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Molecular diagnosis and epidemiology of Mycoplasma Pneumoniae

Dorigo-Zetsma, J.W.

Publication date2000

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Citation for published version (APA):Dorigo-Zetsma, J. W. (2000). Molecular diagnosis and epidemiology of MycoplasmaPneumoniae.

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Download date:26 Apr 2021

CHAPTERR 1

Generall Introduction

r r

Introduction Introduction

GENERALL INTRODUCTION

1.. Description of the organism

1.11 History

Inn 1944 Eaton and colleagues described an agent that passed through filters with pore sizes

thatt retain bacteria and caused pneumonia when inoculated in rodents (1). The agent was

believedd to be a virus as it passed through bacteriologie filters and could be grown in chicken

embryoss only. The relation of the agent to the primary atypical pneumonia (PAP) syndrome

waswas suggested by the fact that human serum from patients recovering from PAP neutralised

thee agent. In the early 1960ies it was established that the organism had many characteristics in

commonn with the pleuro pneumonia-like organisms (PPLO) isolated in 1898 by Nocard and

co-workerss and causing contagious pleuropneumonia in cattle. Its mycoplasmal nature was

establishedd by growing the agent on cell free medium (2), and subsequently it was

appropriatelyy named Mycoplasma pneumoniae.

1.22 Taxonomy

M.M. pneumoniae is one of the currently recognised 102 Mycoplasma species in the order of

Mycoplasmatales.. Thirteen of these 102 Mycoplasma species have been isolated from

humans.. Mycoplasmas are classified in the Class Mollicutes (mollis=soft, cutis = skin) which

namee originates from the lack of a rigid cell wall, separating mycoplasmas from other

bacteria.. They are bounded by a cell membrane containing sterols. Because of their

deformablee membrane and small size (150-250 nm), mycoplasmas are able to pass through

bacteriologiee filters. As mycoplasmas lack a typical bacterial cell wall, they are not visible on

Gramm staining and resistant to cell wall-active antimicrobial agents such as penicillins and

cephalosporins.. Mycoplasmas are able to grow in cell free media and belong to the smallest

self-replicatingg organisms with genome sizes of 580-1350 kb. Recently, sequence data from

thee entire genomes of M. pneumoniae (3) and M. genilalium (4) became available. This

informationn wil l enhance the knowledge of the molecular biological basis of these

microorganisms.. The small genome is remarkable for such successful pathogens. The question

iss whether there is any relation between genome size and the ability of mycoplasmas to grow

inn vitro. Recently, Hutchison el al. sorted out the number of essential genes necessary for

replicationn of M. genilalium (5). Their conclusion was that 140 of the 340 genes, at least under

laboratoryy conditions, are not a prerequisite for the propagation of this smallest-known free-

livingg micro-organism. In addition, obligate intracellular bacteria like Chlamydia pneumoniae,

CoxiellaCoxiella burnetii, and Rickettsia species have genomes larger than many of the self-replicating

mollicutes,, making a direct relation between genome size and the ability to grow in vitro

unlikely.. The genome sequence projects provided the genetic explanation for the requirements

9 9

ChapterChapter 1

off serum, peptone, and yeast extract in nutritious growth media for the in vitro growth of

mycoplasmas.. Only a limited number of genes allows biosynthetic pathways in mycoplasmas

andd no genes are present for amino acid synthesis (3,4). Therefore, M. pneumoniae and M

genitaliumgenitalium are totally dependent on exogenous supply of omino acids.

M.M. pneumoniae is a filamentous organism, about 10x200 nm in size, with a genome of 816

kb.. Mycoplasmal DNA has an exceptional low guanine and cytosine (G&C) content, with M.

pneumoniaepneumoniae showing the highest G&C content of 40 mol% of the entire genome.

Characteristicc for mycoplasmas is the use of TGA as a tryptophan codon instead of a stop

codon.. The flask shaped cells of M. pneumoniae have a terminal tip structure and exhibit

glidingg motility on solid surfaces (6,7). The terminal tip structure is responsible for attachment

off the organism to cell membranes. Of the surface proteins required for cytadherence, the 169

kDaa protein PI has been most extensively studied (8-11).

1.33 Habitat

Humann mycoplasmas colonise mucous membranes in the ororespiratory or genitourinary

tract,, where some species behave as commensals. Under immune suppression, for example

afterr organ transplantation, in AIDS patients or in patients with hypogammaglobulinacmia,

isolationn of mycoplasmas from joints, pericardial tissue or from respiratory tract tissue

differentt from their usual habitat has been described (12,13).

Humann mycoplasmas have been shown to be taken up by polymorphonuclear leukocytes

(PMNL)) and macrophages (14), but whether mycoplasmas can enter epithelial cells is unclear.

M.M. fermentans and M. penetrans have been found in nonphagocytic cells in AIDS patients

(15,16).. The mechanism of cell entry is not fully understood. The specialised tip structure of

M.M. genitalium and M. penetrans seems to play a role in cell entry but also mycoplasma species

lackingg a tip structure like M. fermentans and M. hominis have been found intracellular by

electronicc microscopy (17).

Whetherr mycoplasmas replicate intracellular̂ remains to be resolved. There are

indicationss for cell lysis of human lung fibroblasts after infection with M. genitalium (18) and

celll disruption after invasion with hi. penetrans (16). The intracellular localisation can protect

mycoplasmass against the host immune system and antibiotics and may be responsible for

latentt or chronic infection states.

M.M. pneumoniae has been shown to parasitize cell surfaces of mammalian cells and to enter

thee intracellular environment where it is located throughout the cytoplasm and perinuclear

regions.. It can persist intracellularly for at least 7 days (19). Baseman isolated M. pneumoniae

mutants,, which are able to cytadhere but not to invade cells, suggesting separate mechanisms

forr adherence and invasion (19).

10 0

Introduction Introduction

2.2. Epidemiology

2.11 Epidemiology

M.M. pneumoniae causes usually a mild respiratory disease. Most cases wil l not lead to a

doctor'ss visit and confirmed diagnosis is often not accomplished in daily practice. Available

dataa on morbidity and mortality due to M. pneumoniae infection from regular sources

(statisticss of death due to pneumonia, statistics on hospitalisation due to pneumonia or

laboratoryy data on M. pneumoniae infection) thus wil l reveal only a minor part of actually

occurringg M. pneumoniae infections.

Thee way diagnosis of a M pneumoniae infection is established wil l influence the outcome

off epidemiological studies. Most studies on M. pneumoniae epidemiology performed in the

past,, made use of data obtained by serological testing or by culture of the organism. A

deficientt knowledge in parameters influencing the antibody response exists. Persistent

antibodiess and antibody response after reinfection may differ and probably depend on the age

andd immune status of the people under investigation. Although isolation of M. pneumoniae is

tediouss and takes several weeks to complete, culture of M. pneumoniae has provided

additionall information for epidemiology of the organism only in settings in which special

attentionn to culture was paid. Prolonged shedding of the micro-organism has been described in

certainn populations and is probably related to several factors like the immune status of the

host,, the age of the host, intervention with antibiotics, and may be, the virulence of the strain.

However,, the asymptomatic carrier state is believed to be rare, although contradictionary

resultss have been reported (20,21).

Manyy studies have been performed in outbreak investigations of respiratory disease due to

M.M. pneumoniae, for example among military and in boarding schools (22-24). Long term

studies,, as performed in civilian populations in Seattle (1962-1975)(25) and Tecumseh (26),

havee provided data on community infection rates and epidemiological patterns.

M.M. pneumoniae infection occurs in temperate climates throughout the year, but the absolute

numberr of infections is higher during the winter season. Because most respiratory infections

duee to other pathogens such as rhinovirus, respiratory syncytial virus and influenzavirus occur

inn wintertime, the proportion of M. pneumoniae infection among respiratory infections is

highestt in summer.

Althoughh M. pneumoniae is endemic in most civilian populations where the infection has

beenn sought for, an epidemic pattern is evident from long-term population studies (27-32).

Epidemicss occur with intervals of 4-5 years. However, a change in this epidemic pattern has

beenn observed in Denmark, possibly related to a change of protective immunity in children,

whichh can be a result from increased use of day-care facilities (27). As can be concluded from

longg term population studies among patients with pneumonia (25) and data from laboratories

registeringg M. pneumoniae infection (29)(this thesis), the more severe M. pneumoniae

11 1

ChapterChapter 1

infections,, occur predominantly in school-age children, with another incidence peak in young

adults.. Although these data suggest that the overall incidence is lowest in the over 40 age

group,, it is likely that there is an underestimation of M. pneumoniae infection occurring in

peoplee older than 40 years. Antibody titers due to M. pneumoniae infection in older people are

significantlyy lower compared to younger people (29; this thesis, Chapter 3), implying that M.

pneumoniaepneumoniae infection in the older age groups is not diagnosed in case only serological assays

aree performed. In studies performed among outpatient populations with respiratory tract

infection,, in which diagnosis of M. pneumoniae infection was made by PCR, no difference in

incidencee between the different age groups is found (33; this thesis, Chapter 5).

2.22 Results of registration o/M. pneumoniae from Laboratory Reporting Systems in Europe

Too gain insight into the epidemiological pattern of M. pneumoniae in Europe, the data from

nationall diagnostic laboratory reporting systems in different countries have been analysed.

Onlyy countries having data reported per quarter or per month for at least 10 years were

included.. These countries were: Finland (1972-1998)(34), England and Wales (1975-

1998X35),, the Netherlands (1981-1998)(36) and Denmark (1972-1999)(37). Data from

Icelandd (1987-1996) reported earlier (29), were also included in the analysis. As M.

pneumoniaepneumoniae infection is not a notifiable disease, the information available is based on

volunteerr reporting systems (Finland, England/Wales, the Netherlands) or on data from the

nationall laboratory performing M. pneumoniae tests (Denmark, Iceland). The reported data

obtainedd from laboratory diagnosed M. pneumoniae infection, most often by antibody

detectionn in serum (CFT and/or EUSA), are presented as absolute numbers.

Inn those countries for which monthly data were available, most infections occurred during

thee winter period (November until March)(Fig 1). In epidemic episodes, however, the

infectionn is diagnosed during the whole year, without preference for the winter period (Fig 2).

Thee data showed that endemic cases occur on a regular basis with epidemic outbreaks every 4

too 5 years. This pattern is most regular in England/Wales and possibly in Iceland, for which

countryy only 10 years data are available. Epidemic outbreaks in the different countries do not

alwayss coincide (Fig 2), most likely because of the protective immunity built up in a certain

population. .

Thee laboratory reporting systems from England/Wales and the Netherlands also register sex

andd age of the patients diagnosed with M. pneumoniae. M. pneumoniae infection occurred

slightlyy more frequent in male patients with an exception for the 16 to 40 years age group in

whichh more female patients were diagnosed with M, pneumoniae infection (Fig 3). In both

countriess there was a peak incidence of infections in young children (4 to 10 years) and a

smaller,, second peak in adults of 30 to 40 years old (Fig 4).

12 2

Introduction Introduction

England/Wales s (1975-1998) )

3000 0

2500 0

2000 0

1500 0

1000 0

500 0

0 0

44 week periods

Thee Netherlands (1981-1999) )

1200 0

1000 0

800 0

600 0

400 0

200 0

0 0 5 5

jann feb mrch apr may June July aug sept oct nov dec

Finland d (1972-1999) )

1600 0

1400 0

1200 0

1000 0

800 0

600 0

400 0

200 0

0 0

jann feb mrch apr may June July aug sept oct nov dec

Denmark k (1990-1999) )

2000 0

1500 0

1000 0

500 0

0 0 l.n.n.n.nn n I: II m I m I HM I na I i

jann leb mrch apr may June July aug sept oct nov dec

Figuree 1 Seasonall distribution of M. pneumoniae in 4 Northern European Countries (data derived from (34-37))

13 3

ChapterChapter I

MpMp reports &igland/Wales (per month)

(n=24,239) )

400 0

755 77 79 81 83 85 87 89 91 93 95 97

Mpp reports the Netherlands (per month)

(n=9672) )

250 0

200 0

150 0

100 0

50 0 v l h M u ^ l i ^ ^ ^ 811 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

MpMp reports Finland (per month)

(n=12,105) )

722 74 76 78 80 82 84 86 88 90 92 94 96 98

MpMp reports Denmark (per quarter)

(n=29,333) )

1500 0

833 86 89 92 95 98

MpMp reports beland (per quarter)

(n=762) )

60 0

40 0 30 0 20 0 10 0 iMii lAiiil i

93 3

Figuree 2 Endemicc and epidemic periods of M. pneumoniae as registered by clinical diagnostic laboratories in 5 Northernn European countries (data derived from(29;34-37))

14 4

Introduction Introduction

Agee and Sex distribution rVfc patients

England/Waless 1990-98 (n=12,943) )

2500 0

2000 0

1500 0 number r

1000 0 500 0 AA AA II I

II male

II female

0-44 5-15 16-25 26-40 41-60 > 60

agee category

1000 0

800 0

600 0 number r

400 0

200 0

0 0

Agee and Sex distribution N̂> patients thee Netherlands 1986-94

(n=4892) )

j j j fa fa 1 1 11 1 r?f l I—HL IJIJtlLB i i

Ell male

female

0-44 5-15 16-25 26-40 41-60 >60

agee category

Figuree 3 Agee and sex distribution of patients diagnosed with M. pneumoniae infection in England/Wales and the Netherlandss (data derived from (35;36))

15 5

ChapterChapter I

Mpp infections by age (England/Wales 75-98) (n=28,609) )

IlllllllMlMUl l 00 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

age e

4000

num

ber

r

OO

O

100 0

Mpp infections by age (the Netherlands 81-96) (n=7020) )

I I

III I lilllllllllllllillllllllllllllllllliii ..i,inil,in....,i,l.iM. 33 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

age e

Figuree 4 Agee distribution of patients diagnosed with M. pneumoniae infection in England/Wales and the Netherlands (data derivedd from (35;36))

16 6

Introduction Introduction

2.33 Transmission

Transmissionn of respiratory pathogens can occur by aerosols (e.g. influenza virus,

BordetellaBordetella spp.), by personal contact (e.g. Epstein-Barr virus) or even via contaminated

objectss (respiratory syncytial virus). In the case of M. pneumoniae it is uncertain whether the

spreadd is primarily by droplets, or by direct or indirect contact, or by all of these three routes.

Infectionn with M. pneumoniae with small-particles (2-3um) in the hamster model resulted in

bothh upper and lower respiratory tract infection, whereas infection after exposure to large-

particlee aerosols was limited to the upper respiratory tract (38).

Thee incubation period of M. pneumoniae is approximately 3 weeks. Spread of a M.

pneumoniaepneumoniae infection in the community is slow. However, within communities,

microepidemicss can arise, in which spread probably occurs from person to person. Only a few

commonn source outbreaks of M. pneumoniae have been reported (39-41).

3.. Clinical disease

3.11 Respiratory infection

M.M. pneumoniae infection should be suspected in patients with a respiratory infection with

clinicall symptoms as seen with influenza-like illnesses such as fever, cough, headache,

malaise,, and myalgia although the onset of an infection with M. pneumoniae is usually less

abruptt compared to an influenza virus infection. Also in people with milder respiratory

infectionss M. pneumoniae may be the cause of such a disease. Non-productive cough is a

characteristicc sign in M. pneumoniae infection. In children cough can be paroxysmal. In an

estimatedd 5-10% of patients M. pneumoniae infection progresses to tracheobronchitis or

pneumonia.. Sputum may be produced in such cases. Gram staining of sputum specimens

revealss only inflammatory cells. Pneumonia is usually mild and self-limited, resolving even

withoutt antibiotic treatment (42). However, severe and fulminant cases have been reported

(43,44)) and may be underdiagnosed.

3.22 Complications

AA variety of complications of M. pneumoniae infection have been described, but their

frequenciess are rarely known (45). Skin rashes, one of the commonest complications occurred

inn 10-20% of the recognised M. pneumoniae infections (46,47). The Stevens Johnson

syndromee (erythema multiforme) although rare, can be a life threatening complication. Other

non-pulmonaryy common complications comprise haemolytic anaemia, thrombocytopenia,

thrombosiss and disseminated intra-vasculair coagulation. Furthermore, neurological

complicationss like encephalitis, meningitis and the Guillain Barre syndrome, but also

myocarditiss and arthritis have been described and are believed to be due to circulating

17 7

ChapterChapter 1

immunee complexes. Recovery of the organism from cerebrospinal fluid (48,49), synovial fluid

(50-52)) and the description of mycoplasmal bacteremia as detected by molecular methods

(53,54)) suggest systemic spread of the bacterium.

4.. The bacterium and the host

4.11 Virulence and evasion of the host immune response

Thee clinical picture of mycoplasma infections is more suggestive of damage due to host

immunee and inflammatory responses rather than to direct effects by mycoplasma cell

components.. M. pneumoniae can be considered as a surface parasite of the respiratory

epithelium.. The adhesion is essential for colonisation and infection as the loss of adhesive

capacityy results in loss of infectivity. For M. pneumoniae and M. genitalium the mycoplasma

adhesins,, their genes (PI, P30, and MgPa) and encoded proteins have been studied most

extensively.. The topography of the adhesion molecules with epitope mapping using

monoclonall antibodies against adhesion molecules in situ and against synthetic oligopeptides

providedd insight into the conformation of the adhesins in the membrane (55). However, in M.

pneumoniaepneumoniae a number of accessory membrane proteins are involved in the process of gliding

motilityy and concentration of the adhesin molecules at the attachment tip organelle. Some of

thee genes and encoded proteins of the M. pneumoniae cytoskeleton have been identified (30,

400 & 90 kDa, P65 and HMW1 and HMW3) and are shown to be proline rich and possess

otherr characteristics of eukaryotic cytoskeletal proteins (56-59).

Forr persistence in the host, pathogenic bacteria develop mechanisms to deal with the host

immunee response. One of these mechanisms consists of variation in antigenic structure in

orderr to avoid host immune recognition. For M. hominis deletions in a repeat-containing gene

encodingg a surface localised antigen resulted in antigenic variation (60). Also in the case of M.

genitaliumgenitalium regions in the MgPa adhesin gene encoding adherence mediating epitopes have

beenn described to be highly variable, thus leading to avoidance of the host immune response

(61).. For M. pneumoniae, deletion of repeated sequences in the gene encoding the 30 kDa

proteinn of the M, pneumoniae attachment organelle, resulted in a hemadsorption negative

mutantt of M. pneumoniae (62). Regions of the PI cytadherence gene which are recognised by

cytadherence-inhibitingg anti PI monoclonal antibodies have been shown to vary, at least

betweenn PI group I and II M. pneumoniae isolates (63,64).

4.22 Host susceptibility to M. pneumoniae infection

Peoplee with congenital or acquired immunodeficiencies are recognised to be more

susceptiblee for a variety of mycoplasma infections. The number of recurrences of M.

pneumoniaepneumoniae infection in such patients compared to people with normal immunity is also

18 8

Introduction Introduction

higher.. In hypogammaglobulinaemic patients and in patients undergoing organ transplantation

andd immunosuppressive therapy, dissemination of mycoplasmas such as M. pneumoniae, M.

hominishominis and Ureaplasma urealyticum to joint and bone tissue has been reported (50,65-69).

Primaryy infection with M. pneumoniae does not protect against a subsequent infection with

thee bacterium as follows from clinical studies (70) and animal studies (71). Reinfected

animalss show more lymphocyte infiltration than primary infected animals, indicating that

specificc cell-mediated host response might play a role in the pathology of a reinfection.

Experimentall reinfections of mice showed elevated mRNA expressions for proinflammatory

cytokiness (72). In humans, the difference in clinical disease between a mild primary infection

andd more severe manifestations after a second infection with M. pneumoniae (73), might be

duee to enhanced immune response as found in the reinfected animals (74).

5.. Diagnosis

Diagnosiss of M. pneumoniae infection relies mainly on laboratory tests, as discrimination

off M pneumoniae from other (mostly viral) respiratory pathogens on clinical parameters only,

iss difficult (75). Specific laboratory diagnosis can be made directly, by culture or detection of

antigenn or DNA of the organism, or indirectly by detection of antibodies.

5.11 Culture

Culturee of M. pneumoniae is labour intensive and requires specific media and experienced

personnell to perform. M. pneumoniae is a relatively slow growing bacterium. After adaptation

too artificial media the mean generation time is 6 hours (76). In clinical practice this means that

incubationn from 1 week to 4 weeks is necessary for propagation of the micro-organism, which

makess culture inappropriate for the routine diagnosis in a clinical setting. If culture is

attempted,, the transport medium should contain peptide broth, serum albumin and antibiotics

too retard overgrowth by other bacteria. Throat swabs, nasopharyngeal aspirates and sputum

sampless are all suitable for culturing of M. pneumoniae, provided they have been suspended in

appropriatee transport medium. Inoculation should be performed within 24 h or, if not possible,

sampless should be stored at -70 °C. Culture media to be used for M pneumoniae have to

containn beef heart infusion broth, supplemented with fresh yeast extract and horse serum

providingg sterols and nucleic acid precursors. Inoculation is performed using both solid and

liquidd medium, the latter containing glucose and phenol red. M. pneumoniae ferments

glucose,, which is detected by a colour change of the medium from red into yellow. On solid

mediaa M. pneumoniae appears in 'mulberry' colonies (Fig 5), which are able to hemadsorb

chickk and guinea pig erythrocytes. Specific antisera are required to provide definite

identification,, although close serologic relationship between M. pneumoniae and M.

19 9

ChapterChapter I

genitaliumgenitalium may encounter difficulties in identification (77). Although the usefulness of

culturingg M. pneumoniae in daily practice is limited, culture remains important as a standard

inn studies in which non-cultural methods are evaluated. Also the development of antimicrobial

drugg resistance and antigenic variation can only be studied if clinical isolates of M.

pneumoniaepneumoniae are available.

Figuree 5 MM pneumoniae colonies on agar (40x magnification) (courtesy to AF Angulo, RIVM Bilthoven, The Netherlands) )

5.22 Direct detection of M. pneumoniae

Ass an alternative for culture, direct antigen tests have been developed. A species specific

probee test (Gene-Probe), an EIA and immunoblot assays making use of monoclonal antibodies

againstt M. pneumoniae proteins have been applied in direct detection of (antigen of) the

micro-organismm in nasopharyngeal specimens (78,79).

Inn the last decade studies using amplification of species specific fragments of the M.

pneumoniaepneumoniae genome by PCR have been published. Target sequences used for molecular

detectionn of M. pneumoniae are mostly chosen in the PI cytadhesin gene (80-85) or in the 16

SrRNAA gene (86,87).

Forr preparation of clinical samples before amplification, various procedures have been

described.. Since mycoplasmas lack bacterial cell wall material, DNA can be made available

byy relative simple procedures. A boil-freeze method (thermal shock) has been applied to

nasopharyngeall aspirates (85) and also lysis with proteinase K on aspirates (88),

bronchoalveolarr lavages (84), and throat swab specimens (75,89) has been applied. For

preparationn of tissue or blood specimens a more elaborate DNA extraction is required. The

20 0

Introduction Introduction

usee of an internal process control is necessary in order to avoid false negative results

(82,85,90).. Up to 20% of the throat swab samples after a proteinase K lysis showed inhibition

off the amplification process (90) and up to 25% of nasopharyngeal aspirates after freeze-

boilingg showed inhibition of the PCR (85), as detected by the lack of the internal control band

afterr gelelectrophoresis. Using 6 globine primers, a more roughly way to test for inhibition, up

too 10% of the aspirates showed inhibition after proteinase K lysis (88). Identification of the

PCRR products has been performed by (microliter well) hybridisation (89,91) as well as by a

nestedd PCR (90). Detection limits of M. pneumoniae by PCR in clinical samples have been

describedd to vary between 1.0-50 colony forming unit (CFU), 10-100 colour changing units

(CCU)) and 5-50 fg of template DNA (53,82,87,90,92). Only a few studies compared

reproducibilityy of PCR results obtained with different primersets in different laboratories

(75,85). .

5.33 Serology

Inn routine laboratories antibody detection in serum is the most commonly used technique in

diagnosiss of M. pneumoniae infection. The first serological assay performed was the cold

hemagglutininn (CA) test. Cold agglutinins are non-specific IgM class antibodies against the I

antigenn of erythrocytes. The specificity of the test can be increased when a cut-off titre of >40

iss used as diagnostic criterion (93). As more specific tests became available, the CA test is not

commonlyy used anymore.

Thee most widely used serological assay is the complement fixation test (CFT) in which CF

antibodiess against M. pneumoniae, in conjunction with antibodies against other, mostly viral,

respiratoryy pathogens are determined. The sensitivity of this assay depends on whether the

firstt serum sample is collected early or late after onset of illness and on the availability of

pairedd sera collected with an interval of 2 to 3 weeks. On interpretation of positive CF titers

onee has to bear in mind that the membrane glycolipids-based antigen from M. pneumoniae is

notnot highly specific. Cross reactivity with e.g. vegetable lipids (94) and human brain tissue

antigenss has been demonstrated (95).

ELISAA tests have been developed to detect specific immunoglobulins of different classes,

mostt of them using a crude M. pneumoniae antigen, which includes the cross reacting

glycolipidd fraction. More specific test results can be obtained by using isolated PI protein as

antigen,, which is one of the major virulence factors of M. pneumoniae (96). Proteins derived

fromm parts of the PI gene have also been applied as antigen in an ELISA test (97).

Commerciall availability of these tests sofar is limited. Immunoblotting, to confirm positive

resultss from the less specific serological assays, have shown strong reactivity against the 169

kDaa PI protein of M. pneumoniae (98), but reactivity against this protein among clinically

healthyy persons interferes with conclusive interpretation. Recently, a commercially available

21 1

ChapterChapter I

immunoblott making use of various recombinant proteins based on M. pneumoniae genome

sequencee data (3) has been developed.

6.. Treatment and prevention

6.11 Treatment

Upperr respiratory tract infection due to M. pneumoniae is mostly undiagnosed and is not

treatedd with antibiotics. Also pneumonia is often a self-limiting disease, but appropriate

antimicrobiall treatment shortens the duration of the illness (99; 100). Respiratory infection

causedd by M. pneumoniae is generally treated with tetracycline or erythromycin. As

mycoplasmass lack a cell wall, M. pneumoniae is unaffected by B-lactam antibiotics such as

penicillinn and cephalosporins. Clinical improvement upon institution of appropriate

antimicrobiall therapy is not always accompanied by early eradication of the organism from the

respiratoryy tract. The capacity of M. pneumoniae to reside intracellular̂ may be responsible

forr this. Also the newer macrolides (clarithromycin and azithromycin) and to lesser extent

fluoroquinoloness have been found to have in vitro and in vivo activity against M. pneumoniae

(101). .

6.22 Prevention

Inn terms of transmission, the respiratory route is the least subject to effective control of

infectiouss diseases. In non-respiratory transmission such as vehicle-borne, gastro-intestinal

infections,, intervention is possible in the food chain or in drinking water systems (102; 103).

Inn case of vector borne infections the vector can be eliminated (104) and in case of genital

infectionss change in behaviour wil l reduce transmission of the causative micro-organism

(105).. For some respiratory pathogens control is possible by intervention in the environment.

Thiss is the case for example with Legionella pneumophila (106) or Chlamydia psittaci (107).

However,, for many respiratory pathogens, including M. pneumoniae, environmental control is

notnot feasible and the only way of controlling these infections is through immunisation of the

hostt or providing chemoprofylaxis.

Preventionn of M. pneumoniae infection through vaccination has been studied as early as the

1960ies,, the time that M. pneumoniae was recognised as a respiratory pathogen. These early

vaccinee studies made use of an inactivated M. pneumoniae vaccine after administration of

whichh volunteers were challenged with agar-grown mycoplasma. Those responding

serologicallyy to the vaccination showed protection to respiratory infection. In volunteers who

failedd to respond to the vaccine with antibody development, more severe disease developed

afterr challenge compared with the controls (108). In larger vaccine trials among military

personnell this sensitisation effect could not be confirmed and the protective efficacy of the

22 2

Introduction Introduction

vaccinee against development of M. pneumoniae pneumonia was about 66% in the first year

followingg vaccination (109). However, the overall success of formalin inactivated vaccines

hass been disappointing and no acceptable live vaccine has been developed for human use.

Thee molecular characterisation of the major adhesin PI of M. pneumoniae, prompted trials

too use purified PI as vaccinogen. The results of immunisation with PI in animal studies were

howeverr disappointing (110), showing the same lymphocyte infiltration in lungtissue after

challengee with M. pneumoniae in immunised animals compared to control animals. The

conclusionn is that until now, no useful vaccine (inactivated or live) for M. pneumoniae has

beenn developed. DNA vaccines, having the advantage of inducing both humoral and cell

mediatedd response, have been applied successfully in animals preventing mycoplasmal disease

(M.(M. pulmonis, M. hyopneumoniae) (111). This generation of vaccines may become useful in

humann mycoplasma disease in the future as well.

Onlyy limited data is available on the effect of prophylactic antibiotic use. In a long-term

caree facility prophylactic antibiotic usage has shown to significantly reduce the secondary

attackk rate of M. pneumoniae infection (112).

23 3

ChapterChapter I

Scopee of the thesis

MycoplasmaMycoplasma pneumoniae is a common respiratory pathogen in humans. The usual mild

clinicall presentation of the infected patient does not encourage laboratory diagnosis to be

performed.. However, in case antibiotic treatment is instituted this should be different from the

firstt choice recommended antibiotics for respiratory infections in general practitioner (GP)

settings.. As the organism is notorious for its difficult laboratory diagnosis, molecular

techniquess should enable fast diagnosis and subsequently appropriate antibiotic treatment.

Currentt knowledge of the epidemiology of M. pneumoniae is based on data predominantly

derivedd from studies using serology or culture for diagnosis.

Inn this thesis the value of molecular techniques in diagnosis of M. pneumoniae in both

childrenn and adult patients with a respiratory infection is investigated (Chapters 2 and 3). For

reliablee detection of M. pneumoniae DNA by PCR, control for inhibition of the amplification

reactionn is required. Therefore, we developed an amplification control (AC) to detect

inhibition,, which is described in chapter 4, This AC was applied to clinical specimens,

submittedd for M. pneumoniae PCR. Furthermore, we applied a semiquantitative PCR to

enablee discrimination of strong and weak positive PCR results in specimens obtained from

hospitalisedd and non-hospitalised patients with a respiratory tract infection (Chapter 4).

Too expand knowledge of epidemiological patterns of M. pneumoniae, the PCR was applied

too nose/throat specimens obtained from patients with acute respiratory infection, identified by

GPss in a routine surveillance for respiratory pathogens. M pneumoniae transmission was

studiedd in the households of the M, pneumoniae positive patients (Chapter 5).

Molecularr epidemiology of M. pneumoniae is hampered, as variation in its genome allows

thee distinction of only two types. In order to refine molecular typing, clinical isolates of M.

pneumoniaepneumoniae collected over time in Denmark and the Netherlands, were subjected to various

molecularr typing methods (Chapter 6). For identification of targets, which should enable

directt genotyping of M. pneumoniae in clinical specimens, PI genes were sequenced. In

addition,, these sequences were analysed to uncover possible mechanisms generating PI gene

sequencee variation (Chapter 7).

24 4

Introduction Introduction

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31 1