) were moderately resistant (MIC> 0.12-1.0 micro gram/ml). The remainder (88%) were sensitive to penicillin (MIC<0.06 micro gram/ml) (Saha et al., 1991). However, drug resistant pattern of pneumococci other than penicillin is not yet known in Bangladesh.

1.9 Prevention by vaccineDespite, effective antimicrobial agents severe pneumococcal infections are common and an excessive number of deaths still occur. As a result, there is an urgent need for vaccines that reduce the mortality associated with infections caused by S. pneumoniae (Frank Shann 1995). Polysaccharide vaccineA polyvalent polysaccharide vaccine was developed for the prevention of serious pneumococcal infections.Its wide spread use in adults “at risk” for pneumonia, immunocompromised with chronic diseases or over 65 years age can be expected to reduce mortality, morbidity and associated health care costs (Carol Brignoli Gable et al., 1990). The currently available pneumococcal vaccine contains purified capsular polysaccharide antigens of 23 serotypes of S. pneumoniae. Each 0.5 ml dose of vaccine contains 25 microorganisms of each polysaccharide antigen, compared with 50 micrograms of each in the 14-valent vaccines, which was replaced in 1983 (Mclyntyre et al., 1997).

Efficacy of the vaccine may decrease with increasing age time since vaccination. Among older adults, antibody levels may decrease after 6 years to levels that are not protective (Saha et al., 1996). Almost no response is seen in individuals with leukemia lymphoma or Hodgkin’s disease. Even infants below one year of age will respond to few serotypes but the antibody does not persist (Mclyntyre et al., 1997). A maternal immunization study by Staid et al., has shown that maternal antibody is passed on to the fetus in concentrations two to three fold higher than those of control infants. The burden of pneumococcal disease in developing countries is greater in children than 6 months os age, and therefore because these children are unlike to be fully immunized with pneumococcal conjugate vaccine before 4 months of age and the route of maternal immunization is one that deserves greatest attention (Fedman & Klugman, 1997).

1.10 Conjugate pneumococal vaccine Coupling of pneumococcal polysaccharides to protein has been shown to enhance the immune system response to the polysaccharide moiety in animals, therapy resulting in protective immunity against S. pneumoniae. The immunological basis for the increased immunogenicity of polysaccharide-protein conjugates is related to the T-cell dependent character of these conjugates. Upon repeated immunization increased number of activated protein-specific thymic cells is thought to provide help to polysaccharide-specific B cells, resulting in their differentiation towards memory or plasma cells. Pneumolysin is to date the best studied pneumococcal protein. Virtually all clinical isolates of S. pneumoniae produce pneumolysin. Its primary structure is remarkable as well as being independent of capsular serotypes, geographic area, and time of isolation. Although some native pneumolysin has strong toxic effects several derivatives, which are nontoxic but retain immunogenicity and protective activity of the native protein has been engineered. These constructs therefore seem to meet many criteria for inclusion in a pneumococcal conjugate vaccine. The conjugate induced substantially higher antibody responses than did the polysacchiride alone. This strongly suggested that the carrier protein (Velasco et al., 1995) activate thymic cells.

1.11 Pneumococcal infection in BangladeshS. pneumoniae is the most prevalent cause of community acquired bacterial pneumonia, and of other respiratory infections throughout the world and in Bangladesh, being a developing country, is not an exception. Although not much work has beendone on this important pathogen in Bangladesh, the limited data suggested that it is commonest cause of child mortality and morbidity due to acute respiratory infections. One hundred sixty-five invasive S. pneumoniae strains were isolated from children under five years of age at Dhaka Shishu (children) Hospital during the period 1992 to 1995. Ninety-four (57%) of the strains were isolated from 412 pyogenic cerebrospinal fluid (CSF), and 71 (43%) were from blood of 531 pneumonia patients. The number of male and female patients 111 and 54, respectively, a ratio of 2.05 (Saha et al., 1997). More than 91% of the strains were isolated from patients aged 24 months or less. Eighty –nine percent (146 of 165) of the strains isolated were isolated from patients in the 2 to 24 months age group, and 56% (93 of 165) were from the patients aged 6 to 24 months. Only 3% (5 of 165) of the isolates were from the patients in the neonatal age group (0 to 30 days), and 8.5% (14 of 165) were from the patients 2 years old or older (Table 1.2). predominant serotypes were, in descending order, 7F, 12F, 14, 15B, 4, 23F, 18, 5 and 22A (Saha et al., 1997).Table 1.2 Major Serogroups and types of pneumococcal isolates from patients of different age groups (Saha et al., 1997).Age(Month)

Serotypes Total no. of isolates

Cumulative no. of isolates (%)

1 4 7F 12F 14 15B 23F other

0-1 2 0 0 1 0 0 0 2 5 5(3.0)1-2 0 1 1 0 0 2 1 2 7 12(7.3)2-6 5 0 11 8 6 5 0 11 46 58(35)6-12 2 3 10 9 9 7 3 24 67 125(75.7)12-24 0 4 4 3 2 2 1 10 26 152(91.5)

24-60 2 0 7 1 0 1 1 2 14 165(100)

1.12 As a cause of meningitisS. pneumoniae is the second most common cause of bacterial meningitis in Bangladesh. A laboratory–based study of diagnosed bacterial meningitis in the national pediatric hospital identified 587 culture positive cases from 862 cases of meningitis in 8-years period (Saha et al., 1996). According to the scientists, H. influenzae was the most frequent cause (47%) for meningitis followed by S. pneumoniae (32%). This study also indicated that S. pneumoniae was the prominent organism for meningitis until 1987, a 70% increase in the H. influenzae isolates was obtained among patients with acute bacterial meningitis (Saha et al., 1996).

1.13 HAEMOPHILUS INFLUENZAE 1.13.1 Introduction Invasive bacterial infections are a leading cause of childhood morbidity and mortality worldwide, and H. influenzae is one of the most important bacterial pathogens. It is the most common cause of bacterial meningitis in many developed and developing countries. In developing countries the case fatality rate for this disease is as high as 40%. H. influenzae also is responsible for sepsis, acute lower infections (ALRIs) are the leading cause of death for young children, and H. influenzae causes a substantial proportion of severe ALRIs.Several promising H. influenzae type b vaccines that induce protective immunity in young infants are now available. Unfortunately, the spectrum and incidence of invasive H. influenzae in developing countries is not well characterized, and the exact role that type b strains play in the etiology of pneumonia remains unclear. The potential benefit provided by the use of new H. influenzae type b vaccines in such settings has yet to be determined. This article will review the role played by H. influenzae in serious infections in developing countries and the prospects for prevention of disease by immunization with H. influenzae type b conjugate vaccine. (Funkhouser et al., 1991)

1.13.2 MicrobiologyThe pathogenicity of H. influenzae is determined in large degree by structure on the surface of organism. For encapsulated strains, the outermost structure is the polysaccharide capsule, which consists of one of six antigenically and biochemical distinct types (a-f), the type b polysaccharide consist of a repeating polymer of ribosyl and ribital phosphate (PRP), this capsule antigen is released in vivo but is not degraded and can be detected in body fluids by a variety of immunologic techniques, which often are useful for rapid diagnosis. Considerable evidence suggests that PRP is the major virulence factor of the organism. Antibody to PRP capsule enhance activation of compliment, opsonization, phagocytosis and killing and thereby limits bacteremia and death in animals challenged with H. influenzae type b. In human study, antibody to the PRP capsule has been both therapeutic and protective.Several outer membrane proteins (OMPs) of the H. influenzae cell wall are also being considered as potential vaccine immunogens for the prevention of both type b and non type b disease. OMPs have used to characterize the genetic heterogenecity of type b isolate in different geographic regions. Other constituents of the organism’s cell wall, such as lipopolysaccharide (i.e., endotoxin) and pili appear to be important determinants of pathogenicity but do not appear to be promising vaccine immunogens because of toxicity, poor protective efficacy, and heterogenicity among strains.Colonization of the upper respiratory tract (Funkhouser et al., 1991) ,H. influenzae, particularly non-encapsulated strains, is ubiquitous organisms that colonize the upper respiratory tracts of most humans without causing illness. Studies of healthy infants in developed countries have fund that by the age of 5 years virtually all children have been colonized with one or H. influenzae strains. In contrast, one study of healthy infants in Papua New Guinea showed that almost all infants were colonized with at least one strains of H. influenzae by 3 months of age. Another study in the same country found that 96% of children 6 month to 5 years of age were colonized with at least one strains of H. influenzae and 74% of the isolates were non-capsulated.H. influenzae type b strains also colonize the upper respiratory tract of healthy children but are less prevent than non capsulated strains. In one study in Alaska, the prevalence of colonization with H. influenzae type b was

found to increase from 0 during the first year of life to 3% to 5% by 3 year of age. A longitudinal study in the United States showed that 40% of children carried H. influenzae type b in their upper respiratory tract during the first 5 year of life. Some evidence suggests that in developing countries rates of colonization with type b may be higher. A study of prevalence of colonization in healthy children <5 years of age in Papua New Guinea found that 10% were colonized with H. influenzae type b, in another prevalence study, the rates of colonization with type b among healthy Gambian children were found to be as high as 33% during the first 5 years of life. Colonization rates as high as 50% are seen in individuals who have been in close contact with children with invasive H. influenzae type b disease in settings such as households, day care centers, and orphanages. (Funkhouser et al., 1991)

1.13.3 MeningitisThe epidemiology of H. influenzae has been characterized more fully than has the epidemiological of ALRI and other invasive disease. Because the epidemiologically of meningitis has been easier to establish than the etiology of pneumonia, meningitis meningitis might severe as a marker for an overall incidence of invasive H. influenzae disease. H. influenzae is the leading cause of bacterial meningitis in many areas of the world, including most of Europe, North America, India, Papua New Guinea, the Gambia and Chile. In coutries where disease due to Neisseria meningitides predominates, H. influenzae is the second most common cause of meningitis. More complete and reliable incidence data for H. influenzae meningitis are available from developed countries and are reviewed elsewhere (Funkhouser et al., 1991).The reported incidence of H. influenzae meningitis in some developing regions is very high. The case fatality rates for H. influenzae are also much higher in most developing countries. Studies in Africa and Papua New Guinea have found case fatality rate for H. influenzae meningitis of 20% -40%, whereas in Alaska, elsewhere in the United States and in Europe, the rate is 5%. Several studies have shown various patterns in the epidemiology of H. influenzae meningitis among different ethnic groups in the single geographic area. The incidence of H. influenzae disease is much higher among the Bedouins than among the Jews in Israel.In meningitis the serotypes of the H. influenzae isolates obtained from spinal fluid are almost always type b. Of the isolates from individuals from Papua New Guinae and Alaska from Australian aboriginals and from U.S. Apachs, 80% - 95% are type b, the remaining few isolates are serotype a or non- encapsulated strains(Funkhouser et al., 1991).

Table: Etiology of bacterial meningitis in children <5 years of age.Percentage of cases of meningitis with indicated etiologyStudy site H. influenzae S. pneumoniae N. meningitidis OtherEuropeEngland 37 12 48 3France 41 19 37 3North AmericaUnited States 70 17 10 3AfricaSenegal 41 39 10 10Nigeria 38 39 8 15Cameroon 42 43 3 12Egypt 25 36 20 19Ethiopia 41 22 25 12AsiaPapua New Guinea

49 43 5 3

India 40 20 40 18South America

Chile 65 30 6 2

1.14 NEISSERIA MENINGITIDIS 1.14.1 Introduction N. meningitides is a major cause of epidemic and endemic meningitis in developing countries, particularly in sub-Saharn Africa in what is known as the meningitis belt (Stretching from Senegal, the Gambia and Guninea Bissau in the west Ethiopia in the east) epidemics occur in the dry season often with high loss of life, particularly among children, and young adults. In recent year, major’s epidemics have also been reported from Kenya, Tanzania, Mozambique and South Africa.Up to 90% of outbreaks are due to group A meningococci, Group C has been reported as causing epidemics in Africa, Asia, and South America. Outbreaks due to group B occur in Cuba, and South America and endemic meningitis due to group B occurs in Africa and elsewhere. Outbreaks due to group W135 occur only occasionally. Knowing the serogroup in an epidemic is important in planning vaccination.N. meningitides causes pyogenic (purulent) meningitis, usually following bacteraemia. It often has a sudden onset with intense headache, vomiting, and a stiff neck. N. meningitides occurs as a commensal in the nasopharynx of up to 25% or more of healthy people. Meningococcal septicemia, a severe and often fatal condition with high fever and characterized by rapid circulatory collapse and a haemorragic rash. Petechiae can often be detected in the conjunctivae.Chronic meningococcal arthritis, an uncommon condition. (Monica cheesbrough et al) During epidemics, children and young adults are most commonly affected, with attack rates as high as 1000/100000 population, or 100 times the rate of endemic or sporadic disease occur in children less than 2 years of age. In developed countries, endemic disease is generally caused by serogroup B and C. epidemics in development countries are typically caused by serogroup C although epidemics due to serogroup B have also occurred in Brazil, Chile, Cuba, Norway, and more recently in New Zealand. The risk of secondary cases of meningococcal disease among close contacts (i.e. household members, day care centre contacts, or anyone directly exposed to the patient’s oral secretions) is high. Antimicrobial chemoprophylaxis with a short course of oral rifampin, a single oral dose of ciprofloxacin, or a single injection of ceftriaxone is effective in eradicating nasopharyngeal carriage of N. meningitides. Although very effective in preventing secondary cases, antimicrobial chemoprophylaxis I not an effective intervention for altering the course of an outbreak. In epidemics, mass chemoprophylaxis is not recommended (Popovic, et al. 1999)

1.14.2 MorphologyN. meningitidis is a non motile Gram negative diplococcus (with joining sides flattened), often seen in groups. In smear from specimens, meningococci are found insides pus cells (intracelullar). A few organisms may also e seen lying free (extracellular), particularly when pus cells have been damaged when spreading the smear. Although meningococci are capsule, are not evident. In smear from cultures, meningococci appear as gram negative cocci. N. meningitidis produce transparent or grey, shiny, 1-2 mm colonies after incubation in carbon dioxide. Group A and Group C meningococci produce larger and more mucoid colonies than Group B strains. The colonies of Group B meningococci often appear grey yellow.

1.14.3 CultureN. meningitidis is an aerobic with primary culture growing best in a moist carbon dioxide enriched atmosphere. The temperatutre range of growth is 25-420C with an optimum of 35-37oC. Enriched media are required for isolation. Specimens should be cultured as soon as possible after collection.1.14.4 SerologyMeningococcal capsular polysaccharides antigens can be found in CSF in serum and urine. Direct latex agglutination and coagglutination slide antigen tests are available to detect antigens to the main groups of meningococci. Such tests are of value in epidemics when microbiological laboratory facilities are not available.

N. meningitidis antisera are expensive. Whenever possible public health laboratories should stock N. meningitidis Group A antiserum and if required also Group C antiserum to enable the cause of a meningococcal epidemic to be determined with the minimum of delay.

1.14.5 Antimicrobial sensitivityMost strains of N. meningitidis are sensisitive to penicillin, ampicillin, chloramphenicol, rifampicin, and ceftriaxone. Penicillin- resistant strains of meningococci have been reports from South Africa and elsewhere, and occasionally also resistance to rifampicin.Vaccines are available against meningococcal groups A, C, Y, W135 as monovalent preparations. A vaccine against Group B meningococci has been developed in Cuba and other B vaccines are under development. Protection provided by the vaccine is group specific and lasts for about 3 years. Polysaccharide vaccines are generally poorly immunogenic in children under 2 year. Recently developed polysaccharide protein conjugated vaccines are immunogenic in very young children and provide long lasting protection. (Monica cheesbrough et al., 2000)

1.14.6 Bacterial meningitis in BangladeshFrom 1993 to 2005 gradually isolates many meningitis causing organisms were reported to the Dhaka Shishu Hospital. I am working and analyzed data in Hune 2004 to June 2005 from Dhaka Shishu Hospital Four hundred cerebrospinal fluid (CSF) samples are collected from meningitis patients from the children of urban Hospitals of Bangladesh were studied to find out the etiological changes in CSF. Cell count was found to be directly propotional to the turbidity of the CSF and the amount of sugar lesser in more turbid CSF, whereas the protein concentration increased with the increased in turbidity of the samples (Setarunnahar, et al. 1988). H. influenzae was the most frequent cause of bacterial meningitis (40.8%), followed by N. meningitidis (13.3%), S. pneumoniae (35.7%). Over all attack rates were highest for males were greater than female’s ratio.Attack rates are highest in children under one year of age. Case fatality ratio was highest for Gram negative and miscellaneous cases of bacterial meningitis and lowest for meningitis caused by Haemophilus influenzae. Neisseria meningitidis and Streptococcus pneumoniae meningitis occurred predominantly during the winter; while Haemophilus influenzae meningitis had peak activity in the spring and fall. Ampicillin resistance among Haemophilus influenzae increased from 1993 to 2005. Serogroup S. pneumoniae (1, 4, 7 f, 14, 15b, 23f), Haemophilus influenzae type b, N. meningitidis a are most predominant in Bnagladesh. It is invasive strains that are collected from CSF and blood culture in meningitis cases.

1.16 CONVENTIONAL METHODS FOR PROCESSING AND CULTURING CSF1.16.1 Concentration:The probabilities of detecting bacteria by culture and staining techniques are increased by concentrating the bacteria in a CSF specimen. The number of bacteria present in a CSF specimen from a patient with meningitis may be as few as 10 CFU/ml (Washington, 1991). In addition, approximately 50% of patients with meningitis receive antimicrobial therapy before CSF specimens are collected (Dalton et al., 1968); antibacterial therapy may reduce the number of bacteria in the CSF by 102_ to 106-fold (Murray et al., 1980). Generally, when <0.5 ml of CSF is received into a microbiology laboratory, the entire unconcentrated specimen is used for microscopic examination and culture. When >0.5 ml of CSF is available for microscopic examination and culture, the specimen should be concentrated by centrifugation for at least 15 min at 1,500 to 2,500 x g ( Isenberg et al., 1991, Isenberg, H. D. et al., 1991, Murray et al., 1980 and Ray et al., 1982). A centrifugal force of 10,000 x g has been recommended to sediment bacterial CSF pathogens (Smith, 1973), but such force has been demonstrated to be unnecessary (Murray et al., 1980). A significant number of positive CSF specimens may be missed if the laboratorian uses a sterile pipette to remove the sediment from underneath the entire volume of supernatant (Baron et al., 1990). The supernatant should be decanted or carefully removed into a sterile tube, leaving approximately 0.5 ml behind to be used to suspend the sediment. Thorough mixing of the sediment after removal of the supernatant is essential. Mixing the sediment with the aid of a vortex mixer or forceful aspiration with a sterile pipette will be adequate to dislodge bacteria that may have adhered to the

bottom of the tube following centrifugation. The sediment should be used to inoculate culture media and prepare smears for staining. If a grossly bloody specimen is received, smears for stains can be prepared before and after centrifugation to decrease the likelihood of erythrocytes obscuring bacteria in the sediment of a centrifuged specimen (Dalton et al., 1986).

1.16.2 CultureThe media routinely used for bacterial culture of CSF are 5% sheep blood agar, enriched chocolate agar, and an enrichment broth (e.g., thioglycolate, Columbia, brucella, supplemented peptone, or eugonic). The culture plates should be incubated for at least 72 h at 37°C in an atmosphere containing 5 to 10% CO2. A candle jar can be used if a CO2 incubator is not available. The enrichment broth, with the cap loosened, should be incubated at 37°C in air for at least 5 days. If the Gram stain demonstrates the presence of gram-negative rods resembling members of the family Enterobacteriaceae, a MacConkey agar plate can also be inoculated (Baron, et al., 1990). If the Gram stain reveals organisms that morphologically resemble anaerobic bacteria or if the patient is known to have an underlying condition predisposing the patient to an anaerobic infection (such as chronic otitis media, a pilonidal sinus, or dermal sinus), an anaerobic blood agar plate can be added to the routine culture media, and the plate should be incubated at 37°C in an anaerobic atmosphere (Baron et al., 1990, Givner et al., 1983 and Odugbemi et al., 1985) If possible, a portion of each CSF specimen should be stored temporarily at -70°C, room temperature, or 37°C for potential reculture. If antigen detection tests are anticipated, the specimen should be stored at <40C because bacterial polysaccharide antigens often tend to break down faster at room temperature and 37°C than at ≤ 4°C. Cultures should be examined daily. Gram stain results of colonial or broth growth should be telephoned to a physician caring for the patient. Although bacterial concentrations of .107 CFU/ml of CSF have been correlated with increased morbidity and mortality (Feldman, 1977), quantitative culturing of CSF specimens is not a common or practical procedure. Growth of normal skin flora should raise suspicion of contamination, especially when there is minimal growth on the solid media or growth on a single plate or in the broth only. Culture plates with no growth may be discarded after 72 h, and negative enrichment broths may be discarded after 5 days of incubation. The authors of Cumitech 14 suggest incubating negative cultures that have positive Gram stain findings for an additional 4 days before the cultures are discarded as negative ( Ray, et al.,1982).

1.16.3 Antimicrobial Susceptibility Testing In general, complete antimicrobial susceptibility testing should be performed on all clinically relevant bacteria isolated from CSF. H. influenzae should be tested for the production of 1-lactamase by a chromogenic or acidometric assay (Doern et al., 1991, Morello et al., 1991, Skinner et al., 1977 and Thornsberry et al., 1974). In addition, an assay for the detection of chlorampheniicol acetyltransferase may be used to assess the clinical utility of chloramphenicol (Doern et al., 1991 and National Committee for Clinical Laboratory Standards. 1990). N. meningitidis should be tested for, B-lactamase production when the isolate is from a patient who is not responding well to antimicrobial therapy (Morello et al., 1991, Skinner et al., 1977 and Washington, 1991). S. pneumoniae initially should be tested by the oxacillin agar screen method to screen for frank resistance and relative resistance to penicillin (Doern et al., 1991 and National Committee for Clinical Laboratory Standards, 1990). The agar screen method detects both types of resistance but does not differentiate between them. If an isolate produces as 19-mm zone of inhibition in the screen test, the isolate is either frankly resistant or relatively resistant to penicillin. Subsequently, dilution (MIC) testing should be performed to confirm resistance (relative resistance, MIC of 0.12 to 1.0, ug/ml; frank resistance, MIC of >1.0, ug/ml), because the prevalence of both types of resistance in the United States is so low that the predictive value of a resistant screen result is also low (Doern et al., 1991 and Jorgensen et al., 1990). S. pneumoniae isolates from the CSF of patients with meningitis and that are confirmed by dilution testing to be relatively resistant to penicillin should always be reported as resistant, because such isolates probably will not respond clinically to penicillin (Doern et al.,1991).

1.17 RAPID METHODS FOR DETECTING BACTERIA AND COMPONENTS OF BACTERIA IN CSF1.17.1 Microscopy

Samples of all CSF specimens should be stained with the Gram stain (or other comparable stain) and examined microscopically. Because the diagnostic usefulness of staining procedures depends on the concentration of bacteria in the CSF of patients with bacterial meningitis (10 to 109 CFU/ml), all CSF specimens of sufficient quantity should be processed to concentrate pathogens prior to microscopic examination and culture (Feldman, 1977, Fung et al., 1981, Isenberg et al.1991, Isenberg et al., 1991, Murray et al., 1980, Ray et al., 1982 and Washington, 1991

1.17.2 Gram stain. The Gram stain is a simple, rapid, accurate, and inexpensive method for detecting bacteria and inflammatory cells in CSF from patients with bacterial meningitis. Seventy-five to 90% of CSF culture-positive specimens are Gram stain positive (Karandanis et al., 1976 and Lauer et al., 1981); the percentages decrease to 40 to 60% in patients who have received antimicrobial therapy prior to lumbar puncture ( Dalton et al., 1968 and Jarvis et al., 1972). The Gram stain is generally accepted to be most reliable at detecting .10' bacteria per ml of body fluid (Coovadia et al., 1985, Isenberg et al., 1991, Rubin, 1987 and Tinghitella, 1991). This fact has been demonstrated for CSF by La Scolea and Dryja (La Scolea et al., 1984), who showed that 25, 60, and 97% of CSF specimens with <103, 103 to 104, and > 105 CFU/ml, respectively, were positive by Gram stain. The clinical utility of the Gram stain apparently depends on the bacterial pathogen. Bacteria have been observed in 90% of cases of meningitis caused by S. pneumoniae and Staphylococcus spp., 86% caused by H. influenzae, 75% caused by N. meningitidis, 50% caused by gram-negative bacilli, and <50% caused by L. monocytogenes and anaerobic bacteria ( Greenlee, J. E. 1990). Some workers prefer to use basic fuchsin as the Gram counterstain to provide better staining of organisms such as Haemophilus spp. and Fusobacterium spp., which stain poorly with safranin ( Ray et al.,1982).

1.17.3 Acridine orange stain. Stains other than the Gram stain can be used to screen smears of CSF for bacteria. Acridine orange is a fluorochrome stain that can intercalate into nucleic acid. At a low pH (4.0), bacteria and yeasts appears bright red, and leukocytes appear pale apple green. In one study, the acridine orange stain was slightly more sensitive than the Gram stain (82.2% compared with 76.7%) and was capable of detecting bacteria at concentrations of > 104CFU/ml, a concentration 10-fold lower than that detectable by the Gram stain ( Lauer et al., 1981). Work by Kleiman et al. suggests that a major advantage of acridine orange is that it is more sensitive than the Gram stain in detecting both intra- and extracellular bacteria in CSF from patients who have received antimicrobial therapy (Kleiman, M. B. et al., 1984). Kleiman et al. found the Gram stain to be positive in 0 of 47 and the acridine orange stain to be positive in 45 of 47 (96%) CSF specimens obtained from patients who had been given antimicrobial agents for .18 h prior to collection of CSF. Another advantage of the acridine orange stain is a reduction in the time devoted to examining a CSF smear. This reduction results from the striking contrast between the bright bacteria and the dark background and the use of only x400 magnification to examine most smears. Acridine orange-positive smears must be Gram stained to verify the presence of bacteria and to determine the Gram reaction of the bacteria (Ray et al., 1982). Fortunately, acridine orange-stained smears can be Gram stained without prior decolorization of the acridine orange (McCarthy et al., 1980)

1.17.4 Wayson stain. The Wayson stain appears to be a simple and sensitive stain for screening smears of CSF for bacteria. The components of the stain are basic fuchsin, methylene blue, ethanol, and phenol. Daly et al. found the Wayson stain to be more sensitive (90%) than the Gram stain (73%) and to be as specific (98%) as the Gram stain (99%) in the detection of bacteria in smears of CSF (Daly et al., 1985). In Waysonstained preparations, bacteria appear dark blue, proteinaceous material appears light blue, and leukocytes appear light blue and purple. In the opinion of Daly et al., the contrast between bacteria and background is more pronounced with the Wayson stain than with the Gram stain, which enables the laboratorian to spend less time examining CSF smears (Daly et al., 1985). However, Wayson-stained smears cannot be Gram stained. A second smear must be Gram stained when bacteria are detected in Wayson-stained smears of CSF.

1.17.5 Quellung procedureThe quellung capsular reaction is rarely used; however, it can be used to confirm the presence of organisms with morphology typical of S. pneumoniae, N. meningitidis, or H. influenzae type b. In the quellung reaction procedure, antisera specific for the capsular polysaccharides of each of these three bacteria are mixed with separate portions of clinical specimens. Specifically, a drop of CSF, a loopful of specific antiserum, and saturated methylene blue can be mixed on a microscope slide, covered, and examined under an oil objective. The formation of antigen antibody complexes on the surfaces of these bacteria induces changes in the refractive indices of their capsules. Microscopically, the capsule appears to be clear and swollen. The test requires highly specific antibody at high titer and a laboratorian with expertise in the method. Details concerning the methodology of the test can be found in the fifth edition of the Manual of Clinical Microbiology (Facklam et al., 1991).

1.18 Methods of Detecting Bacterial AntigensCounterimmunoelectrophoresis (CIE), coagglutination (COAG), and latex agglutination (LA) have been adapted for the rapid and direct detection of soluble bacterial antigens in CSF of patients suspected of having bacterial meningitis. These tests are widely used in clinical microbiology laboratories and can be important supplements to the culture and Gram stain of CSF specimens. Rapid antigen detection tests may provide true-positive results when culture and Gram stain results are negative for meningitis patients who have received antimicrobial therapy (Gilligan et al.,1989, Marcon, 1990 and Tilton et al.,1984). In addition, the results of these rapid tests can prompt a physician to implement early and specific antimicrobial therapy rather than the broad-coverage therapy that is usually instituted until culture and antimicrobial susceptibility results are available, in 18 to 24 h.The most common central nervous system pathogens to which antigen detection tests have been applied are H. influenzae type b, S. pneumoniae, Streptococcus group B, and N. meningitidis serogroups A, B, C, Y, and W135. Except for Streptococcus group B, these bacteria possess soluble, type-specific, capsular polysaccharide antigens that are released into surrounding body tissues and fluids as the bacteria proliferate (Coonrod, 1983.). Streptococcus group B possesses a soluble type-specific cell wall polysaccharide antigen. In bacterial meningitis patients successfully treated with antimicrobial agents, bacterial antigens are detectable in body fluids for many days after the CSF becomes sterile. H. influenzae, N. meningitidis, and S. pneumoniae antigens have been detected by COAG and LA in the CSF and serum for 1 to 10 days after the initiation of treatment with antimicrobial agents (Habte-Gabr et al., 1987; Suksanong et al., 1977 and Thirumoorth et al., 1979). Thirumoorth and Dajani used COAG and LA to detect higher concentrations of H. influenzae type b antigen in urine and serum than in CSF of patients who had received 1 to 3 days of treatment with antimicrobial agents (Thirumoorth, et al., 1979). Kaldor et al. found that the H. influenzae type b antigen titer in concentrated urine from children often increased on the second day of therapy and slowly decreased thereafter (Kaldor et al., 1973). H. influenzae antigen was detected in the urine as long as 18 days (mean, 10 days) after the initiation of therapy. With the use of COAG and LA, Riera reported the persistence of H. influenzae antigenuria to be a mean of 19.9 days in patients recovering from H. influenzae meningitis (Riera, 1985). Baker et al. used CIE to detect bacterial antigen in the urine of survivors of Streptococcus group B meningitis for as long as 75 days (mean, 22.4 days) after the initiation of appropriate therapy with antimicrobial agents’ (Baker et al., 1980).

1.18.1 Counterimmunoelectrophoresis (CIE)CIE was once an important and rapid diagnostic method for the laboratory diagnosis of bacterial meningitis. In CIE, the application of an electric current to immuno diffusion agar accelerates the diffusion of antigen and antibody toward each other in the agar and enables any subsequent immuno precipitation to be completed in 30 to 60 min. The introduction of commercially available COAG and LA reagents for the detection of CSF pathogens has made CIE a test performed in only a few laboratories. CIE is less sensitive (by a factor of 10) than COAG and LA in the detection of bacterial antigens in CSF and urine (Fung et al., 1985); however, CIE has excellent specificity (Fung et al., 1985, Granoff et al., 1986). CIE is

used only rarely today because it requires high-quality antisera, stringent quality control, special equipment, and an experienced laboratorian to obtain optimum sensitivity. In addition, CIE is cumbersome and slow when compared with COAG and LA.

1.18.2 Enzyme immunoassays (EIA)EIAs for the detection of bacterial antigens in CSF use specific (primary) antibodies bound to a solid support such as a plastic microwell tray or tube or polystyrene beads. If homologous bacterial antigen is present in a CSF specimen, the antigen will be bound by the immobilized primary antibody. An enzyme-labeled (secondary) antibody with specificity for the bacterial antigen of interest detects the antigen bound to the primary antibody. The addition of a substrate for the enzyme results in the production of a colored product if specific bacterial antigen was present in the CSF specimen and bound to the primary antibody.EIAs have been evaluated for their abilities to detect H. influenzae type b, S. pneumoniae, and N. meningitidis antigens in CSF (Beuvery et al., 1979, Drow et al., 1983, Sippel et al,. 1984, Sugasawara et al., 1984 and Yolken et al., 1984). The sensitivities and specificities of these tests have been reported to be 84 to 100% and 89 to 100%, respectively. The tests can detect bacterial antigens in concentrations as low as 0.1 to 5 ng/ml. currently; commercially available EIAs for the detection of bacterial antigens in CSF are available only in Europe (Behring, a biotin-avidin procedure; Pharmacia, a horseradish peroxidase procedure). EIAs generally take several hours to complete and require multiple controls. For these reasons, EIAs are better suited for testing specimens in a batch mode than for testing of individual CSF specimens as they are received into the laboratory, usually on a stat basis

1.18.3 LAL AssayThere are three Food and Drug Administration-approved methods for the LAL assays (Prior, 1988). The simplest LAL assay is the gel endpoint method. This test is performed by incubating 0.1 ml of lysate with 0.1 ml of fluid specimen for 1 h at 37°C and inverting the mixture 180° to determine whether a clot has formed. The determination of whether a clot has partially or fully formed is subjective and can make endpoints difficult to read. Patients with untreated meningitis commonly have at least 105 CFU per ml of CSF (Tinghitella et al., 1991). The turbidometric LAL assay involves the use of a spectrophotometer to measure the change in optical density that occurs during the gelation reaction. In the chromogenic substrate LAL assay, a synthetic color-producing substrate (which contains a chromogenicp-nitroanilide group) and a modified LAL assay are used. The formation of a clot as an endpoint is eliminated to a large degree and is replaced by the production of a yellow color. In all three LAL assays, the use of pyrogen-free laboratory ware is imperative.The LAL assay is a very sensitive and specific assay for the detection of endotoxin in CSF. A correctly performed LAL assay can detect approximately 103 gram-negative bacteria per ml of specimen (Tinghitella et al., 1991). Nachum reviewed 4,884 CSF specimens, which had been examined by LAL assays and calculated the overall sensitivity and specificity of the LAL assay (Nachum, 1990). Compared with cultures for gram-negative bacteria, LAL assays have a sensitivity of 93% and a specificity of 99.4%. Bacteria that have been detected in CSF by LAL tests include H. influenzae type b, N. meningitidis, E. coli, Pseudomonas spp., Serratia marcescens, Klebsiella pneumoniae, and other gram-negative bacilli (Nachum, 1990).Not all reports give the LAL test the stamp of approval for diagnosis of gram-negative meningitis. McCracken and Sarff reported a sensitivity of 71% for the detection of neonatal gram-negative meningitis in CSF specimens with positive cultures and a false-positive rate of 14% in CSF specimens with negative cultures (McCracken et al., 1976). These results led McCracken and Sarff to conclude that the LAL test was not sensitive enough to serve as a screening procedure for the diagnosis of gram-negative meningitis in neonates. The LAL test has not found widespread use as a diagnostic tool for meningitis because the test detects only gram-negative bacteria and does not differentiate between different gram-negative bacteria.

1.18.4 Gas-liquid chromatography (GLC)

GLC was first used in clinical microbiology for the identification of anaerobic bacteria. This technique facilitates the separation, quantitation, and identification of several (often trace) constituents of physiological fluids (LaForce et al., 1979). The application of GLC for the detection and identification of microorganisms in CSF is still in the developmental stages. Craven et al. presented data that demonstrated that GLC can be used to differentiate among cryptococcal, tuberculous, viral, and parasitic infections of the central nervous system (Craven et al., 1977). Brice et al. used GLC techniques to establish chromatography patterns for the following five common bacterial agents of meningitis: S. pneumoniae, H. influenzae, N. meningitidis, S. aureus, and E. coli (Brice et al., 1979). Lipid, carbohydrate, and lipopolysaccharide components served as characteristic markers for the identification of these organisms. Brice et al. concluded that GLC might be a useful assay for the rapid laboratory diagnosis of bacterial meningitis. GLC has been reported to be potentially useful in the detection of bacteria in CSF. LaForce et al. found that CSF from patients with meningitis caused by H. influenzae and S. pneumoniae showed fatty acid and carbohydrate GLC profiles that were clearly different from those of normal VOL. 5, 1992 CSF (LaForce, et al., 1979). In addition, GLC profiles of H. influenzae- and S. pneumoniae-infected CSF was different from each other. These investigators suggested that, at least theoretically, prior treatment of a patient with antibacterial agents would not be expected to interfere immediately with GLC results because antibacterial agents would not alter fatty acid or carbohydrate components of infecting bacteria.GLC has not been widely used for the diagnosis of bacterial meningitis for several reasons. The technique requires equipment that is relatively expensive, and the methodology is much more technically demanding than the antigen detection assays in use in most laboratories. Computer-assisted evaluation of results might be needed to aid in the interpretation of GLC results because misleading backgrounds and artifacts can cause difficulty in identification (Drow, 1988.).

1.18.5 Polymerase chain reaction (PCR)The PCR has been used recently in the early detection of N. meningitidis in CSF from a patient with meningitis (Kristiansen, 1991). The patient's blood cultures were positive for N. meningitidis, but culture, Gram stain, and acridine orange stain of CSF did not detect bacteria in the CSF. The CSF was purulent, with 48,000 polymorphonuclear leukocytes per, ul. The patient had received intravenous penicillin 30 min before the CSF specimen was obtained. Use of the PCR and nucleic acid probes could have provided an early definitive diagnosis of meningococcal meningitis in this patient if the test had been performed on CSF when the patient was admitted to the hospital. The authors concluded that the PCR is a rapid method for the amplification of DNA and can be extremely useful in the early laboratory diagnosis of meningitis caused by N. meningitidis even when the patient has received prior antibiotic therapy. They also stated that, in principle, meningococcal meningitis could be excluded on the basis of a negative PCR result.

1.19 AIM OF THE STUDYEven with the advances in the development of many powerful antimicrobial agents, bacterial meningitis still remains a serious cause of morbidity and mortality in childhood. Many epidemiologic and microbiologic investigations have been reported the variability in disease risk in different populations and races. In USA, Haemophilus influenzae was the most common cause of bacterial meningitis, with an annual incidence of 45-67/100,000 children less than 5 years of age prior to the routine immunization with H. influenzae type b (Hib) conjugate vaccine (Kim, et al., 1998). However, in some European countries, the annual incidence of meningococcal meningitis is higher than that of H. influenzae meningitis. In other parts of the world, few prospective population-based studies on the epidemiology of bacterial meningitis have been carried out. Despite the general consensus of the importance and seriousness of bacterial meningitis in children in Bangladesh, there is little information on the incidence, causative organism, age distribution and mortality rate.Specific aims of the StudyThe specific aims of this study are:To isolate and identify the aetiological agents of acute bacterial meningitis from the CSF by culture.

To assess the prevalence and pattern of antimicrobial resistance of the isolated aetiological agents for proper selection of antibiotic therapy.To improve the clinical diagnosis and management of meningitis.To determine the burden of meningitis disease in Bangladesh.To evaluate the usefulness of various laboratory procedures in diagnosis bacterial meningitis.Rapid, accurate and inexpensive diagnosis of acute bacterial meningitis.

2. MATERIAL AND METHODS

2.1 Study PopulationThis study was conducted over a period of one year from August 2010 to August 2011. Three hundred and seventy one individuals with suspected meningitis of children were included. The patients were clinically diagnosed of having bacterial meningitis by physicians.

2.2 Sampling SitesSamples were collected from different hospitals and diagnostic centres located at Dhaka city in Bangladesh including Popular Diagnostic Centre Ltd; Dhaka Medical College Hospital; Central Hospital Ltd; Square Hospital Ltd and IBN Sina Diagnostic Centre Ltd.

2.3 Clinical SpecimensChildren with suspected to suffer from acute meningitis were collected from hospitals and diagnostic centres. Single serum and cerebrospinal fluid (CSF) specimens were collected for evaluation by routine bacterial culturing and serum and CSF analyses. CSF and blood specimens were collected aseptically in sterile containers by experiences personnel. The CSF was collected by lumber puncture from 3rd and 4th lumber region by needle aspiration by physicians. The specimens were stored at -700C until further use. Each specimen was divided into 2 or 3 aliquots for bacteriological, microscopic and biochemical analyses.2.4 Collection of the SampleFor the purpose of collection of the samples, few screw capped test tubes were cleaned and sterilized in the autoclave at 121oC and 15 lbs pressure for 30 min. The tubes were carried within a thermostat containing ice and some without ice. After collection of each sample the test tubes were properly capped and labeled. Care was taken to avoid any contamination. Maintaining the aseptic precautions and following the above procedures a number of samples were collected at different times from different places.

2.5 Preservation of Sample As meningitis caused by different types of microorganisms, the collected samples were inoculated in Chocolate agar media both aerobically and an aerobically, Mac-Conkey agar media, Blood agar media, Lowenstein Jensen media. This was to isolate the colonies of the causative organisms as soon as the sample brought to the laboratory. In case of delay it was kept in refrigerator because the cells deteriorate rapidly.

2.6 Routine CSF MicroscopySmears were made from fresh un-centrifuged CSF that appeared cloudy or from the sediment of centrifuged CSF. Smears were stained by Gram stain procedure. Total leucocyte count in CSF was made by direct microscopic quantitative method. The greater than 500 cells/mm3 was as significant for bacterial meningitis.

2.7 Biochemical Analysis of CSFCSF protein and glucose concentrations were determined by using Vistor 250 System (WS-JJV250, USA) (Fig 2.1). CSF protein concentration greater than 100 mg/dL and sugar level below 40 mg/dL were considered as suspected bacterial meningitis.

Fig 2.1: Ortho Clinical Vitros 250 Chemistry System (WS-JJV250)

2.8 Estimation Serum C-Reactive Protein (CRP)Serum CRP level was estimated by using CRP latex agglutination kit (Chrono Lab., UK). On the provided slide a drop of test materials was mixed with latex CRP reagent and agglutination reaction was seen after 2 min. The presence of agglutination was considered as positive test. CRP level >40 mg/dL in serum was taken as significant

2.9 Microscopic Examination for Total Cell Counts of CSFIn case of acute bacterial meningitis, the CSF was turbid and a great increase in the number of leucocytes was found. Total number of cells in CSF was counted as soon as the sample brought to the laboratory because the cells deteriorate rapidly. The method of counting was varied with the number of cells expected. Upon several hundred cells per mm3 was a common findings and early in the disease almost all of them were polymorphs.

2.9.1 Methods for Low White Cell Count(a) One drop of well mixed undiluted fluid was transferred to a WBC counting chamber. All the cells were counted in nine squares. (b) The number was represented a value of 9/10 mm3. So the result was multiplied by 10/9 to obtain the number of cells/mm3.

2.9.2 Methods for Moderate Cell Count(a) The specimen was mixed thoroughly. If not very cloudy or bloody, the CSF was draw to mark 1, in a white cell counting pipette. Then the diluting fluid was draw to mark 11, producing a dilution of 1:10.(b) It was mixed thoroughly and one third of the mixture was discarded. The one drop of the mixture was transferred on the slide of the blood counting chamber as in the method for leucocyte counting.(c) Four squares were counted, 1 mm2 each (4 corner square), on each side of the counting chamber and the results of all 8 squares counted were added.(d) The total number of cells was divided by 8 to obtain the number per single large squares of cells in 0.1mm3. It was then multiplied by 10 which converted the number found in 1mm3. Second time it was multiplied by 10 which was the dilution factor. If the total cell count was low or moderate, a rough estimate of the differential count can be obtained, by classifying the cells seen in the counting chamber.

2.9.3 Methods for High White Cell Count(a) In case of highly turbid CSF the erythrocyte pipette was used. The CSF was draw to 1 mark in an erythrocyte counting pipette. Then the diluting fluid was draw to mark 101, producing a dilution of 1:100. It was mixed thoroughly and 1/3 of the mixture was discard. Then one drop of the mixture was transferred on the slide of the counting chamber in the usual manner.

(b) Under low power objective, all the cells resting on 4 large squares (corner mm squares) were usually counted.(c) Number of cells per mm3 = number of cells counted 1/volume in which cells were counted x dilution.(d) Four “large squares” were counted, each of which had a volume of 0.1 mm3 .Thus the volume in which the cells were counted was 4 x 0.1 or 0.4 mm3. The dilution was 1 to 100. Therefore the number of cell per mm3 = number of cells counted in 4 large squares X 2.5 X 100.

2.10 Direct Microscopy for OrganismsAs the sample was draw from a patient, it was necessary to study the morphology of organisms as quickly as possible. So the CSF was centrifuged at the speed 3000 rpm for 30 min. It was for the precipitation of microorganisms from the suspending fluid. The fluid was then separated as the upper supernatant and lower precipitates. The precipitate of the sample was used for direct staining and the supernatant for biochemical tests. In respect of staining much importance was given on the slides. For good staining of bacteria the slides were extremely cleaned.The following steps were followed in the preparation of fixed stained smear(a) Preparation of the smear: A portion of the precipitates after centrifugation of the CSF was taken out by a Pasteur pipette in the sterile way on a slide. A very thin film was made which was allowed to dry in air. The method was followed in almost all types of staining.(b) Fixation of smear: The smear was fixed by slightly heating the slide over a sprit lamp.(c) Application of stains: The fixed smears were stained mainly by the differential staining i.e. Gram staining method.

2.10.1 Gram Staining for the Identification of BacteriaThis is one of the most important and widely used differential staining techniques. This method, which is a modification of Burke’s (1922) method, was recommended for general use. In this process the fixed bacterial smear was subjected to a number of solutions. A presumptive diagnosis of bacterial meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis Escherichia coli, Klebsiella pneumoniae and Salmonella non-typhi group D can be made by Gram stain by CSF sediment or by detection of specific antigens in the CSF by a latex agglutination test. Positive results of either or both tests can provide evidence of infection, even if cultures fail to grow.Procedure1. The CSF was centrifuged for 30 min at 3000 rpm.2. Smear was prepared by placing 1 or 2 drops of sediment on an alcohol rinsed and dried slide, allowing drop to form one large drop. 3. The slide was air dried in a biosafety cabinet.4. The slide passed quickly through a flame three times to fix smear. Alternatively, fixation by methanol (95% -100%) can be used.5. The smear was flooded with ammonium oxalate crystal violet and let stand for 1 min.6. Then gently rinsed with tap water and drained off excess water.7. The mear was flooded with gram’s iodine solution and let stand for 1 min.8. Gently rinsd with tap water and drained.9. The smear was decolorized with 95% ethyl alcohol (5-10 second may be enough).10. Counterstain with safranin for 20-30 seconds or with carbol fuschin 10-15 seconds.11. Rinsed the slide with tap water and blot dry.12. Examined the stained smear microscopically, using a bright field condenser and an oil immersion lense.N. meningitis may occur intra or extracellular in the polymorphonuclear leucocytes and appear as Gram-negative, coffee-bean- shaped diplococci. S. pneumoniae are lenceolate, Gram positive diplococci sometimes ocuring in short chains. H. influenzae are small, pleomorphic Gram negative rods or coccobacilli with random arrangement. Other manuals should be consulted for Gram stain reactions of other bacteria.

2.10.2 Acid Fast StainingFor acid fast staining, Zeihl-Neelson method (1883) was followed. Cerebrospinal fluid was allowed to stand in a stopper tube for an hour or longer, when a “spider web” coagulum formed in fluid. The dot was carefully transferred to a slide. The preparation was dried and fixed by heat and it was then stained by the Zeihl-Neelson method. In the absence of clotting the fluid was centrifuged, the deposit usually small was taken up in a Pasteur pipette. It was dropped without spreading on a slide and slowly dried and then stained.

ProcedureStaining: The slide was flooded with carbol fuchsin solution (1 gm basic fuchsin in 10 ml absolute alcohol and 100ml of 5% phenol solution).Mordanting: It was heated intermittently short of boiling and allowed to act for 5 minutes.Decolourization: The slide was covered with 20% sulphuric acid (H2SO4) and waited for 1 minute and washed with water. The process was repeated several times.Counter staining: After washing it was counter stained with Loffler’s methylene blue for 10-30 seconds and washed with water and blotted and dried in air and then gradually observed under microscope by oil immersions lense. The result was recorded as acid fast and non acid fast staining.

2.10.3 Leishman Staining: For the Leishman staining dry unfixed films was used. At first the undiluted stain (by dissolving 0.15 g of Leishman powder in 100 mL pure alcohol) was used and the methyl alcohol fixed the film. The stain was then diluted with distilled water.Procedure(1) The undiluted stain was poured on the unfixed film and allowed it to act for 1 minute.(2) By means of pipette and rubber teat a double volume of distilled water was to the slide and diluted the stain. The diluted stain was allowed to act for 10 minutes.(3) The slide was flooded gently with distilled water for 30 seconds.(4) The excess water was removed with blotting paper and dried in air.(5) The prepared slide was the observed under microscope and different types of cells of the CSF were counted. Differential count was done directly from the CSF in cases where the total cell count was high.2.11 RAPID DETECTION METHOD OF ACUTE BACTERIAL MENINGITIS2.11.1 Immunochromatographic Test (ICT) The Bionex NOW Streptococcus pneumoniae antigen test is an in vitro rapid immunochromatographic (ICT) assay for the detection of Streptococcus pneumoniae antigen in CSF specimens from patients with symptoms of pneumonia. It is intended to aid in the presumptive diagnosis of pneumonia in conjunction with culture and other methods.

Reagents and materialsClock, timer, or stopwatch, standard CSF collection containers.Test devices: A membrane coated with rabbit antibody specific for Streptococcus pneumoniae antigen and with goat anti-rabbit antibody IgG is combined with rabbit anti Sterptococcus pneumoniae antigen conjugate in a hinged test device.Reagent A: Citrate / Phosphate buffer with sodium lauryl sulphate, Tween 20, and sodium azide. Sample swabs: Designed for use in the Bionax NOW Streptococcus pneumoniae CSF antigen test. Positive control swabs: Heat inactivated Streptococcus pneumoniae positive CSF dried onto swabs.Negative control swab: Heat inactivated normal human CSF dried onto swabs. Procedure for Patient Samples(1) The patient CSF are taken at room temperature (59-860F, 15-300C), then swirled gently to mix. Removed device from its pouch just before use and lay flat.(2) Diped a Binax swab into the CSF sample to be tested, completely covering the swabs head.

(3) There were two holes on the inner panel of the device. Inserted swab into the bottom hole. Finaly pushed upwards so that the swab tip is fully visible in the top hole. (4) The reagent A vial is holded vertically, 1 to 1.5 inches above the device. Then slowly added three drops of reagent A to the bottom hole.(5) Immediately peeled off adhesive liner from the right of the test device. Closed and securely sealed the device. Read result in windows 15 minutes after closing the device. Results read in beyond 15 minutes may be inaccurate. However, some positive patients may produce a visible sample line in less than 15 minutes.

Interpretation of resultsA negative sample will be given a single pink to purple colored control line in the top half of the window, indicating a presumptive negative result. This control line means that the detection part of the test done correctly, but no Streptococcus pneumoniae antigen was detected.A positive sample will be given two pink to purple colored lines. This means that antigen was detected. Specimens with low levels of antigen may give a faint patients line. Any visible line is positive. If no lines are seen, or if just the sample line is seen, the assay is invalid. Invalid tests should be repeated call Binax Technical services at (800) 323-3299.

Precautions (1) Invalid results, indicated by no control line, can occur when an insufficient volume of reagent A is added to the test device. To insure delivery of an adequate volume, hold vial vertically and drops slowly.(2)The test device is sealed in a protective foil pouch. Do not use if pouch is damaged or open. Remove test device from pouch just prior to use. Do not touch the reaction area of the test device.(a) Did not use kit past its expiration date.(b) Did not mix components from different kit lots.(c) Swabs in the kit are approved for use in the Binax.(d) Solution used to make the control swabs are inactivated using standard methods.(e) CSF specimens used for the test may not be appropriate for bacteriological culture.

LimitationsThe Binax NOW Streptococcus pneumoniae CSF antigen test has been validated using CSF sample only. Other samples that may contain Streptococcus pneumoniae antigen have not been evaluated. A negative antigen result does not exclude infection with Streptococcus pneumoniae. The result of this test as well as culture results, serology or other antigen detection methods should be used in conjunction with clinical findings to make an accurate diagnosis. The Binax NOW Streptococcus pneumoniae CSF antigen test has not been evaluated on patients taking antibiotics for greater than 24 hours or on patients who have recently completed an antibiotic regimen.

2.11.2 Latex Agglutination Tests for Bacterial AntigensLatex agglutination test is used for identification of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus group B and E.coli. For best results, test the supernatant of the centrifuged CSF sample as soon as possible. If immediate testing is not possible, the sample can be refrigerated (between 20C and 80C) upto several hours, or frozen at 200C for longer periods. Reagents should be kept refrigerated between 20C and 80C when not in use. Product deterioration occurs at higher temperatures, especially in tropical climates, and test results may become unreliable before the expiration date of the kit. Latex suspension never is frozen.Performance of the test(1) The supernatant of CSF was heated in a boiling water bath for 5 min.(2) The latex suspensions were shaked gently until homogenous.(3) Placed one drop of each latex suspension on a disposable card.(4) Added 30-50 micro of the CSF to each suspension.

(5) Rotated by rotator for 2-10 min. Reading the test resultsNegative reaction: The suspension remains homogenous and slightly milky in appearance.Positive reaction: Agglutination of the latex particles occurs within 2 min.

2.12 ISOLATION PROCEDURE For the isolation of organisms the culture of the CSF was necessary. It was for definite diagnosis of the organisms. As a number of morphologically and physiologically different types of microorganisms are responsible for meningitis, different types of media ware required and different techniques were followed. So for culture of single sample different types of media were taken simultaneously. Specifically to isolate pneumococci blood agar (aerobically and anaerobically) medium was used. Meningococci also required enriched media for growth and chocolate agar medium was satisfactory. Growth was facilitated by the presence of 10% CO2 (Carbondi-oxide). To culture Escherichia coli, Klebsiella spp. Blood agar and MacConkey media were used. Lowenstein–Jensen media were used to culture Mycobacterium tuberculosis, which is one of the severe causes of meningitis.

2.12.1 Composition and Preparation of the Media For the cultivation of causative organisms of meningitis five different culture media were used including Chocolate agar, Muellar-hinton agar, MacConkey media, Blood agar media and Lowenstein-Jensen media. The composition of all above mentioned media according to Cruickshank et al (1973) are given in the appendix section. A number of steps followed for the preparation of media are given below: (1) Adjustment of pH of the MediaAfter mixing the components of the media, the pH was adjusted to 7.4 - 7.6 and by adding N/10 KOH and Hcl alternately. (2) Sterilization of the Media After adjusting the pH of the media, they were sterilized in autoclave at 1210C temperature and 15 lbs pressure per square inch. In case of blood agar media 5-10% sterile sheep blood was added to the sterilized blood agar base. (3) Preparation of Plates After autoclaving the media, they were poured into sterile petri plates and allowed solidifying. In that way replicate plates were prepared from each medium.2.12.2 Inoculation of the CSF to the Appropriate Media In this procedure selection of proper portion of the specimen and selection of the proper medium were important. For this procedure the sediment of the centrifuged sample was inoculated to a number of media by simple streaking method according to Harrigen et. al (1966).2.12.3 Incubation of the Inoculated PlatesThe inoculated plates were incubated at optimum temperature (370C) for 24 to 72 hours.

2.12.4 Anaerobic CultureIt was made by the displacement of oxygen with other gases like carbondioxide (CO2), was employed. A popular method was the candle jar. The inoculated plates were placed inside a large airtight container and a lighted candle was kept in that before the jar was sealed. The burning candle used up all the oxygen inside before that was extinguished. The candle jar provided a concentration of carbon dioxide, which stimulated the growth of most bacteria. Meningococcal were incubated an aerobically in an atmosphere of excess CO2.

2.12.5 Isolation of Colonies After 24 hours to 72 hours incubation of the plates, several colonies were found to develop in the plates. The pneumococci, Meningococci colonies showed rose pink colour. In Mueller-Hinton agar it showed opaque white colonies. Klebsiella showed iregular mucoid colonies in MacConkey plate.

2.13 IDENTIFICATION PROCEDURE

The colonies developed in the plates were selected and their pure culture was made to get homogeneous population. Then morphological cultural and biochemical tests were done for identification of each species. 2.13.1 Study of Bacterial Morphology

Morphology and staining characteristics help in the preliminary identification. The morphology of the organism depend on number factors such as the strain studied, nature of the culture medium, temperature and time of incubation, age of the culture and the number of sub cultures. The shapes of the vegetative cells of the selected organisms were determined. The arrangement of the cells, whether present singly, in chains or in clusters were also observed. For staining reactions the age of the culture was important. In older culture, staining characteristics either varied or not brought out well. Simple stains brought out morphology best. Gram stain divided bacteria into Gram positive and Gram negative; Zeihl-Neelson stain into acid fast and non acid fast. The steps of fixed stained smears were discussed earlier in this section (direct microscopy).

2.13.2 Study of the CultureCultural characteristics provided additional information for the identification of bacteria. The characters revealed in different types of media were noted. Solid Media Culture When the microbial colonies grown in solid media they were studied on the basis of following characters, shape, size, elevation, margin, surface, etc. Broth Culture During the period of this study nutrient, glucose and serum broth were used, The characteristics noted were the degree of growth, presence of turbidity and its nature, presence of deposit and the nature of surface growth. Slant Culture The characteristics noted were the degree of growth, their nature their surface, coloure, structure, odour and changes in the medium.

2.14 PHYSIOLOGICAL STUDIES OF SELECTED STRAINS Studies made on the requirement of oxygen, the need for CO2, capacity to form pigments, and power of haemolysis, which helped in the identification of the species. Tests for following physiological activities were done to identify the organisms (Cowen and steel, 1975).

2.14.1 Oxidase ReactionThe ability of the organism to produce cytocrome oxidase was determined by the addition of the test reagent, tetramethyl-p-phenylenediamine dihydrochloride (TMPD). The isolated strains were cultivated on fresh media and incubated at 370C, under aerobic conditions for 24 hours. A piece of filter is soaked with a few drops of oxidase reagent. A colony of the test organism is then smeared on the filter paper. Oxidase positive bacteria turned the filter paper into deep purple color within 2 minutes. 2.14.2 Catalase TestHydrogen peroxide breaks down to form oxygen and water with the help of catalase. When an organism come in contact with hydrogen peroxide and release bubble of oxygen, it indicates that organism produce catalase. Required Hydrogen per-oxide (H2O2) -3% (10 volume solution) Procedure(a) 2-3 ml of H2O2 solution was taken in a test tube.(b) Several colonies were removed from suspected colonies and immersed into the H2O2 solution. (c) Looked for immediate bubbling. Result Active bubbling : Positive catalase test No bubbling : Negative catalase test 2.14.3 Carbohydrate Utilization Test

For carbohydrate utilization test cystine trypticase agar is used. Carbohydrate utilization test was used to validate the identification of a strain as N. meningitidis. To confirm a culture of N. meningitidis a set of four tubes, such containing a sugar (glucose, maltose, lactose and sucrose) was used. With an inoculating loop, a small amount of growth was taken up from an overnight culture on chocolate agar plate. The inoculum was mixed into the medium. The caps of the tubes were fastened and placed in a 370C incubator (without CO2). The culture was incubated at least 72 hours before discarding as negative. Development of visible turbidity and a yellow colour indicates growth and the production of acid.2.14.4 Kligler Iron Agar (KIA) TestIt contains glucose, lactose, and sucrose, phenol red to demonstrate fermentation, and ferrous sulfate to demonstrate hydrogen sulfide production. KIA was used to differentiate gram negative enteric organisms on their ability to ferment dextrose and/ or lactose and their ability to produce H2S. KIA is poured in test tubes so that there is a “butt” and a “Slant”. A stab inoculation is done with a sterile straight wire in the butt and zigzag inoculation over the surface of the stant. Then incubated the tube for overnight at 370C. Red colour indicates alkaline reaction and yellow colour indicates acid reaction.Interpretation(a) Red stant, yellow butt indicate fermentation of glucose. (b) Yellow stant, yellow butt indicate fermentation of lactose, sucrose or both. (c) Red stant, Red butt indicate no fermentation.(d) Cracks and bubbles in medium indicate gas production from glucose.(e) Blackening of the butt, hydrogen sulphite (H2S) is produced.2.14.5 Citrate Utilization TestThis test was used to differentiate among enteric organisms on the basis of their ability to ferment citrate as a sole carbon source. Simmons citrate agar slant was used. The medium was inoculated by means of a stab and streak inoculation. A very small amount of 24 hours old culture was inoculated in citrate medium and incubated for 24 hours at 370C. Citrate positive culture was identified by the presence of growth on the surface of the slant, which was accompanied by blue colouration after 24 hours incubation at 37oC. Appearance of change of colour during growth indicated the utilization of citrate.2.14.6 Motility Indole Urea (MIU) testMotility indole urea semisolid medium was used for this test. One suspected isolated colony was touched with a sterile straight wire loop and stabbed into agar very carefully down the tube, without touching the bottom. A paper strip impregnated with indole reagent (Kovac’s reagent) was inserted between the cotton plug and tube. The tube was incubated at 370C for 18 to 24 hours. Motile property of any organism was detected by the presence of growth along the line of inoculation. Negative reaction against indole can be detected by the absence of discolouration of indole paper or positive reaction by the production of pinkish colour on paper. The presence of urease was detectable when the colour of the medium turned to pink. This happened because the substrate urea is split into its products; the presence of ammonia created an alkaline environment that caused the phenol red to turn into deep pink. Kovac’s reagent: P dimethyl aminobenzaldehyte ………….5 gmAmyl alcohol (Butyl alcohol) ……….75ml Concentrated Hcl ……………………25ml2.15 SPECIFIC TESTS FOR IDENTIFICATION OF PATHOGENS2.15.1 Bile Solubility TestThis is an important test to differentiate Diplococcus pneumoniae from Streptococcus viridans. The bile solubility test was performed by taking several loops of the strains showing alpha-haemolytic colonies on a blood agar plate and making a suspension in a 1.0 ml of sterile saline adjusting to 0.5 McFarland density standards (Popovic et al., 1994). The suspension of cells was divided into two equal volumes (0.5ml per tube). An amount of 0.5 ml of saline was added into one tube and 0.5 ml of 2% sodium deoxycholate (bile salts) was added to the other. Then the tubes were gently shaken and incubated at 370C for up to two hours. Then the tubes were periodically examined for lysis of the cells in the tube containing bile salts. A clearing of the

suspension or loss of turbidity indicated the positive result. It can be performed using either the “tube method” or the “plate method.”Tube Method for the Performance of the Bile Solubility TestTwo tubes are required for bile solubility testing of each suspect strain of S. pneumoniae.a) A loop of the suspect strain was taken from fresh growth on a blood agar plate and prepared a bacterial cell suspension in 0.5 ml of sterile saline. The suspension of bacterial cells should be cloudy, similar to that of a 0.5 or 1.0 McFarland turbidity standard. b) The suspension was divided into two equal amounts (i.e., 0.25 ml per tube). Added 0.25 ml of saline to one tube and 0.25 ml of 2% sodium desoxycholate (bile salts) to the other.• A 2% concentration of bile salts was made and then added 0.2 g of sodium desoxycholate to 10 ml of saline.c) The tubes were gently shaken and incubated at 35°– 37°C for up to 2 hours.d) The tubes were examined periodically for lysis of cells in the tube containing the bile salts. A clearing of the tube, or a loss in turbidity, is a positive result.• Strains that yielded clearing of the suspension in tube in the bile solubility test should be reported as “bile soluble.”• Strains for which the turbidity in the tube remains the same as that in the saline control tube are reported as negative for bile solubility (or “bile insoluble” or “bile resistant”).2.15.2 Optochin Susceptibility TestSuspected alpha-haemolytic colony was touched with a sterile bacteriological loop and streaked onto a blood agar plate. After the loop was streaked across the plate, an optochin disc with a diameter of 6 mm (containing 5µg ethyl hydrocupreine) was placed aseptically on the streak lines where the loop was first placed. The plates were incubated in a candale-extinction jar, overnight at 370 C. Zone of inhibition of the alpha-haemolytic growth having greater than 14 mm diameter was identified as Streptococcus pneumoniae (Popovic et al., 1994). The optochin susceptibility test is performed with a 6-mm, 5-μg optochin disk and is used to differentiate between S. pneumoniae and viridans streptococci. Optochin-susceptible strains can be identified as S. pneumoniae.Performance of the Optochin Susceptibility Testa) The suspect α-hemolytic colony was touched with a sterile bacteriological loop and streaked for isolation onto a blood agar plate in a straight line. Several strains can be tested on the same plate at once, streaked in parallel lines and properly labeled.b) Aseptically placed an optochin or “P” disk with a diameter of 6 mm on the streak of inoculum, near the end where the wire loop was first placed. c) The plates were incubated in a CO2 incubator or candle-jar at 35°C for 18–24 hours.d) Read, record, and interpret the results.2.15.3 Test of X and V Factors RequirementsSmall, gram negative bacilli or coccobacilli which require X and V factors grow on chocolate agar plate (CAP) and not on blood agar plate (BAP), have a pungent indol smell, do not group with meningococcal antisera, may be Haemophilus influenzae. In the absence of vaccination, almost all cases of Haemophilus influenzae meningitis are caused by serotype b.Identification of H. influenzae SerotypePerfomance of the test is similar to that described for N. meningitis except for the choice of antisera. Suspected Hib isolates should be tested against H. influenzae type b antiserum, an antiserum to one of the other groups, and saline. A strongly positive reaction with b antiserum and no reactivity with an antiserum to one of the other groups and saline is presumptive evidence of Hib. If the isolate is non-reactive with the b antiserum, test it with a polyvalent antiserum. If positive, the isolate must be tested with the remaining antisera (a, c, d, f) to determine its serotype. If negative, it is probably non-typable. Determination of X and V factor requirements is necessary to confirm the identity of the isolate as H. influenzae or another Haemophilus species. Perfomance of the testA milky suspension of cells was made from an overnight culture on the CAP or the Haemophilus ID plate in 10 micro litre of 0.5% formalinized saline.

For the agglutination reaction, transfered a loopful (5 micro litre) of the cell suspension to an ethanol washed slide divided into three sections.To each section, respectively, added an equal volume of Hib antiserum, a different type of specific antiserum, and salineMixed with a toothpick and gently rock the slide for up to a minute.Reading of test resultsOnly strong agglutination reactions are used as positive. In a strong reaction, all the bacterial cells will be clumped and the suspension fluid will apear clear. When a strain reacts with more than one antiserum, the result is recorded as non-typable.Identification of X and V Factor RequirementsX, V and XV paper disks are used. H. influenzae is a fastidious organism requiring media cpntainig haemin (X factor) and nicotinamide adenine dinuceotide (NAD, V factor). Growth occurs on CAP because of haemin released during the heating process in the preperation of chocolate agar. Haemin is available from non haemolyzed as well as haemolyzed cells. Alternatively NAD is added as an Iso Vitalex. H. influenzae is identified on the basis of its growth requirements for X and V factors.

Identification of Haemophilus species by their growth requirementsSpecies X factor V factor ß-haemolysis on sheep blood agarH. influenzae + + _H. parainfluenzae _ + _H.haemolyticus _ + +

Performance of the TestA heavy suspension of cells was prepared from a primary isolation plate in a suitable broth of peptone water. If the primary isolation plates contain insufficient growth or are contaminated, make a subculture on a CAP. Caution should be used in preparation of broth to avoid transfer of media to the broth; even the smallest sample of agar will affect the test and may lead to misidentification of the bacteria.Inoculated a heart fusion or tryptic soy agar plate. A sterile swab of the suspension is streaked over one half of the plate, and paper disk containing X, V, and XV factors are placed on the inoculated plate after the inoculums has dried. When two bacterial strains are tested on the same plate, the disks must be placed on the manner shown.Reading the Test ResultsH. influenzae grew only around the disk containing both X and V factors. Alternatively, the prophyrin test of kilian can be used. This determines the X factors requirement of the isolate while avoiding the problem of X factor carry over from primary culture media and X factor contamination of test media (Manual of clinical microbiology). Paper disc for the Detection of β-lactamase EnzymeCefinase discs are intended for use in rapid testing of isolated colonies of N. gonorrheae, Staphylococcus species, H. influenzae, enterococci and anaerobic bacteria for the production of β-lactamase. Material and ReagentsMaterials provided Cefinase discs, 50 discs per cartage.Reagents: Discs impregnated with nitrocefinProcedure: Using forceps moisten disc with one drop of processed water and then wipe across the colony. Result: A positive reaction will show a yellow to red color change on the area where the culture was applied. A negative result will show no color change on the disc.2.16 DETERMINATION OF ANTIMICROBIAL SUSCEPTIBILITY BY DISC DIFUSSION METHOD Antimicrobial susceptibility tests were performed to measure the ability of an antibiotic to inhibit bacterial growth in vitro by disc diffusion which was a modified by Kirby-Bauer method (Bauer et al., 1966). Following media and antibiotic discs were used for determining antimicrobial susceptibility tests.2.16.1 Media

Chocolate agar media supplemented with NAD (5μg/ml) and 1% haemoglobin (Haemin 0.04%), (CA-Hg-NAD) was used for H. influenzae. Mueller-Hinton agar media supplemented with 5% sheep blood was used for Streptococcus pneumoniae. Chocolate agars used for N. meningitidis and Mueller-Hinton agar, Mac-Conkey’s media were used for others.2.16.2 Antibiotic Discs UsedFollowing antibiotic discs were used to determine the susceptibility of identified microorganisms:Ampicillin (AMP) (10 μg)Chloramphenicol (C)(30 μg)Trimethoprim/Sulfamethoxazole (SXT)(25 μg)Ceftriaxone (CRO)(30 μg)Erythromycin (E) (15 μg)Penicillin (P)(10 μg)Meropenem(MEM) (10µg)Imipenem(IPM),10µgCephradine(CE) (30µg)Cefixime(CFM)(5µg)Ceftazidime(CAZ)(30µg)Cefepime(FEP) (30µg)

2.16.3 Preparation of Inoculum for Susceptibility TestThe inoculum for seeding the susceptibility media was prepared from fresh pure cultures of the organism. A cell suspension of the bacteria was prepared in Muellar-Hinton broth and adjusted as equal to a density of a Mc-Farland 0.5 standard (1 x 108 CFU/ml). When the proper density was achieved, a sterile cotton swab was submerged in the suspension, lifted out of the broth and excess fluid was removed by pressing and rotating the swab against the wall of the tube.2.16.4 Inoculation of the Susceptibility Test MediaThe swab was then used to inoculate the entire surface of the respective agar plate three times, rotating the plates at 600C between each inoculation. The inoculated plate was allowed to dry (usually taking only a few minutes but no longer than 15 minutes) before the discs were placed on the plate. Commercial antibiotic discs were placed on the agar plates. The plates containing the discs were incubated at 370 C for 16 to 18 hr in an inverted position in a candle extinction jar.

2.16.5 Estimation of Susceptibility of the IsolatesAfter overnight incubation, the diameter of each zone of inhibition was measured with a scale (in mm)

3. RESULTS

3.1: Study PopulationCerebrospinal fluid samples (n = 371) were collected from different patients with suspected meningitis aged between 2 months and 12 years from different hospitals and diagnostic centres at Dhaka city in Bangladesh. After collection, the physical appearances of the CSF were noted. They were grouped as crystal clear, moderately turbid and highly turbid CSF according to their turbidity. The CSF was used for latex agglutination test, microscopy, culture and biochemical analyses.

The rapid diagnosis of bacterial meningitis in this study included (1) Latex for bacterial antigens for Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus group-B and Escherichia coli, and (2) Immunochromatography test (ICT) for Streptococcus pneumoniae. The total and differential leucocytes counts were done. The concentrations of glucose, protein and C-reactive protein were measured and the CSF specimens were also subjected to microbiological analyses.

In addition to CSF samples, blood samples were also collected for biochemical and microbiological studies. General information and distribution of age and sex of meningitis patients are mentioned (Table 3.1). Among the 371 patients, 63% (234/371) were male and 37% (137/371) were female.

Table 3.1: General information of the study children with suspected meningitis.Parameters FindingAge group of children 2 months-12 yearsTotal children with suspected meningitis 371Total male children patient 234Total female children patient 137Male to female ratio 1.7:1Total latex positive cases 52Total culture positive cases 45Total Gram stain positive cases 48

3.2: Cytological StudyThe total numbers of leucocytes in the cerebrospinal fluid were counted as soon as the samples were brought to the laboratory. The crystal clear CSF showed 0-45 cell/mm3, moderately turbid CSF showed 46-500 cells/mm3. The highly turbid CSF was collected from cases of acute bacterial meningitis. In the later cases as great increases in the number of leucocytes were found and the range was 501 to >10,00 cells/mm3. Total number of leucocytes counted from different CSF is given in table (Table 3.2). Table 3.2 Appearance of CSF in relation to number of total leucocyte cell count (per mm3)Appearances of CSF

Range of total cell count/mm3

Total number of casesNegative (n ═ 319)

Positive(Latex & culture)(n ═ 52)

Crystal clear <5 219 005-45 51 02

Moderately turbid

46-100 33 05101-500 10 04

Highly turbid501-1000 05 25

>1000 01 16In 73.3% (272/371) CSF was colorless and 3.8% (2/52) with CSF positive cases the appearance of CSF was colorless. In 96.2% (50/52) with a CSF positive cases the appearance of CSF was milky that was highly turbid.

Most of the meningitis positive cases showed increased total leucocytes cells counts (TLC) and proteins and a decreased serum sugar concentration. 86.5% (45/52) of culture positive cases show that TLC was higher than 100/mm3 but 13.5% (7/52) of culture positive cases was show that TLC was less than 100/mm3 and 5.0% (16/319) of negative cases showed that TLC was higher than 100/mm3 but 95% (303/319) of negative cases showed that TLC was less than 100/mm3 (Table 3.2 and Fig 3.1).

Growth and TLC

13.5

86.595

5

0

20

40

60

80

100

TLC 0-100 TLC >100

Perc

ent (

%)

Growth No Growth

Fig 3.1: Percentage of growth and TLC (Total Leucocytes Cell) between TLC 0-100 and TLC >100/mm3

3.3 Results of Gram Stain of the CSF SamplesThe precipitate of the CSF sample was used for direct microscopy using Gran staining reaction for the preliminary identification of the organism. After staining bacteria were found in 48 (12.9%) cases. No Gram positive rod bacteria were found from any sample. The type of organisms found were Gram negative coccobacilli, Gram positive cocci, Gram negative cocci and Gram negative bacilli (rod shaped). Table 3.3 represents summarized findings of Gram stain of the CSF samples.

Table 3.3: Morphology and staining properties of bacteria found in the CSF (n = 48)Morphology Staining property Number of positive casesCocci (Streptococcus pneumoniae)

Gram positive 23 (48.0%)

Coccobacilli (Haemophilus influenzae type b)

Gram negative 10 (20.8%)

Cocci (Neisseria meningitidis )

Gram negative 8 (16.6%)

Rods(Escherichia coli)

Gram negative 7 (14.6%)

Fig 3.2: Gram-positive diplococci of Streptococcus pneumoniae in the CSF under microscope

3.4 Biochemical StudyAfter total cell counting, the CSF samples were centrifuged. They were then differentiated into lower precipitates and upper supernatants. The total amount of protein and glucose were measured from the supernatant portion. The amounts were varied according to the turbidity of the CSF.

3.4.1 Protein ConcentrationIn this study, 100% (52) of positive cases showed that protein was more than 100 mg/dL, while 75.2% (240/319) and 22% (70/319) of the negative cases had a protein level under 45 mg/dL and 100 mg/dL respectively and 2.8% (09/319) of the negative cases patients had a protein level higher than 100 mg/dL (Fig 3.3). Mean CSF protein level in the positive group was 315 mg/dL, which was more than the 117 mg/dL of the negative group.

0 0

100

75.2

22

2.80

20

40

60

80

100

120

<45 45-100 >100

Protein concentration (mg/dl)

Perc

ent (

%)

Positive cases Negative cases

Fig 3.3: Distribution of protein concentration between positive cases and negative cases in suspected with meningitis children

3.4.2 Glucose ConcentrationCSF glucose level was also estimated in this study. CSF glucose level <40 mg/dL was found in about 94.2% (49/52) within 52 of latex agglutination positive cases meningitis children, and only 5.8% (3/52) of positive cases had normal level. Among the negative cases, CSF glucose level <40 mg/dL was found in only about 11.6% (37/319) of cases, while the glucose level was above >40 mg/dL is 88.4% (282/319) cases samples (Fig 3.4). Mean CSF glucose level in the positive culture group was 22.5 mg/dL which was lower than the 53.3 mg/dL of the culture negative group. This difference was statistically significant.

41

83

11

26

0102030405060708090

100

<20 20-40 >40

Glucose concentration (mg/dl)

Num

ber o

f cas

es

Positive cases Negative cases

Fig 3.4: Distribution of glucose concentration between positive cases and negative cases with suspected meningitis children

3.4.3 C- Reactive Protein (CRP)

Fig 3.5 shows the level of serum C-reactive protein (CRP) among culturally positive and negative suspected meningitis cases. Serum CRP level was high (>40 mg/dL) among 86.5% (45/52) of the positive bacterial

meningitis children and only 24.8% (79/319) children with suspected non-bacterial meningitis had positive serum CRP test. Only 13.4% (7/52) of the positive cases exhibited negative serum CRP, while 75.2 % (240/319) of negative cases were also negative for serum CRP.

13.4

86.5

75.2

24.8

0

10

20

30

40

50

60

70

80

90

100

CRP < 40 CRP >40

Serum CRP level (mg/dL)

Perc

ent (

%)

Positive Negative

Fig 3.5: C-Reactive protein (CRP) level of positive and negative suspected meningitis cases

3.5: Result of Rapid Diagnosis3.5.1 Immunochromatography Test (ICT)Serological test is very important for diagnosis of bacterial meningitis from patient’s clinical samples of CSF. The ICT test is a more specific and more sensitive than CSF culture. Out of 52 latex agglutination CSF positive specimens, ICT was positive for 42% (n = 22), and among the positive ICT cases only 32% (n = 14) were culturally positive for Streptococcus pneumoniae.

Fig 3.6 Immunochromatography test (ICT) positive of Streptococcus pneumoniae

3.5.2 Latex Agglutination Test (LAT)Latex agglutination test is another important rapid serological test that showed more sensitivity than culture results. 14.2% (52) of Latex agglutination test (LAT) were positive for 14.2% (52) CSFs. Out of 52 CSF specimens that exhibited positivity for bacterial meningitis using LAT, only 12.1% (n = 45) showed positive results in culture (Fig: 3.7 and Fig 3.8). This result showed that serological test was more significant for diagnosis of acute bacterial meningitis than culture (p = 0.86).

Fig 3.7: Latex agglutination test positive for Streptococcus pneumoniae on the top right first one and all are negative.

Fig 3.8: Latex agglutination test for Neiseria meningitidis

Specimens of CSF from children patients with microbiologically confirmed meningococcal infection tested using Wellcogen test-card latex agglutination. Patients tested with (1) N. meningitidis ACYW135 test reagent (+ve); (2) N. meningitidis B/E. coli K1 test reagent (–ve); and (3) N. meningitidis ACYW135 control reagent (–ve)

Fig 3.9: Positive and negative agglutination reactions on a slide: grouping antisera and saline control with Neisseria meningitidis

3.6 Isolation and Identification of Microorganisms from CSFFor isolation of microorganisms from CSF, the specimens were immediately inoculated in two types of media such as blood agar and chocolate agar and incubated at 370C for 24 to 72 hours. Out of 371 CSF samples collected from suspected meningitis children, 45 cases were culture positive.

3.6.1 Isolation of Different Types of MicroorganismsThe colonies were isolated from the culture plates. The morphological, cultural and biochemical properties of the isolated organisms were studied respectively. Identification of the organisms was done by justifying the findings with standard references. Table: 3.4 and Table1: 3.5 represent the characterization and identification of the isolated organisms.

Table 3.4: Morphological and cultural characteristics of the isolatesIsolated organisms Streptococcu Haemophil Neisseria Escherich Klebsiella Salmonella

s pneumoniae

us influenzae

meningitidis

ia coli pneumoniae group D

Vegetative cells

Cocci, lanceolate, arranged in pairs with bases opposed to each other

Slender, short coccobacilli

Oval, diplococci with flattened surfaces apposing each other

Rod shaped

Rod shaped Rod shaped

Gram reaction Gram positive

Gram negative

Gram negative

Gram negative

Gram negative Gram negative

Oxygen relationship Facultative anaerobic, 5% CO2 facilitated growth

Aerobic, facultative anaerobic, 5% CO2 facilitated growth

Aerobic, facultative anaerobic, 10% CO2 facilitated growth

Aerobic or facultative anaerobic

Aerobic Aerobic or facultative anaerobic

Colonyappearance

On blood agar

Small, initially dome-shaped and later ‘draughtsman’ colonies, glistening, translucent, mucoid colonies surrounded by a greenish

No growth Large, translucent, round with regular edge without haemolysis

Large, greyish-whitish, opaque, moist, circular colonies

Large and mucoid

Large, grayish-whitish,

On chocolate agar

Small, glistening, mucoid colonies surrounded by a greenish zone of alpha haemolysis

Large, colourless to grey, opaque, no haemolysis was present

Large, translucent, and round with regular edge

Large, translucent, and round with regular edge

Large and raised whitish colonies

Circular dome-shaped, smooth, opaque and mucoid colonies

Table 3.5: Biochemical characteristics of the isolatesIsolated organisms

Streptococcus pneumoniae

Haemophilus influenzae

Neisseria meningitidis

Escherichia coli

Klebsiella pneumoniae

Salmonella group D

Catalase test

Negative Positive Positive Negative Negative Negative

Oxidase test

Negative Positive Positive Negative Negative Negative

Pigment Not Not Not Not Not Not

produced produced produced produced produced producedUtilization of carbohydrate

Not done Not done All strains oxidizes both glucose and maltose with acid production but no gas

Ferment glucose with production of acid and gas

Ferment glucose with acid and gas produced

Ferment glucose with acid but no gas

H2S production

Not done Not done Negative Negative Not done Positive

Indole production

Not done Not done Not done Negative Not done Negative

Citrate utilization

Not done Not done Not done Negative Positive Negative

Motility Non-motile

Non-motile

Non-motile

Motile Non-motile

Motile

X and V factors requirements

Not done All strains showed growth around the XV factor strip

Not done Not done Not done Not done

Optochin sensitivity

Sensitive to optochin

Not done Not done Not done Not done Not done

Bile solubitity

Soluble in bile salt solution

Not done Not done Not done Not done Not done

Out of 371 specimens’ cultured, 45 (12%) yielded growths of Streptococcus pneumoniae (SPN), Neisseria meningitidis (NM), Haemophilus influenzae type b (HI), and other organisms such as Salmonella-non typhi group D (SALD), Klebsiella pneumoniae (KP) and Escherichia coli (EC). Latex agglutination positivites was observed with 14% (52 cases) comprising culture positivity in 87% (45/52) and culture negativity in only 13% (7/52) cases (Fig 3.17).

Frequency of pathogen occurred among the children

52 45

326

0

50

100

150

200

250

300

350

Latex positive Culture positive Culture negative

Num

ber o

f cas

es

Fig 3.10: Show correlation between latex agglutination and culture

Growth distribution according to by age was very important in this research. It was observed that during the 0-4 year of children meningitis cases are very severe disease but more than 4 years, their severity was less. On total 52 culturally positive and latex positive cases children, 67.3% (35/52) cases was grow among 0-4 years and 26.9% (14/52) was grow 5-9 years but other age groups of positive cases was very low that was 5.8% (3/52) cases. Among the 2-12 months, Streptococcus pneumoniae and Haemophilus influenzae type b was very high but more than 12 months, N. meningitidis growth rate was very high (Table 3.6 and Fig 3.11).

Table 3.6: Causes of bacterial meningitis distribution by age and latex for bacterial antigens and cultural positive cases (n = 52)

Distribution, No. (%) Organism 0-4 5-9 10-12 TotalYears Years YearsStreptococcus pneumoniae 18 (34.6) 6 (11.5) 2(3.8) 26 (50.0)Haemophilus influenzae 8 (15.3) 2 (3.8) - 10 (19.2)Neisseria meningitidis 6 (11.5) 3 (5.7) - 9 (17.3)Escherichia coli 3 (5.7) 1(1.9) 1(1.9) 5 (9.6)Streptococcus Group B 1(1.9) - - 1(1.9)Total 37 (71.1) 11(21.1) 3 (5.7) 52 (100)

18

8

6

3

1

6

23

122

0 01 1

0

2

4

6

8

10

12

14

16

18

20

SPN HI NM EC Others

Bacterial pathogens

Num

ber o

f the

chi

ldre

n

0-4 y5-9 y10-12 y

Fig 3.11: Distribution of the pathogens among various age groups

There were 45 culture positive cases, of which, S. pneumoniae (22, 48.9%), N. meningitidis (8, 17.8%), and H. influenzae (7, 15.6%), E.coli (3, 6.7%), and other pathogens (4, 11.1%). Higher positivity was found with latex

agglutination test showing 52 positive cases including S. pneumoniae (26, 50.0 %), N. meningitidis (9, 17.3%), and H. influenzae (10, 19.2%), E.coli (5, 9.6%), and others pathogens (1, 1.9%) (Table 3.6 and Fig 3.11).

S. pneumoniae 50%

N. meningitidis17%

H.influenzae19%

E.coli8%

Others6%

Fig 3.12: Prevalence of acute bacterial meningitis in children in Bangladesh (n = 52)

26

10 9

4 3

22

7 8

3 4

0

5

10

15

20

25

30

SPN HI NM EC Others

Acute bacterial pathogens

Freq

uenc

y, N

o.

Latex positive Culture positive

Fig 3.13: Distribution of pathogens in culture and latex positive cases in meningitis children. SPN = Streptococcus pneumoniae; HI = Haemophilus influenzae; NM = Neisseria meningitidis and EC = Escherichia coli

Male cases outnumbered the female in this study.The increase of the prevalence of meningitis in female in the age group under one year was recognizable. There was a seanonalites in the occurance of bacterial meningitis been highest during March-April, which accounted for about 55.8% (29/52) cases. Lower occurance was observed in winter season 5.8% (3/52) and in the late autumn, there was no positive meningitis cases (Fig 3.14).

5.89.6

55.8

13.4

0

15.4

0

10

20

30

40

50

60

Winter Spring Summer Autumn LateAutumn

Moonsoon

Seasons in Bangladesh

Isol

atio

n ra

te (%

)

Fig 3.14: Seasonal prevalence of acute meningitis in children in Bangladesh

3.6.2 Identification of Different Types of MicroorganismsDifferent types of culture plates were used for identification of Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis, E. coli and other pathogens. Streptococcus pneumoniae isolate grew on blood agar and chocolate agar media.On blood agar medium both S. pneumoniae (Fig 3.15) and viridans streptococci produced discrete colonies with greenish colouration around the colonies. However, the colony morphology of S. pneumoniae and viridans streptococci is different, which is depicted in Fig 3.16. S. pneumoniae isolates were confirmed by bile solubility test (Fig 3.17) and optochin sensitivity test (Fig 3.18 and 3.19). Haemophilus influenzae type b and Neisseria meningitidis were grown on only chocolate agar media and identified by oxidase test and other biochemical parameters.

Fig 3.15: A properly streaked blood agar plate with pneumococci and viridans streptococci

S.pneumoniae

Fig 3.16: The S. pneumoniae has a depressed center at 24 – 48 hours incubation, whereas the viridans streptococci retain a raised center.

Fig 3.17: Positive and negative results of the bile solubility test

Fig 3.17 The bile solubility test for two different strains. A strain 1 was not S. pneumoniae as both tubes are turbid. There was a slight decrease in turbidity in the tube containing bile salts for strains 1 (2nd tube from the

left) but the tube was almost as turbid as the control tube (1st tube on the left). Strains 2 were S. pneumoniae. The tube on the far right was clear, all the turbidity due to the cells has disappeared and the cells have lysed; by contrast the control tube (3rd tube the left) is still very turbid.

Fig 3.18: The optochin susceptibility test for Streptococcus pneumoniae.

Fig 3.18 The optochin susceptibility test for S. pneumoniae uses P-disks (optochin disks); this laboratory manual presents guidelines for interpretation of the optochin susceptibility test based on a 6-mm, 5-μg optochin disk. The strain in the top streak grew up to the disk: it is resistant to optochin and therefore is not a pneumococcus. The strains in the center and lower streaks are susceptible to optochin and appear to be pneumococci.

Fig 3.19: Antibiogram sensitivity pattern of Streptococcus pneumoniae showing optochin disc susceptibility (Optochin disc sensitive zone 20mm of 6mm disc & Optochin disc resistant zone 00mm of 6mm disc)

H. influenzae is a fastidious organism requiring media containing haemin (X, factor) and nicotinamide adenine dinucleotide (NAD, V factor) for growth. H. influenzae is identified on the basis of its growth requirements for X and V factors. H. influenzae can be differentiated from most other species of Haemophilus by its requirement for both X and V factors for growth. H. haemolyticus is the only other species requiring X and V factors but this species differs from H. influenzae by producing hemolysis on sheep blood agar. H. influenzae grew only around the XV disk (i.e., the disk containing both X and V factors), as shown on the upper half of the plate (Fig 3.20).

OP

Optochin susceptibility

Fig 3.20: Growth factor requirements: X and V factors on paper disks for identification of H. influenzae

Fig 3.21: Slide agglutination test for detecting H. influenzae type b.

Fig 3.22: A properly streaked MacCkonkey’s agar plate with E.coli

3.7 Results of Antimicrobial Susceptibility Test by Disc diffusion MethodAppropiate antibiotic therapy is a critical aspect of management of bacterial meningitis. Antibiotic choice is empirical, based on age at onset, likely pathogens, and antibiotic susceptibility patterns. In this study, most of the isolates on N. meningitidis, H. influebzae type b and S. pneumoniae were found sensitive to moderate spectrum penicillins (ampicillin), two broadest spectrum beta-lactam antibiotics of carbapenems group, namely, imipenem and meropenem and most are also sensitive to other antibiotics of quinolone (ciprofloxacin) and cephalosporin (ceftriaxone). Most of the isolates showed resistance to cotrimoxazole and gentamicin. Moreover, some isolates also exhibited resistance to chloramphenicol and erythromycin. S. pneumoniae, N.

meningitidis and H. influenzae type b isolates were also 100% sensitive to penicillin G, imipenum and meropenum. Among 22 isolates of Streptococcus pneumoniae which was exhibited less sensitive to first line of drugs including gentamicin (8%) and ciprofloxacin (83%), but moderately sensitive to chloramphenicol (93%), erythromycin (95%) and highly sensitive (100%) to ampicillin, penicillin-G, imipenum and meropenum and higher resistance antibiotic of co-trimaxazole (40%) and gentamicin (92%) (Fig 3.23).

100 10083

8

93 100 100

60

100 95

0 017

92

70 0

40

0 50

20

40

60

80

100

120

AMPCRO CIP CN C IM

PMEM

SXTP-G E

Antibiotic

Perc

ent (

%)

Sensitive Resistance

Fig 3.23: Percentage of Susceptibility and non susceptibility of Streptococcus pneumoniae

Among 7 isolates of Haemophilus influenzae type b, were which exhibited good sensitive to ampicillin (91%), ceftriaxone (96%), ciprofloxacin (85%), gentamicin (30%), chloramphenicol (90%) and erythromycin (60%). Higher resistance antibiotic of co-trimaxazole (75%) and gentamicin (70%) (Fig 3.24 and Fig 3.25). H. influenzae was β-lactamase positive 70% and negative 30%. About 95% of β-lactamase positive strains were ampicillin resistant (Fig.3.26).

Fig 3.24: Determination of antibiotic susceptibility patterns of H. influenzae by disc diffusion technique using different antibiotics.

91 9685

30

90100 100

25

100

60

9 415

70

100 0

75

0

40

0

20

40

60

80

100

120

AMP CRO CIP CN C IMP MEM SXT P-G E

Antibiotic

Perc

ent (

%)

Sensitive Resistance

Fig 3.25 Percentage of susceptibility and non-susceptibility of Haemophilus influenzae type b

70

30

0

10

20

30

40

50

60

70

Percent (%)

Positive Negative

Beta lactamase

Fig 3.26: Percentage of β-lactamase between positive and negative cases for Haemophilus influenzae

Among 8 isolates of Neisseria meningitidis, were which exhibited highly sensitive to ampicillin (88%), ceftriaxone (80%), azithromycin (77%), chloramphenical (88%). Only two drugs are highly resistance such as gentamycin (92%) and co-trimaxazole (89%) (Fig 3.27).

8877 80

100

8

88100 100

11

10094

1223 20

0

92

120 0

89

06

0

20

40

60

80

100

120

AMP AZM CRO CIP CN C IMP MEM SXT P-G E

Antibiotic

Perc

ent (

%)

Sensitive Resistance

Fig 3.27 Percentage of Susceptibility and non susceptibility of Neisseria meningitidis

All Escherichia coli and Klebsiella pneumoniae isolates were MDR and most of them showed resistance to 8 to 12 of the antibiotics used including moderate-spectrum penicillins (amoxicillin), broadest spectrum of beta-lactam antibiotic, quinolones (ciprofloxacin), sulphanimide (cotrimoxazole), cephalosporins (cephradine, ceftriaxone and ceftazidime) and aminoglycosides (gentamicin). However, all the isolates were found to be

sensitive to two broadest spectrum beta-lactam antibiotics of carbapenems group, namely, imipenem and meropenem, most are also sensitive to other antibiotics of quinolone (ciprofloxacin) groups.Among (03) isolates of E.coli, were which exhibited 100% sensitive of ceftriaxone, cefixzim, ceftazidime, cefepime, ciprofloxacin, imipenum and meropenum, Only four drugs are resistance such as ampicillin (100%), cephradine (15%), co-trimaxazole (25%) gentamycin (5%) (Fig.3.28 and Fig 3.29).

Fig 3.28: Determination of antibiotic susceptibility patterns of E.coli by disc diffusion technique using different antibiotics

0

85100 100 100 100 100 100

75

95 100 100100

150 0 0 0 0 0

25

5 0 00

20

40

60

80

100

120

AML CECRO

CFMCAZ

FEPIM

PMEM SXT

CNCIP

LEV

Antibiotic

Perc

ent (

%)

Sensitive Resistance

Fig 3.29 Percentage of Susceptibility and non-susceptibility of Escherichia coli.

Only one isolate of K. Pneumoniae were which exhibited sensitive to imipenum (100%), meropenum (100%), ciprofloxacin (81%) gentamycin (88%), co-trimaxazole (74%) and ampicillin (96%), ceftriaxone (80%), azithromycin (77%) (Fig 3.30).

4

37

7081

88100 100

74

96

63

3019

120 0

16

0

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40

60

80

100

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AMP AZM CRO CIP CN IMP MEM SXT

Antibiotic

Perc

ent (

%)

Sensitive Resistance

Fig 3.30 Percentage of Susceptibility and non susceptibility of Klebsiella pneumoniae

3.8 Combined Results of Different Diagnostic Methods of Meningitis in ChildrenThree hundred and seventy one CSF samples were collected from meningitis children patients. Fifty two cases were diagnosed as bacterial meningitis on the basis of latex agglutination test and within the fifty two, forty five cases were diagnosed as bacterial meningitis on the basis of cultural, cytological and biochemical findings of the CSF. Out of 52 latex positive CSF samples, only 45 (12.1%) were culture positive and remaining 7 (1.8%) samples cases gave positive result in latex agglutination test. The results of different diagnostic methods are mentioned in the Table 3.7.

Table 3.7: Different diagnostic methods of meningitis in childrenGram staining of CSF

Culture of CSF

Glucose concentration<40 mg/dL

Protein concentration>100 mg/dL

WBC count>100/mm3

Latex agglutination

+ ve - ve + ve - ve

92.4% 100% 88.9%

+ ve - ve4812.9%

32387.1%

4512.1%

32687.9%

5214.0%

31986.0%

4. DISCUSSION

In the study presented here, 371 children suspected for suffering from meningitis between the age of two months and twelve years were included. Most of them belonged to poor families. They were the victim of malnutrition and suffering from different diseases due to the deficiencies of vitamins.

During the one year of study period, 371 CSF sample were collected from the patients with suspected meningitis in addition of blood sample from children the different hospitals located at Dhaka city at the age group of 12 years, randomly male and female. Among the 371 children patients, 63 %( 234/371) were male and 37 % (137/371) were female. The nature of the sample studied was the cerebrospinal fluid (CSF) which was collected from the affected children admitted in different hospitals. The most commonly presenting signs and symptoms were-Abnormal temperature.Pain in neck and back.Stiffness of neck.Kernig’s sign.Vomiting.

Most of this appeared after the initial onset of illness in the infants. Neurological signs and symptoms were exhibited by the patients from the beginning of their illness. Acute bacterial meningitis continues to be a significant health concern, with a fatality rate of more than 30% in some studies. Although the face of acute bacterial meningitis has changed substantially over the past 15 years, this disease still causes significant mortality (particularly in developing countries) and neurological sequelae (Nathan and Scheld, 2000). Early diagnosis and prompt treatment are very important in the management of meningitis. However, it is not always easy to diagnose meningitis because of its early clinical manifestations are often not specific, especially in babies and small children (Pusponegora et al., 1998). After the patients’ arrival in the hospital diagnosis was confirmed by lumber puncture and immediate examination of the cerebrospinal fluid. The cerebrospinal fluid was examined for cells, protein level and sugar content. In every case a stained film was examined microscopically for organisms and culture was done.

Among total 371 samples, 73.3% (272/371) were crystal clear, 14% (52/371) were moderately turbid and remaining 12.7% (47/371) were highly turbid. The cell counts of the C.S.F were proportionate according to the turbidity. The crystal clear CSF showed 0-45 cell/mm3, moderately turbid CSF showed 46-500 cells/mm3. The highly turbid CSF was collected from cases of acute bacterial meningitis. In the later cases as great increases in the number of leucocytes were found and the range was 501 to >10,00 cells/mm3. In these cases differential counts when performed demonstrated polymorphonuclear predominance.

All of the culture positive CSF specimens, in this investigation, were found to be more or less turbid, and the turbidity in most cases was found proportional to the total blood cell count. Normal CSF is crystal clear. However, as few as 200 white blood cells (WBCs), mostly lymphocytes, per mm3 or 400 red blood cells (RBCs) per mm3 will cause CSF to appear turbid (Seehusen DA, et al., 2003). In this study, most of the culturally-proven meningitis cases showed an increased cerebrospinal fluid (CSF) total WBC count and protein concentration, and a decreased sugar concentration. In 73.3 % (272/371) CSF was colorless and 3.8% (2/52) with CSF positive cases the appearance of CSF was colorless. In 96.2% (50/52) with a CSF positive cases the appearance of CSF was milky that was highly turbid. Most of the meningitis positive cases showed increased total leucocytes cells counts (TLC) and proteins and a decreased serum sugar concentration. 86.5% (45/52) of culture positive cases show that TLC was higher than 100/mm3 but 13.5% (7/52) of culture positive cases was show that TLC was less than 100/mm3 and 5.0% (16/319) of negative cases showed that TLC was higher than 100/mm3 but 95% (303/319) of negative cases showed that TLC was less than 100/mm3 (Table 3.2 and Fig 3.1). Organisms were recovered from the turbid C.S.F. where polymorphonutrophils are predominant, protein concentration increased greatly with the reduction of sugar concentration. The CSF contained >100 WBC/mm3 with a majority of polymorphonuclear cells and /or a glucose concentration <30 mg/dL is also suggestive for bacterial meningitis (Fraser, 1973).

During the course of this study, cerebrospinal fluid (CSF) examined by latex agglutination test in 371 cases showed that positive was 14.2 % (52/371) cases and CSF smear examined by Gram stain showed that the presence of bacterial organisms only in 12.9% (48/371) cases and no organisms could be identified from the remaining 1.0 % (4/371) of latex positive specimens but organisms were isolated by culture of 86.5% (45/52) cases that latex positive was 52 cases and no organisms could be isolated from the remaining 13.5% (7/52). Possibly this was because of earlier antibiotic therapy and delay in culturing or lesser number of organism present in C.S.F. Jervis and Sexena (1972) reported that 135 children between one month and 12 years of age with bacterial meningitis were treated with some types of antibacterial drugs before the diagnosis was made. The CSF smears were positive for bacteria in 68.4% of the ‘treated’ and 81% of the untreated patients.

The precipitate of the CSF sample was used for direct microscopy using Gran staining reaction for the preliminary identification of the organism. After staining bacteria were found in 48 (12.9%) cases. No Gram positive rod bacteria were found from any sample. The type of organisms found were Gram negative

coccobacilli, Gram positive cocci, Gram negative cocci and Gram negative bacilli (rod shaped). Table 3.3 represents summarized findings of Gram stain of the CSF samples.In this study, Gram stain of CSF was positive in 100% of culturally positive cases of bacterial meningitis and none of the culturally negative CSF specimens exhibited microorganisms on direct microscopy. Therefore, direct microscopic examination by Gram staining of CSF could be considered as a reliable method of diagnosing bacterial meningitis. Several factors influence the sensitivity of Gram stain. Laboratory techniques used to concentrate and stain CSF can greatly influence reliability (Kaplan SL. 1999). Greater numbers of colony-forming units (CFU)/mm3 of CSF increase the likelihood of a positive result. Staining will be positive in 25% of cases if fewer than 1,000 CFU/mm3 is present, and in 75% of cases if more than 100,000 CFU/mm3 are present (Lyons Mk and Mcyer FB. 1990). Lastly, the experience of laboratory personnel is very important. Up to 10% of initial Gram stains are misread (Pruitt AA. 1998). Tunkle and Scheld (1995) reported that the sensitivity of the CSF Gram staining could decrease from 75% to 50% in patients who have already been given antimicrobial therapy.

In this study, 100% (52) of positive cases showed that protein was more than 100 mg/dL, while 75.2% (240/319) and 22% (70/319) of the negative cases had a protein level under 45 mg/dL and 100 mg/dL respectively and 2.8% (09/319) of the negative cases patients had a protein level higher than 100 mg/dL (Fig 3.3). Mean CSF protein level in the positive group was 315 mg/dL, which was more than the 117 mg/dL of the negative group. Low CSF protein levels can occur in conditions such as repeated lumbar puncture or a chronic leak, in which CSF is lost at a higher than normal rate (Dougherty JM, et al., 1986). Low CSF protein levels also are seen in some children between the ages of six months and two years, in acute water intoxication, and in a minority of patients with idiopathic intracranial hypertension (Seehusen DA, et al., 2003). The protein level of 2.8% of culturally negative cases was also high (>100 mg/dL) in this study. Elevated CSF protein has also been seen in other infections, intracranial haemorrhages, multiple sclerosis, Guillain Barre Syndrome, malignancies, some endocrine abnormalities, certain medication use, and a variety of inflammatory conditions (Seehusen DA, et al., 2003; Fishman RA., 1992 and Dougherty JM, et al., 1986).

CSF glucose level was also estimated in this study. CSF glucose level <40 mg/dL was found in about 94.2% (49/52) within 52 of latex agglutination positive cases meningitis children, and only 5.8% (3/52) of positive cases had normal level. Among the negative cases, CSF glucose level <40 mg/dL was found in only about 11.6% (37/319) of cases, while the glucose level was above >40 mg/dL is 88.4% (282/319) cases samples (Fig 3.4). Mean CSF glucose level in the positive culture group was 22.5 mg/dL which was lower than the 53.3 mg/dL of the culture negative group. This difference was statistically significant. A true normal range cannot be given for CSF glucose (Seehusen DA, et al., 2003). As a general rule, CSF glucose is about two thirds of the serum glucose measured during the proceeding 2 to 4 hours in a normal adult. This ratio decreases with increasing serum glucose levels. CSF glucose levels generally do not go above 300mg/dL (16.7 mmol/L) regardless of serum levels doughery. Glucose in the CSF of neonates varies much more than in adults, and the CSF –to-serum ratio is generally higher than in adults (Conly JM, et al., 1983). In case of bacterial meningitis, the CSF glucose (reference range is 40-70 mg/dl) is <40 mg/dl in 60% of patients (Razonable RR, et al., 2006). Previous studies demonstrated that CSFglucose concentration <45 mg/dL and CSF protein concentrations >50 mg/dL are suggestive of bacterial meningitis (Slesinger, 2001).

Fig 3.5 shows the level of serum C-reactive protein (CRP) among culturally positive and negative suspected meningitis cases. Serum CRP level was high (>40 mg/dL) among 86.5% (45/52) of the positive bacterial meningitis children and only 24.8% (79/319) children with suspected non-bacterial meningitis had positive serum CRP test. Only 13.4% (7/52) of the positive cases exhibited negative serum CRP, while 75.2 % (240/319) of negative cases were also negative for serum CRP.

The level of serum C-reactive protein (CRP) among culturally positive and negative suspected meningitis cases. Serum CRP level was high (>40 mg/dL) among 86.5% of the positive bacterial meningitis, and only 24.8%

children with suspected non-bacterial meningitis had positive serum CRP test. Only 13.4% of the positive cases exhibited negative serum CRP, while 75.2% of negative cases were also negative for serum CRP. There is no uniform agreement about the sensitivity of CSF-CRP levels in predicting pyogenic meningitis (Tankhiwale SS, et al., 2001). High sensitivity rates were found in some studies (Corrall JC, et al., 1981 and Abramson JS, et al., 1985), whereas low levels were reported in other studies (Tankhiwale SS, et al., 2001; Philips AGS, et al. 1983 and Benzamin DR, et al., 1984.). This continues to be a diagnostic dilemma that should be further explored.

Laboratory investigations of CSF specimens in suspected acute meningitis are extremely important for prompt recognition of the nature of the infecting organism as management and therapy of the patient depend on this information (Panjarathinam R ,et al., 1993 and Williams GR, et al., 1988). Primary care physicians frequently perform lumbar puncture, because cerebrospinal fluid (CSF) is an invaluable diagnostic window to the central nervous system (Wiswell TE, et al. 1995; Visser VE, et al., 1980 and Shattuck KE, et al., 1992). Commonly performed tests on CSF include protein and glucose levels, cell counts and differential, microscopic examination, and culture (Razonable RR, et al., 2006 and Seehusen DA, et al., 2003).CNS infections can cause lowered CSF glucose levels, although glucose levels are usually normal in viral infections (Niu MT, et al., 1990). CSF glucose level <40 mg/dL was found in about 61% culturally positive cases. Among the culturally-negative cases, while CSF glucose level <40 mg/dl was found in only about 12% of cases. A previous study in Bangladeshi population showed CSF glucose level <40 mg/dL in 81% cases of culture-proven meningitis cases (Alam MR, et al., 2006). However, normal glucose levels do not rule out infection, because up to 50% of patients who have bacterial meningitis would have normal CSF glucose levels (Dougherty JM, et al., 1986).C-reactive protein (CRP) is a non-specific acute-phase response molecule produced by hepatocytes in reaction to most forms of inflammation, infection, and tissue damage (Tankhiwale SS, et al., 2001). CRP levels in serum and cerebrospinal fluid (CSF) have been shown to be increased as a result of invasive central nervous system infection (Peltola Ho. 1982), and also to differentiate between bacterial and viral meningitis, since the CRP levels have been found to be significantly lower in cases of viral meningitis (Corrall JC, et al., 1981 and Debcer FC, et al., 1984). Isolation of aetiological agent by culture is a time consuming process while estimation of CRP is a rapid diagnostic procedure (Tankhiwale SS, et al., 2001).

Serum CRP values appeared to be more sensitive in differentiating bacterial and non-bacterial meningitis than the usual parameters measured in CSF like WBC count, protein and sugar (Vaidya AK, et al., 1987). The present findings also correlate with findings of others (Corrall JC, et al., 1981; Debcer FC, et al., 1984 and Vaidya AK, et al., 1987). Unfortunately, viral aetiological agents responsible for meningitis could not be detected in this study. Thus, CRP detected is a helpful screening test to differentiate bacterial and non-bacterial meningitis at the bedside and CRP detected patients should be considered to have bacterial meningitis until proven otherwise (Vaidya AK, et al., 1987).C-reactive protein (CRP) is the classic acute phase reactant (Tankhiwale SS, et al.2001). Microbial infection stimulates hepatocytes in liver to produce CRP (Vaidya AK, et al., 1987). CRP levels in serum and cerebrospinal fluid (CSF) have been shown to be increased as a result of invasive central nervous system infection (Vaidya AK, et al., 1987). Isolation of aetiological agent by culture is a time consuming process, while estimation of CRP is rapid diagnostic procedure. The diagnostic utility of CSF- CRP levels was also evaluated in the present study. In our study, CRP values were found <40 mg/dL in both culturally positive and negative cases.

CSF cytology, in our study, showed cell counts of total leucocytes >5,00/mm3 in about 91.1% (41/45) culture-positive cases and only about 8.9% (4/45) culture-negative cases. The values of total leucocytes have also been reported to predict invasive disease in pneumococcal, H. influenzae type b and Neisseria infections (Peltola V, et al., 2006 and Baker RC, et al., 1989.). In the present study, a strong association with the diagnosis of bacterial meningitis was noted with the diagnostic criterion of CSF protein (Fig 3.3). CSF protein concentration >100

mg/dL was found among 83% of culturally positive cases and only about 32% of culturally negative cases. This is in agreement with that reported by Alam et al (Alam MR, et al., 2006) in Bangladesh.

Serological test is very important for diagnosis of bacterial meningitis from patient’s clinical samples of CSF. The ICT test is a more specific and more sensitive than CSF culture. Out of 52 latex agglutination CSF positive specimens, ICT was positive for 42% (n = 22), and among the positive ICT cases only 32% (n = 14) were culturally positive for Streptococcus pneumoniae.

Latex agglutination test is another important rapid serological test that showed more sensitivity than culture results. 14.2% (52) of Latex agglutination test (LAT) were positive for 14.2% (52) CSFs. Out of 52 CSF specimens that exhibited positivity for bacterial meningitis using LAT, only 12.1% (n = 45) showed positive results in culture (Fig: 3.7 and Fig 3.8). This result showed that serological test was more significant for diagnosis of acute bacterial meningitis than culture (p = 0.86). In 1992, Gray and Federco reported that latex agglutination test could detect the presence of antigen even though antibiotics have been given and culture is negative. The results found from this study also reflect this.

Male cases outnumbered the female in this study. The increase of the prevalence of meningitis in female in the age group under one year was recognizable. There was seasonality’s in the occurance of bacterial meningitis been highest during March-April, which accounted for about 55.8% (29/52) cases. Lower occurance was observed in winter season 5.8% (3/52) and in the late autumn, there was no positive meningitis cases (Fig 3.14).

The prior antibiotic therapy would alter the laboratory C.S.F. findings sufficiently to make specific diagnosis more difficult (Harter, 1963; Quaade and Kristensen et al., 1962; Heycock, 1959. Heycock (1964) also reported that partial treatment of meningitis before admission into the hospital may make the diagnosis more difficult. The chance of finding the causative organism in the partially treated cases are greatly diminished and and this can complicate the treatment in hospital.All the organisms isolated were at first tested for their morphological, cultural and biochemical characters. On such basis they were matched with standard description in the Burgey’s manual (8th ed). The biochemical tests were performed according to Cruickshank et al., 1975.

Meningitis continues to be a formidable illness with high morbidity and mortality in Bangladesh (Saha SK, et al., 1997; Rahman MF, et al., 1990; Saha SK, et al., 2003; Saha SK, et al.2005 and Hoque MM, et al.2006). Gram-positive coccus Streptococcus pneumoniae and Gram-negative bacillus Haemophilus influenzae have been incriminated as major bacterial aetiological agents of pyogenic meningitis in various studies (Saha SK,et al. 1997 & Rahman MF, et al.1990). In spite of potent antibiotics and improved management of the critically ill, there is a significant risk of death or severe neurological sequelae following bacterial meningitis in childhood (Grimwood K, et al. 2000.). A meta-analysis in a developed country found that 4.5% died and at least one major adverse outcome (severe intellectual disability, epilepsy, spasticity, deafness) was present in 16.4% of survivors (Grimwood K, et al., 1995).

During the 12-month study period, a total 371 cerebrospinal fluid specimens from suspected meningitis children of age below 12 years were analyzed for aetiological diagnosis of bacterial meningitis. There were 45 culture positive cases, of which, S. pneumoniae (22, 48.9%), N. meningitidis (8, 17.8%), and H. influenzae (7, 15.6%), E.coli (3, 6.7%), and other pathogens (4, 11.1%). Higher positivity was found with latex agglutination test showing 52 positive cases including S. pneumoniae (26, 50.0 %), N. meningitidis (9, 17.3%), and H. influenzae (10, 19.2%), E.coli (5, 9.6%), and others pathogens (1, 1.9%) (Table 3.6 and Fig 3.11). This finding is similar to the study reported by Bohme et al.,(1993), Carroll (1993), Akpede (1994, Kanra et al.,(1996), Fernandez-Jaen et al.,(1998), Campagne et al.,(1999). In 1998, Kim et al. reported that the most common organisms of culture-proven bacterial meningitis in the last 10 years in Korea have been S. pneumoniae, H. influenzae and N. meningitidis in order of frequency. The widespread use of the H. influenzae type b conjugate vaccine

dramatically decreased the incidence of meningitis caused by H. influenzae type b, S. pneumoniae is the leading cause of bacterial meningitis in the US (Short and Tunkle, 2000).

Typing of H. influenzae isolates during the study period showede that all isolates were type b. In 1997 Saha et al. reported that typing of the strains of H. influenzae in Bangladesh revealed that 98% were type b. In this study, most of the cases were found to be caused by H. influenzae type b and S. pneumoniae in the first year of life. This finding is similar to that of Saha et al., (1997).

A number of bacteria have all been grouped together as “other pathogens” for the purpose of statistical analysis. As causative agents of bacterial meningitis, this group of bacteria was higher in frequency than that of N. meningitides, S. pneumoniae and H. influenzae with a total of 52 confirmed cases by latex agglutination test for bacterial antigen and culture. The strongest association with the diagnosis of other pathogens was patient age, 1 year. This is a significant finding for the pre-diagnosis of uncommon bacterial meningitis (Karanika M, et al.2009). In contrast, the three most commonly presented bacterial pathogens amongst infants and young children during the last decades can be narrowed down to N. meningitides, S. pneumoniae and H. influenzae (Peltola H.2000; Centres for disease control and prevention (CDC),2002 and Theodoridou MN, et al., 2007).

Neisseria meningitidis was isolated from 8 (17.8 %) children of 4 months and 6 years of ages. The patients were treated with penicillin and sulphonamide and the patients were cured gradually.

Escherichia coli and Klebsiella pneumoniae were also frequented as causative organism of meningitis. Escherichia coli were isolated 5.8% (3/52) both culture and LAL test but K. pneumoniae was isolated only one cases by culture that was 2.2% and all of them between 2 months and 9 year. From the report of Mangi et al (1975) it was found that Klebsiella spp. infected the children above one year age, which was similar for one case in the study. The present results are also in contrast to our previous report where we found other organisms in only about 11% cases, while the three important pathogens represented the rest of the episodes (Alam MR, et al. 2006). In another study in Bangladesh, Saha et al (Saha SK, et al.1997), also reports the similar findings. One probable explanation for higher isolation rate of other organisms might be due to inclusion of all ages presenting with symptoms of meningitis , and most (ca. 80%) of the patients were aged above 10 years. While the previous investigations (Alam MR, et al., 2006 and Saha SK, et al., 1997), were conducted only in children. Considering the infants and young children, the isolation rate of other organisms, namely, Escherichia coli, Klebsiella pneumoniae, Salmonella Group –G and Pseudomonas aeruginosa was relatively lower than the three common pathogens. N. meningitides and H. influenzae type b were isolated only from infants, while S. pneumoniae was isolated from both infants and children patients.

Appropiate antibiotic therapy is a critical aspect of management of bacterial meningitis. Antibiotic choice is empirical, based on age at onset, likely pathogens, and antibiotic susceptibility patterns. In this study, most of the isolates on N. meningitidis, H. influebzae type b and S. pneumoniae were found sensitive to moderate spectrum penicillins (ampicillin), two broadest spectrum beta-lactam antibiotics of carbapenems group, namely, imipenem and meropenem and most are also sensitive to other antibiotics of quinolone (ciprofloxacin) and cephalosporin (ceftriaxone). Most of the isolates showed resistance to cotrimoxazole and gentamicin. Moreover, some isolates also exhibited resistance to chloramphenicol and erythromycin. S. pneumoniae, N. meningitidis and H. influenzae type b isolates were also 100% sensitive to penicillin G, imipenum and meropenum. Among 22 isolates of Streptococcus pneumoniae which was exhibited less sensitive to first line of drugs including gentamicin (8%) and ciprofloxacin (83%), but moderately sensitive to chloramphenicol (93%), erythromycin (95%) and highly sensitive (100%) to ampicillin, penicillin-G, imipenum and meropenum and higher resistance antibiotic of co-trimaxazole (40%) and gentamicin (92%) (Fig 3.23). Fortunately, the majority of MDR isolates are susceptible to moderate –spectrum penicillins, azithromycin and ceftazidime. Similar

resistance pattern of H. influenzae type b (Saha SK, et al., 2005& Shoma S, et al., 2001) and S. pneumoniae (Saha SK, et al., 2003) has been reported in other studies in Bangladesh.

Examination of cerebrospinal fluid (CSF) via lumbar puncture (LP) is the only way to confirm meningitis as clinical signs are non-specific and unreliable and blood cultures may be negative in 15-55% of cases (Wiswell TE, et al., 1995; Visser VE, et al., 1980 and Shattuck KE, et al., 1992).

Comparison of data from a study of neonatal meningitis conducted in a national paediatric hospital in Bangladesh between 1987 and 1994 suggests that the bacteria responsible for meningitis have changed very little over this decade (Saha SK, et al., 1997). In the 8-year period study there were 587 culture-positive cases, of which H. influenzae (47%) and S. pneumoniae (32%) accounted for 80% (Saha SK et al., 1997).

Until recently, bacterial meningitis was a greatly feared infectious disease because it struck and killed rapidly, many of its victims were children, and as many as 25% of survivors had sequelae such as permanent brain damage, mental retardation, or hearing loss (Quagliarello VJ, et al. 1997). The present study was carried out to evaluate the spectrum of pathogens of patients of two months to 12 years of ages presenting with clinical signs and symptoms of meningitis in Dhaka City. Causes of bacterial meningitis distribution by age and latex for bacterial antigens and cultural positive cases (n = 52) and (n = 45) respectively (Fig 3.10). Highest number of patients belonged to the adult age group (0-4 years), while children (5-9 years) and adolescents (10-12 years representing the lowest patient category. Drug sensitivity pattern of positive cerebrospinal fluid (CSF) cultures was determined. An attempt was also taken to evaluate the usefulness of various procedures in diagnosing bacterial meningitis.

Culture–positive bacterial meningitis was recorded in the presence of a clinical picture compatible with a diagnosis of bacterial meningitis with positive CSF cultures. On the basis of this criterion, only 45 (12.1%) cases out of 371 patients fulfilled for inclusion as cases of bacterial meningitis. Low bacterial isolation rates were reported in two large studies varying between 1.74 and 3.5% (Sonavane AE, et al., 2008; Weber MW, et al., 2002; Akpede GO, et al., 1994 and Molyneux E, et al., 1998). Culture negative bacterial meningitis was scored 326(87.9%) in the presence of a clinical picture compatible with bacterial meningitis accompanied with negative CSF cultures. In spite of the fact that majority of the patients enrolled in this study was children, but the culturally positive bacterial meningitis was mostly occurred among infants of age group 0-4 years but all of them were aged below 1 year (Table 3.6). Only few positive cases (45) of bacterial meningitis were detected among aged between 2 months and 12 years. In this study, 371 children patients aged up to 12 years were enrolled, of them 52 (14.0%) had confirmed bacterial meningitis. The finding is in agreement with our previous study on childhood meningitis where 13.7% CSF positive culture was recorded (Alam MR, et al., 2006). Bacterial meningitis was more commons in males than females with the total male to female ratio was 1.7:1; although the ratio was 1:1 in case of infants. Men outnumbered women by ratio of 1.2:1 in a German study of bacterial meningitis in adults (Pfister HW, et al., 1993). In a study in Taiwan, in tertiary case centre for the entire country, it was found a male predominance of 2:1 (Tang LM, et al. 1999) Male predominance in bacterial meningitis has also been in Bangladesh (Gurley ES, et al. 2009).

In this study, Group B Streptococcus was found and Gram-negative enteric pathogens like Klebsiella pneumoniae 1(2.2%) and Salmonella species 2(4.4%) were isolated from others 11.1% episodes. Gram-negative enteric or related organisms like Klebsiella pneumoniae, E.coli and Pseudomonas aeruginosa had been isolated infrequently from meningitis cases in Bangladesh (Hoque MM, et al., 2006 and Mitra Ak, et al., 1993) while there is paucity of information of Salmonella infection in Bangladesh. In this study Salmonella was isolated from the CSF of 2 (3.6%) patients with acute meningitis. Salmonella meningitis is a rare disease in both developed and developing countries.

It was also found that pneumococcal meningitis was the most fatal and serious type .Escherichia coli were also an infectious agent which was mainly found in the infants less than one year. This might be due to the ignorance of parents about hygiene and child care. So, this study might provide us with proper knowledge about the child care.

Isolation rate of S. pneumoniae and H. influenzae was very high among children of age group 2-12 months, while N. meningitidis was predominant isolate from children above 24 months of age. The results are consistent with the previous studies in Bangladesh (Saha SK, et al., 2003 & Saha SK, et al., 2005). Late onset of meningitis reflects community acquisition of the pathogens. The corresponding organisms are different from developed countries; early onset meningitis is more likely to be caused by group B Streptococcus (GBS), Escherichia coli, and Listeria monocytogenes, while late onset meningitis may be caused by other Gram-negative organisms well as Staphylococcal species (Health PT, et al., 2003).

From the study it was found that meningitis is a serious problem in our country than in the developed countries. Most of the suffering children belonged to lower socio-eonomic class. They lived in crowed and unhygenic conditions. Their parents had little or no knowledge about health and hygiene. None of them were with sound health. They were brought to the hospital in later stage of disease development and as such the mortality rate was high (13, 95%). The susceptibility was gradually decreased with the gradual increase of ages.

Allister (1975) reported an experimental study of pneumococcal meningitis for the characterization and quantitation of the inflammatory process in the meningitis of rabbits. The inflammation increased progressively with time. The identifying tests for Diplococcus pneumoniae were the bile solubility, optochin sensitivity, inulin fermentation and mice pathogenicity. All the isolated strains of pneumococci were soluble in bile, sensitive to optochin, fermented inulin and pathogenic to mice. The tests can differentiate this organism from the alpha- haemolytic streptococci (Choudhury M. R., 1980).

After isolation and identification of the organisms, antibiotics sensitivity test was done. As the antibiotics therapy is vital to combat bacterial meningitis. The patients received several regimens but best results were obtained with a combination of the broad-spectrum ampicillin and Penicillin.Among 7 isolates of Haemophilus influenzae type b, was which exhibited good sensitive to ampicillin (91%), ceftriaxone (96%), ciprofloxacin (85%), gentamicin (30%), chloramphenicol (90%) and erythromycin (60%). Higher resistance antibiotic of co-trimaxazole (75%) and gentamicin (70%) (Fig3.24 and Fig 3.25). H. influenzae was β-lactamase positive 70% and negative 30%. About 95% of β-lactamase positive strains were ampicillin resistant (Fig.3.26).

Among 8 isolates of Neisseria meningitidis, were which exhibited highly sensitive to ampicillin (88%), ceftriaxone (80%), azithromycin (77%), and chloramphenical (88%). Only two drugs are highly resistance such as gentamycin (92%) and co-trimaxazole (89%) (Fig 3.27).

All Escherichia coli and Klebsiella pneumoniae isolates were MDR and most of them showed resistance to 8 to 12 of the antibiotics used including moderate-spectrum penicillins (amoxicillin), broadest spectrum of beta-lactam antibiotic, quinolones (ciprofloxacin), sulphanimide (cotrimoxazole), cephalosporins (cephradine, ceftriaxone and ceftazidime) and aminoglycosides (gentamicin). However, all the isolates were found to be sensitive to two broadest spectrum beta-lactam antibiotics of carbapenems group, namely, imipenem and meropenem, most are also sensitive to other antibiotics of quinolone (ciprofloxacin) groups.Among (03) isolates of E.coli, were which exhibited 100% sensitive of ceftriaxone, cefixzim, ceftazidime, cefepime, ciprofloxacin, imipenum and meropenum, Only four drugs are resistance such as ampicillin (100%), cephradine (15%), co-trimaxazole (25%) gentamycin (5%) (Fig.3.28 and Fig 3.29). Only one isolate of K. Pneumoniae were which exhibited sensitive to imipenum (100%), meropenum (100%), ciprofloxacin (81%)

gentamycin (88%), co-trimaxazole (74%) and ampicillin (96%), ceftriaxone (80%), azithromycin (77%) (Fig 3.30).

All E.coli and K. pneumoniae isolates were MDR and most of them showed resistance to 10 to 14 of the antibiotics used including moderate-spectrum penicillins (amoxicillin), broadest spectrum of beta-lactam antibiotic (azactam), quinolones (ciprofloxacin), sulphanimide (cotrimoxazole), cephalosporins (cephradine, ceftriaxone and ceftazidime) and aminoglycosides (gentamicin). However, all the isolates were found to be sensitive to two broadest spectrum beta-lactam antibiotics of carbapenems group, namely, imipenem and meropenem, most are also sensitive to other antibiotics of quinolone (ciprofloxacin) groups. The emergence of MDR Gram-negative bacteria has led to a concurrent alarming increase in central nervous system (CNS) infections caused by such bacteria (Lu CH, et al.2000; Nguyen MH, et al.1994 & O’Neil E, et al.2006). Particularly the emergence of resistance to third and fourth-generation cephalosporins has resulted in a significant reduction in available treatment options for Gram-negative bacillary meningitis (O’Neil E, et al.2006; Lu CH, et al.1999 & Nunez ML, et al.1998). In addition, the clinical utility of the available antibiotics that remains active in vitro against such MDR bacteria (most often aminoglycosides and polymyxins) in the CNS is further limited by decreased penetration through the blood-brain barrier (Schoenbach EB.1949). Thus, administration of intraventricular or intrathecal antibiotics alone or in combination with systemic antibiotic therapy is sometimes chosen for the treatment of Gram-negative bacillary meningitis (Nguyen MH, et al.1994). The introduction of ciprofloxacin has provided an attractive option for therapy of Gram-negative meningitis. There are no randomized controlled clinical trails comparing the safety and efficacy of the newer carbapenems (imipenem and meropenem), macrolode and quinolone, or their combinations (Health PT, et al., 2003). However, for the reason multiple antibiotic resistances these antibiotics alone or in combination are recommended for therapy in suspected MDR Gram-negative meningitis.

The importance of early recognition of bacterial meningitis through clinical and laboratory findings continues to be a major concern for clinicians (Centres for disease control and prevention (CDC, 2002). Predictive factors that have shown significant interest in the last decade with respect to bacterial meningitis include peripheral blood leucocyte count (>1,000 cells/mm3) (Freedman SB, et al., 2001), raised CSF protein (>100 mg/dL) (Peltola V, et al., 2006.), low CSF glucose (<40 mg/dL) (Conly JM, et al., 1983) and CSF C-reactive protein (CRP, >40 mg/dl) (Tankhiwale SS, et al., 2001). Interestingly, prognostic factors of clinical and laboratory findings have been used as specific prediction rules for the differentiation of bacterial from viral meningitis (Nigrovic LE, et al., 2007).

CONCLUSIONThe present investigation was an attempt to determine the spectrum of pathogens of patients of all ages presenting with suspected meningitis in Dhaka City. Meningitis will continue to see changes in epidemiology of meningitis with the introduction of new antibiotics. Bacterial pathogens remained similar though their isolation rates, and antibiotics resistance patterns, especially among Gram-negative organisms, have been changing. Gram-negative enteric and related pathogens (11.1%) including E.coli, K. pneumoniae, and Salmonella-non typhi group-D outnumbered the common aetiologic agents of meningitis (88.9%) including N. meningitides, H. influenzae type b and S. pneumoniae. The number of available antibiotics with proven safety and efficacy against the latter three agents included moderate-spectrum (amoxicillin and ampicillin) and broadest spectrum (e.g. azactam) of beta-lactam antibiotics, cephalosporins (cephradine, ceftriaxone and ceftazidime), quinolones (ciprofloxacin) has changed very little. These antibiotics, except for azactam, are mostly ineffective against Gram–negative enteric pathogens. For this reason, it is important that we find ways to promptly treat patients as accurately as possible, for the most likely pathogens as soon as they present in hospital, thereby reducing mortality rates. However, the best hope for dealing with meningitis, especially neonatal and childhood cases is in prevention. Diagnostic criteria have been detailed in few studies and we believe that our study will enlighten others to use clinical and laboratory predictors for the predetermination of bacterial pathogens rather than just for mortality.

Bacterial meningitis is an important serious illness worldwide. Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis are the most common causes of bacterial meningitis in children in this study. The aetiological diagnosis of meningitis in developing countries remains a problem in clinical practice. CSF for latex agglutination test for acute bacterial antigens, Gram stain and serum CRP values appeared to be more sensitive in diagnosing bacterial meningitis than the other laboratory parameters measured in CSF like total WBC count, protein, and sugar level. However, culture remains important in assessing the antibiotic susceptibility pattern of the causative organisms

CONCLUSIONS

Majority of the meningitis cases among children less than 5 years of age in Bangladesh, are caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis.Incidence of bacterial meningitis is higher in the first year of life.Surveillance of CSF isolates would be of some value for guiding the treatment of invasive meningitis disease.A combination of Gram stain, culture, serological tests are necessary for detecting acute bacterial agents of meningitis (maximum number of pathogens).Typing of H. influenzae strains revealed that 100% of the isolated strains from CSF sample are type b.Penicillin can be the drug of choice for pneumococcal diseases.Resistance to the first line of antibiotics is observed amongst the majority of Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitides strains isolated from the CSF samples of meningitis patients. Thus surveillance of antibiotic resistance is essential for treatment and control of bacterial meningitis.High resistance (>60%) to cotrimoxazole with elevated MICs, time to revaluate the role of cotrimoxazole as a first-line agent for treatment of true meningitis in the country