Video: ...eta.health.usf.edu/publichealth/PHC4031/S14/Block3/Week15_Protozoa... · falciparum and...

Slide 1

Emerging Infectious DiseasesHSC4933

Lecture 9: Emerging Parasitic Protozoa pt. 2 (Apicomplexans-2: Malaria, Babesia)

Presented by Sharad Malavade, MD, MPH

1

This lecture is the third part of the parasitic protozoa. It represents the second part of the apicomplexan parasites and we will cover malaria and babesia. As you will see this lecture is quite long and heavy on malaria. And that is because malaria is such a huge global health problem.

Slide 2

HSC4933. Emerging Infectious Diseases 2

• Ch.8 (p. 183 [table 8.3], pp. 187-194, special attention to tables)

• Ch. 11 (pp. 310-15)

• Ch. 14 (pp. 412, 422, 425)

Readings-Lecture 9

These are the readings for the lecture. The tables are significant and should be studied well.

Slide 3


• Malaria (Plasmodium spp., Apicomplexan):

Background: http://www.cdc.gov/malaria/about/index.html

Video: http://www.animalplanet.com/tv-shows/monsters-inside-me/videos/malaria-parasite.htm

Babesiosis (Babesia spp., Apicomplexan):

Background: http://www.cdc.gov/parasites/babesiosis/

Video: http://animal.discovery.com/videos/monsters-inside-me-babesiosis.html

Monsters Inside Me

These are some web links for you to get some background information

Slide 4


• Understand the enormous impact malaria has on global health• Know the various species of Plasmodium that can infect man, and differences

between them. • Identify populations that are especially at risk for malaria and babesiosis • Understand the various facets of malaria that make it difficult to eradicate• Know the general life cycles of malaria and Babesiosis-sources of infection,

infective/diagnostic stages, hosts. How are these cycles different from other parasitic protozoa? For malaria, know key diagnostic differences between P. falciparum and P. vivax

• Be familiar with antimalarial drug resistance and where it is a major problem. What drugs are failing in the world and what are viable alternatives?

• Know aspects of malaria and babesia that make them emerging diseases• Describe why vaccines for malaria are elusive

Learning Objectives

These are the learning objectives of the lecture. Most of the lecture covers malaria. Malaria is a global health problem of great proportions. Upon completion of the lecture you will know the plasmodium species that infect an to cause malaria, identify the populations at risk of malaria and babesia. You will understand the facets of malaria that make it difficult to eradicate. You will know the general life cycle of malaria and babesia including sources of infection, infective and diagnostic stages, hosts. How they are different from other parasitic protozoa. For malaria you will know the key diagnostic differences between P.falicparum and P.vivax. You will become familiar with the antimalarial drug resistance and where in the world it is a major problem. Also, what are the alternative drugs available in the arsenal against malaria. You will know aspects of malaria and babesia that make them emerging diseases and learn why vaccines for malaria are so elusive.

Slide 5


• Phylum Apicomplexa pt. 2

– Class Aconoidasida, Order Haemosporida

Plasmodium spp.

– Class Aconoidasida, Order Piroplasmida

Babesia spp.

Lecture 9: On the Menu

This lecture will be the second part of Phylum Apicomplexa. It will cover malaria and babesiosis. The slide shows the taxonomy of these parasites.

Slide 6


Malaria

Lets talk of malaria first. It kills more humans per year than any other parasite. The slides shows graphic that cover the entire spectrum of malaria from clinical cases to diagnostics, treatment and preventive measures. One cannot over-estimate the seriousness of the disease as one child dies per minute due to malaria according to the WHO.

Slide 7

7

• http://video.nationalgeographic.com/video/player/science/health-human-body-sci/health/malaria-sci.html

Malaria Intro Video

Slide 8

• Malaria is the most important parasitic disease affecting man

• Disease of antiquity– First described in Chinese writings in

2700 B.C.

• Approximately 156 species of Plasmodium which infect various species of vertebrates.– Four species are considered true

parasites of humans (int. host)

• Transmitted by Anopheles mosquitoes (def. host)

Malaria: Background

Malaria is a disease known to man since antiquity. Approximately 156 species that infect vertebrates. There are four major species that infect humans and there is one more that is now an emerging problem in Malaysia that we will talk about later. Malaria is transmitted by the mosquitoes of the genus Anopheles. The top picture is an enhanced image showing the erythrocytes. A couple are infected and you can see the tiny parasites in them. One of the infected cells is lysed and is releasing the tiny parasite forms called merozoites. The lower image is of a female anopheles mosquito taking a blood meal.

Slide 9

• World Health Organization estimates– About 3.4 billion people are at risk of malaria– 270 million new infections per year in 2012– 627000 malaria deaths in 2012– 90% of deaths occur in Africa (south of Sahara)

• Occurs in tropical and temperate regions worldwide• Most cases in Africa, followed by Asia and South

America• Dependent upon vector and host, other factors• Global “contraction” from 1900-1950 and

accelerated during 1950-1965

Public Health Impact

As I said malaria is the most important parasitic disease of man. These are some of the numbers for malaria from the WHO. These vary from year to year. The numbers wont be asked in the quizzes. About 3.4 billion people are at risk of malaria. In 2012 there were about 207 million cases of malaria and about 627,000 deaths. About 90% of cases occur in Africa(south of the Sahara). Malaria is found in tropical and temperate regions of the world. After Africa, the highest number of malaria cases occur in Asia and South America. There was a global contraction of the number of cases of malaria following the malaria control efforts from 1900-1950, but the number of cases accelerated from 1950-1965 due to resistance to insecticides amongst other factors.

Slide

10 • P. vivax– Most common, except in Africa

– Temperate and tropical

– 48h cycle, relapse, hypnozoites,

drug resistance

• P. falciparum– Tropical

– Holoendemic in much of Africa

– 48h cycle, mortality, Tropical regions, drug resistance

– Most lethal

Plasmodium spp. Facts

• P. malariae– Global, but patchy 72h cycle

• P. ovale– Mainly in tropical Africa and

Oceania

– 48h cycle, relapse

• P. knowlesi– New emerging species in

humans

– Malaysia, 24 h. cycle

Lets look at some of the facts of the Plasmodium species. Plasmodium vivax is the most common species of Plasmodium that causes malaria except in Africa where P. falciparum is more common. It occurs in temperate and tropical regions and has a 48 hour cycle. It can relapse and is due to hypnozoites which we will discuss in a short while. Drug resistance is a serious problem with P. vivax. The next species is P. falciparum which is endemic in much of Africa. This also has a 48 hour cycle and high mortality. It also has concerns of drug resistance. It is the most lethal form of Malaria. P. malariae is global but a patchy 72 hour cycle. P.ovale is found mainly in tropical Africa and Oceania. It has a 48 hour cycle and causes relapses. P.knowlesi is recently emerging in Malaysia where it has been suspected to have been transmitted from monkeys to humans. It has a 24 hour cycle.

Slide

11


Malaria: Life cycle

See animated life cycle link in BB

Mosquito is

definitive host

Man is

intermediate

host

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells and mature into schizonts , which rupture and release merozoites . (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony ). Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal . The parasites' multiplication in the mosquito is known as the sporogonic cycle . While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes . The zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the mosquito where they develop into oocysts . The oocysts grow, rupture, and release sporozoites , which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle .

Slide

12

12

Worldwide distribution of malaria

From Public health challenges and prospects for malaria control and elimination

Pedro L Alonso & Marcel Tanner Nature Medicine 19, 150–155 (2013) doi:10.1038/nm.3077

This map shows the distribution of malaria in the world. As seen the most critical areas for malaria are South Asia, Southeast Asia and Africa.

Slide

13


• Infants & young children (under 5 yrs. old)• Non-immune pregnant women

– High rates of miscarriage and cause over 10% of maternal deaths (soaring to a 50% death rate in cases of severe disease) annually.

• Semi-immune pregnant women risk severe anemia and impaired fetal growth even if they show no signs of acute disease.

• HIV-infected pregnant women are also at increased risk.

• Non immune (tourist)

Who does malaria affect?

Malaria affects infants and small children more severely than adults. Especially under-five children. Non-immune pregnant women can undergo miscarriage. Pregnant women with partial immunity have anemia when they get malaria. Also at risk are women with HIV. Non-immune travelers and tourists can get infected and carry the parasite to non-endemic regions.

Slide

14


• Costs to individuals and their families - purchase of drugs for treating malaria at home; - expenses for travel to, and treatment at, dispensaries and clinics; - lost days of work; absence from school; - expenses for preventive measures; - expenses for burial in case of deaths.

• Costs to governments include - maintenance, supply and staffing of health facilities; - purchase of drugs and supplies; - public health interventions against malaria, such as insecticide spraying or

distribution of insecticide-treated bed nets; - lost days of work with resulting loss of income; and - lost opportunities for joint economic ventures and tourism.

• Direct costs (for example, illness, treatment, premature death) have been estimated to be at least US$ 12 billion per year. The cost in lost economic growth is many times more than that.

Malaria – the cost

Costs to individuals and their families -purchase of drugs for treating malaria at home; -expenses for travel to, and treatment at, dispensaries and clinics; -lost days of work; absence from school; -expenses for preventive measures; -expenses for burial in case of deaths. Costs to governments include - maintenance, supply and staffing of health facilities; - purchase of drugs and supplies; - public health interventions against malaria, such as insecticide spraying or distribution of insecticide-treated bed nets; -lost days of work with resulting loss of income; and -lost opportunities for joint economic ventures and tourism. Direct costs (for example, illness, treatment, premature death) have been estimated to be at least US$ 12 billion per year. The cost in lost economic growth is many times more than that.

Slide

15


• Substandard, counterfeit drugs

• Drug resistance

• Insecticide resistance

• Deterioration of public health services

• Environmental changes

• No vaccine

Why is it getting worse?

The reasons why malaria is still a problem include substandard drugs or counterfeit drugs, resistance to drugs. This is a serious problem as newer drugs have to be constantly developed for the fight against malaria. Insecticide resistance in malaria has caused the malaria control program to slow down. Newer insecticides are now available that are different from those initially used for vector control. The deterioration of public health services especially in developing countries impedes public heath education and surveillance efforts for the control of malaria. Environmental changes have led to the spread of the vectors and increased risk to naïve populations. Also, as yet there is no effective vaccine against malaria.

Slide

16


Anopheline Mosquitoes• Approximately 430 known species, 30-50 transmit

malaria • Life cycle – 7 to 20 days (egg to adult)

– Females mate once and lay 200-1000 eggs in 3-12 batches over a lifetime

– Find their host by chemical and physical stimuli– Average life span of adult mosquito < 3 week

Malaria development• Development in mosquito (extrinsic incubation

period)=7 to 12 days• Each male & female gametocyte produce >10,000

sporozoites

• Important vector species begin entering houses in the early evening, often at sunset.

• Important vector species often begin leaving houses just before or during sunrise.

Mosquitoes and Malaria

Anopheles mosquito species are the vectors for malaria. Only the females bite humans to take a blood meal as they need the protein for laying their eggs. There are approximately 30-50 species that can transmit malaria. The life cycle of the mosquito is from 7 to 20 days. The females mate once and lay about 200-1000 eggs. Around evening and dusk, the vector mosquitoes begin entering the houses and they leave before sunrise. So the risk of getting a mosquito bite and malaria is highest between dusk to dawn.

Slide

17


• Determines presence of larval habitats and abundance of vectors

• Influences distribution and abundance of humans (mosquito-human-mosquito cycle of transmission)

• Factors that influence presence, abundance and longevity of mosquitoes. – Temperature– Rainfall– Relative Humidity– Topography

Altitude, elevation Soil Type Land cover (vegetation)

Ecology and malaria transmission

The ecological features of the region and the abundance of mosquito breeding habitats determines the abundance of the vector mosquitoes. Amongst factors that influence the presence, abundance and longevity of mosquitoes are temperature, rainfall, relative humidity and topography like altitude, soil type, and vegetation.

Slide

18


Distribution of malaria vectors

This map shows the distribution of the different species of anopheline vectors that transmit malaria.

Slide

19

19

Malaria: 2013

This slide shows the species-wise distribution of malaria in the world. As can be seen from the map > 90% P. vivax infection is singularly found in China, Afghanistan, some countries of Central and West Asia, Argentina, Bolivia and Mexico. Falciparum is found in most of the African countries. Both Falciparum and vivax are co-endemic in India, Southeast Asia, Eastern Africa around the horn of Africa and northern South America along with Madagascar.

Slide

20


This slide shows the malaria elimination that has been achieved country-wise. The green regions have no malaria, the blue regions are working on elimination of malaria while the red ones are currently working on the control of malaria.

Slide

21


• Malaria was endemic in the US until the late 1940's.

• Most of the transmission occurred in the southeastern states.

• Control efforts conducted by the state and local health departments, supported by the federal government, resulted in the disease being eradicated by 1949.

• Such measures included drainage, removal of mosquito breeding sites, and spraying (occasionally from aircrafts) of insecticide

Malaria in the U.S.

While today there is no malaria in the US, our country is no stranger to malaria. Malaria was endemic in the US in the until the 1940’s and most of the transmission occurred in the southeastern states. The control efforts instituted by the local and state health departments along with federal support resulted in the disease being eradicated in the US by 1949. The measures included drainage, removal of mosquito breeding sites and spraying of insecticides.

Slide

22

22

Recent Stats for Malaria in the US- CDC

0

200

400

600

800

1000

1200

1400

1600

1800

U.S. military personnel

U.S. civilians

Foreign residents

Status not recorded

Malaria Surveillance — United States, 2011

Surveillance Summaries. November 1, 2013 / 62(ss05);1-17

This graphic shows the number malaria cases in the US since 1980 to 2011. The number of cases of malaria has increased in the recent years. Foreign residents and US citizens that travel abroad make up most of the cases. So it is necessary to take malaria prophylaxis when visiting the endemic regions.

Slide

23

23

Plasmodium species in malaria cases in the US 2008-2011 (CDC)

0

100

200

300

400

500

600

700

800

900

1000

2008 2009 2010 2011

P. falciparum

P. vivax

P. malariae

P. ovale

P. knowlesi

Mixed

Undetermined



This graph shows the cases of malaria depending on the species of Plasmodium. Falciparum followed by vivax are the most prominent aetiological agents.

Slide

24

24

Number of malaria cases, by state or territory in which case was diagnosed — United States, 2011



This graph shows the state-wise incidence of malaria cases in the US in 2011. NYC leads with 238 cases and this is not surprising the number of immigrants in NYC. It is followed by California, Maryland and Florida.

Slide

25


• Prevention of malaria in travelers• Ensure prompt diagnosis and treatment of

all infected persons• Containment of local outbreaks of malaria• Conduct surveillance of malaria cases• Sensitize local communities to the risk of

malaria• Encourage personal protection measures

to prevent mosquito bites• Conduct vector control activities• During 1963-1999, 93 cases of

transfusion-transmitted malaria were reported in the United States

Minimizing Risk of Re-emerging malaria in U.S.

Lets talk of minimizing the risk the risk of re-emerging malaria in the US. This is focused on prophylaxis for travelers and education for use of mosquito repellants. All infected persons should be promptly diagnosed and treated to minimize transmitting the infection to mosquito vectors. When an outbreak of malaria is reported, it is essential to amp up the mosquito control efforts to eliminate any mosquitoes that may have acquired the parasite. Active surveillance of malaria cases and sensitization of the communities for the risk of malaria. During 1963-1999, there were 93 cases of transfusion transmitted malaria were reported in the United States.

Slide

26


• Infects long-tailed macaques• Previously diagnosed as P. malariae in

humans• 24 hour cycle, higher parasitemia than P.

malariae• Exists primarily in Malaysia• 2008 study: P. knowlesi DNA was detected in

27.7% samples from Sarawak hospitals, 83.7% from Sabah, 5 from Pahang

• Similar infections have been found in Thailand, the Philippines, and Singapore

• Economic exploitation of the forest is perhaps bringing monkeys, mosquitoes and humans into increased contact.

Plasmodium knowlesi

Plasmodium knowlesi is the newest kid on the block for human plasmodial infections. It naturally infects long-tailed macaques in Malaysia. The first cases were initially misdiagnosed as P.malariae, but the parasitemia was higher than in P.malariae and had a 24 hour cycle. In 2008, a study found P.knowlesi DNA in 27.7 % samples from Sarawak hospitals and 83.7% samples from Sabah. Similar infections have been found in Thailand, Philippines and Singapore. Economic exploitation of the forest is perhaps bringing the monkeys, humans and mosquitoes in increased contact.

Slide

27


P. knowlesi: Emerging Disease

Here is an example of acquiring P. knowlesi while traveling abroad. This is a great case to illustrate how infection can be imported into the US and cause a serious outbreak given that we have competent Anopheles vectors here.

Slide

28


• The first symptoms of malaria after the pre-patent period (period between inoculation and detectable stages: when the sporozoites undergo schizogony in the liver) are called the primary attack. It is usually atypical and may resemble any febrile illness.

• Headache• Feeling of weakness and exhaustion• Aching in bones, limbs, or back• Loss of appetite• Desire to stretch or yawn• Nausea and vomiting

MALARIA: Prodromal symptoms

Lets look at the clinical part of malaria. The clinical symptoms of malaria include Headache, malaise, aching bones, limbs or back, loss of appetite, nausea and vomiting. These are not specific and resemble any febrile illness.

Slide

29


PREPATENT PERIOD INCUBATION PERIOD

P. falciparum 11 - 14 days 8 - 15 days

P. vivax 11 - 15 days 12 - 20 days

P. ovale 14 - 26 days 11 - 16 days

P. malariae 3 - 4 weeks 18 - 40 days

Prepatent & incubation periods of human malaria

prepatent period is the period between infection of the host and the earliest time at which the causative agent can be recovered from the patient or, in the case of parasites, eggs or larvae can be recovered from feces, urine or blood. It is usually shorter than the incubation period but may be longer in some parasitic infestations, e.g. hookworm infestation in puppies. The table gives the pre-patent and incubation periods for the different Plasmodium species. Vivax and falciparum have similar prepatent periods but different incubation periods which overlap to some extent.

Slide

30


• The asexual intra-erythrocytic stage causes all pathophysiological processes– Parasite and red cell material released with schizont rupture, resulting in chills

and high grade fever.

• Paroxysms– Episodes of chills and fever – Begins with cold stage of up to 1 hr duration-Profuse sweating as body

temperature falls – Fever stage follows rapidly for 6-12 hr

Nausea, Vomiting, Rapid pulse

• The pattern of intermittent chills/fever mirrors the synchronized parasite development in an infected person’s blood (48 or 72 hours)– Benign tertian: P. vivax– Malignant tertian : P. falciparum– Quartan : P. malariae

• Recrudescences= reinfection as result of parasites not being cleared• Relapses= result of P. vivax and P. ovale stages that hide in liver

Uncomplicated Malaria

The clinical symptoms of malaria are caused by the intra-erythrocytic stages. The parasite and red cell material released release by schizont rupture results in chills and high grade fever. Paroxysms are episodes of chills and fever. It begins with cold stage of upto 1 hour and there is profuse sweating as the body temperature falls. Fever stage follows rapidly for 6-12 hours and is characterised by nausea, vomiting and rapid pulse. Now depending on the pattern of intermittent chills/fever mirrors the synchronized parasite development in an infected person’s blood (48-72 hours) Benign tertian for P.vivax Malignant tertian for P.falciparum Quartan for P.malariae Recrudescence is the reinfection as results of parasites not being cleared. Relapse is the result of P.vivax and P.ovale stages that hide in the liver (hypnozoites)

Slide

31

Severe Malaria and other manifestations

• Cerebral malaria, with abnormal behavior, impairment of consciousness, convulsions, seizures, coma, or other neurologic abnormalities

• Severe anemia due to hemolysis (destruction of red blood cells)

• Hemoglobinuria (hemoglobin in the urine) due to hemolysis (Blackwater fever), renal failure

• Pulmonary edema or acute respiratory distress syndrome (ARDS)

• Abnormalities in blood coagulation and thrombocytopenia (decrease in blood platelets)-bleeding/clotting disorders

• Cardiovascular collapse and shock• Metabolic acidosis• Hyperparasitemia

Severe disease with life-threatening complications- P. falciparum

• Hypoglycemia• Septicemia• Gastrointestinal bleeding• Pneumonia


• The clinical presentation can vary substantially depending on the infecting species, the level of parasitemia, and the immune status of the patient.

Don’t memorize, but know spectrum of

severity and how different from

uncomplicated malaria

Severe malaria occurs when P. falciparum infections are complicated by serious organ failures or abnormalities in the patient's blood or metabolism. The manifestations of severe malaria include: ARDS which may occur even after the parasite counts have decreased in response to treatment. It is important to know cerebral malaria and severe anemia. The hemolysis leads to hemoglobin in the urine and the diseases is then known as blackwater fever. Severe anemia can also lead to cardiovascular collapse and shock. There can be hyperparasitemia in which the parasite number are very high. Severe disease in P.falciparum infection include hypoglycemia, septicemia, GI bleeding and pneumonia. The clinical presentation can vary substantially depending on the infecting species, the level of parasitemia, immune status of the individual. There is an age dependency for disease complications: severe anemia occurs early in childhood; cerebral malaria, later. After repeated infections, children develop anti-malarial immunity that controls parasite growth and limits disease from subsequent infections. Adults living in endemic regions rarely experience severe malaria morbidity.

Slide

32


• Most common clinical presentation of severe malaria in man

• Definition– Parasitized red blood cells (PRBCs) adhere to the

cerebral microvasculature, causing blockage of the blood's pathway - stops blood flow, leading to a shortage of oxygen and nutrients those areas of the brain (hypoxia).

– Unrousable coma– Other causes of encephalopathy have been excluded

(viral infections, head injury, etc)– Mortality rate of ~22% despite treatment

P. falciparum- Cerebral malaria

Lets now look at Cerebral Malaria. It is seen only in infection with P. falciparum. And is the most severe malaria in man. It is seen when the parasitised RBCs adhere to the cerebral vasculature and cause blockage of leading to ischemia in the affected parts of the brain. There is unarousable coma. Differential diagnosis includes different causes of encephalopathy. It has a high mortality despite treatment.

Slide

33


• In sub-Saharan Africa, there are 400,000 cases of severe maternal anemia and 75,000-200,000 infant deaths annually.– 10,000 maternal deaths per year.

• Adverse effects on both mother and fetus, including maternal anemia, fetal loss, premature delivery, intrauterine growth retardation, and delivery of low birth-weight infants.

• It is a particular problem for women in their first and second pregnancies and for women who are HIV-positive.

• High transmission area vs. low transmission area: anemia and birth outcomes differ

Pregnancy and Malaria

Malaria affects pregnancy adversely and can lead to maternal anemia, fetal loss, premature delivery, intrauterine growth retardation and delivery of low birth weight infants. It is a particular problem for women in their first and second pregnancies and for women who are HIV positive. When one compares the high vs low transmission areas, the anemia and birth outcomes differ.

Slide

34


• Elicit a good travel history• Ethnic origin of patient or family

members• History of previous episodes of malaria• Blood transfusions or i.v. drug use• Occupation• Giemsa stained blood smears• PCR• Dipstick antibody tests to differentiate

species• DNA staining

Diagnosis: Important facts

Thick and thin blood smears

Diagnosis depends on lab tests and a good clinical history especially the travel history. This helps in figuring out the infecting species, ethnic history, history of drug use, occupation. Traditionally a thick and thin smears are made, the thick smear is to detect the infection while the thin smear helps identify the microscopic features of the parasite and identify the species. Dipstick antibody tests help to differentiate species.

Slide

35


P. falciparum• Mostly rings or gametocytes present

– Trophozoites and schizonts cytoadherence

• Crescent shaped gametocytes• 24 to 36 merozoites (if schizont present)• No Schuffner’s stippling• Infected erythrocyte not enlargedPlasmodium vivax• All stages present• Infected erythrocyte enlarged• Schuffner’s stippling (dots) • 12 to 24 merozoites• Oval gametocyte almost fills erythrocyte

Diagnostic criteriaRing stages Gametocyte

We will discuss in detail the diagnostic criteria for P. vivax and P. falciparum as they are the most significant species. For P.falciparum mostly the rings forms or gametocytes are present. The gametocytes are crescent shaped and the RBC is not enlarged. If a schizont is present, it has 24-36 merozoites. For P . Vivax all stages are present in the blood and there is stippling (Schuffner’s stippling) of the erythrocytes with enlargement. Schizonts have 12-24 merozoites. The gametocytes are oval and fill the RBC.

Slide

36


P. falciparum vs. P. vivax

Notice the differences?

This slide shows the different stages of P. vivax and falciparum for comparison. As you can see, the stippling is present in vivax and absent in falciparum. The RBC is enlarged in vivax and not in the other. Also note the shape of the gametocytes. They are banana shaped in falciparum and oval shaped in vivax.

Slide

37


• Treatment varies according to the infecting species, the geographic area where the infection was acquired, and the severity of the disease

• Treatment for malaria should not be initiated until the diagnosis has been confirmed by laboratory investigations.

• Treatment of infection– Acute, uncomplicated malaria– Severe or cerebral malaria

• Radical cure• Prophylaxis

– Causal, Suppressive – Post-exposure

• Intermittent Presumptive Treatment (IPT)– Infants (IPTi)– Pregnancy (IPTp)

Antimalarial Treatment

The treatment of malaria differs according to the infecting species and the geographic location where the infection was acquired and the severity of the disease. Treatment of malaria should not be initiated until the diagnosis of the infecting species has been confirmed by the lab investigations. The treatment of malaria has various concepts and it can seem all very confusing. As listed out in the bullets on the slide, the treatment is for two types of malaria : the first is the Acute uncomplicated malaria which is treated with specific drugs depending on the region of the world and the resistance pattern to chloroquine and other antimalarial drugs. The second is the severe or cerebral malaria which is treated with quinidine gluconate and either doxycycline or clindamycin.

Radical Cure - treatment intended to achieve cure of P. vivax or P. malariae malaria. These two species have exoerythrocytic [outside of red blood cells i.e. in the liver] stages. Requires primaquine treatment, which destroys latent exoerythrocytic stage parasites (hypnozoites). Typical case patient: a returned traveler from Central America who has had a relapse of malaria. Prophylaxis for malaria can be of two type. The first is taken prior to travel and continued during travel and the other is post exposure. There is also something called intermittent Presumptive treatment for infants and pregnant women. This is in endemic regions to clear existing parasites and to prevent new infections.

Slide

38


• Choose appropriate antimalarial drug and route of administration

• Follow blood glucose carefully

• Administer I.V. fluids for shock and hypovolemia

• Administer oxygen by nasal catheter

• Consider replacing erythrocytes for hematocrit of 30 or parasitemia > 10%

Treatment: Severe Malaria

Lets look at the treatment of severe malaria. The treatment is based on the following things, An appropriate antimalarial drug and route of administration. Intravenous route is better than oral as the oral bioavailability may be lower than I/V and the patient may be in coma so I/V is the only choice. Monitoring of blood glucose as treatment with quinine can lead to drops in blood sugar and its inherent risks. I/V fluids should be administered for shock and hypovolemia Oxygen is administered by nasal catheter to ensure adequate oxygen delivery. Sometimes patients need replacement of RBCs if their hematocrit is 30 or if the parasitemia is greater than 10%.

Slide

39


• Quinine, intravenous– Slow infusion avoids cardiovascular toxicity and

hypoglycemia

• Quinidine gluconate, intravenous– Very effective, but adverse cardiac events

• Artemisinin derivatives– Rapidly acting– Several derivatives and formulations

Artesunate drug of choice for severe disease– Compassionate use IND in US (via CDC)– >30% more effective than quinine in preventing death in cerebral

malaria

Treatment- Cerebral Malaria

Cerebral malaria is treated with the following drugs. The choice of the drugs depends again on the geographic location and known resistance patterns as we will see in a short while. Quinine is the oldest antimalarial drug and is used as an I/V infusion. The infusion is given slowly to avoid cardiovascular toxicity and hypoglycemia. Quinidine gluconate is also similar but has risk of adverse cardiac events. In fact quinidine is an anti-arrhythmic drug that is used to treat cardiac arrhythmias. Artemisinin derivatives are rapidly acting and there are several derivatives and formulations. In the US, High quality-intravenous artesunate is available only to malaria patients hospitalized in the United States who need intravenous treatment because of: severe malaria disease high levels of malaria parasites in the blood inability to take oral medications lack of timely access to intravenous quinidine quinidine intolerance or contraindications quinidine failure The drug is provided to the hospitals, upon request and on an emergency basis, by the CDC Drug Service or by one of the CDC Quarantine Stations located around the country.

Slide

40


DRUG PROBLEMS

Artemisinin Recrudescence, Neurotoxicity

Atovaquone Resistance

Azithromycin Limited efficacy

Chloroquine Resistance

Doxycycline Phototoxicity, GI intolerance

Fansidar Resistance, Allergic Rxns

Halofantrine Cardiotoxicity

Mefloquine Resistance, Psychiatric effects

Primaquine Therapeutic Index, Resistance?

Proguanil Resistance, Mouth ulcers

Quinidine gluconate Going off the market?

Quinine Resistance, Tinnitus

Problems with antimalarial drugs

This slide summarizes the problems with the different anti-malarials. As we can see, resistance is a major issue with the drugs.

Slide

41

Time to Development of Resistance to Antimalarial Drugs

1940

Chloroquine

16 years

Fansidar

6 years

Mefloquine

4 years

Atovaquone

6 months

1950 1960 1970 1980 1990

This is a great graph that shows the decreasing time interval from discovery of a new drug for malaria and the emergence of resistance.

Slide

42


The problem of resistance is so important that there is now surveillance for emergence of resistance and global bodies to address the issue as seen in this slide.

Slide

43


• Discovered during WW II• Effective against all Plasmodium spp.• CQ is trapped in the parasite food

vacuoles where it is toxic– Inhibits polymerization of hemazoin

and free heme kills the parasite

• ‘Magic bullet’ that was basis of 1960s malaria eradication campaign

• Resistance first emerged in early ’60s in S. America and SE Asia– Resistance conferred by complex

mutations in a Plasmodium falciparum chloroquine resistance transporter gene (pfcrt)

– Big problem in Africa, Asia

Chloroquine (CQ)

Chloroquine was discovered during WW II. It was the magic bullet effective against all species. However, now there is widespread resistance. It acts by accumulating inside the erythrocyte in food vacuoles where it is toxic. It prevents polymerisation of hemazoin. The parasite makes hemoglobin less toxic by converting it to hemazoin, chloroquine inhibits this process and effects death of the parasite. But resistance to CQ emerged in the early ‘60’s in South America and SE Asia. The resistance was due to complex mutations in the parasite chloroquine resistance transporter gene and now is a huge problem in Africa and Asia.

Slide

44


Spread of chloroquine resistant Plasmodium falciparum

This shows the spread of CQ resistant P.falciparum. It was first in Colombia in 1959 and in Thai-Cambodia in 1957. It is theorized that the first resistant in 1957 spread later to other parts of the world making CQ useless.

Slide

45


• Prophylaxis - in CQ & multi-drug resistant regions

• Treatment - in CQ & multi-drug resistant regions

– Combined with artesunate to treat mefloquine resistant malaria in SE Asia

– For P. vivax or P. falciparum

Mefloquine (Lariam)

Mefloquine developed by the US Army. It is useful in regions where CQ resistance is present but recent reports have indicated resistance to it. It is also used in combination with Artemisinin. It has started to lose effectiveness against P. falciparum

Slide

46

Artemisinin (Qinghaosu)

• Harvested from the Chinese herb Artemesia annua (sweet wormwood)

• Many derivatives

• 200 BC-China: recognized as herbal remedy of hemorrhoids and fever

• 1972 identified as active constituent of Artemesia annua

• 1999 Artemisinin combination therapy (ACT) O

O

O O

O

C15H22O5

Artemisinin is from the herb Artemesia annua. Originally used by Chinese for treatment of fever and is known by the name of ‘qinghaosu’. There are many derivatives that are now used for treatment of malaria. The structure show the endoperoxide bridge that is responsible for the key activity. In 1972 they identified the active compound and in 1999 ACT was started for treatment of malaria.

Slide

47 • Rapid relief from symptoms and fast clearance times• Broad stage specificity with gametocidal activity• Cheap alternative to other drugs • Not recommended for prophylaxis because of short

elimination time• Animal studies: neurotoxicity at high doses and also death of

embryos, and morphological abnormalities in early pregnancy• Can be combined with other drugs for effective treatment

(ACT)• Only treatment for severe malaria besides quinine• Resistance reported in 2009-2010.

Artemisinin Treatment of Malaria

Benefits of artemisinin is that it kills the gametocytes unlike some other drugs. It is a s cheap alternative compared to other drugs but could still be expensive for poorer countries. It gets eliminated very fast by the body so combination with another drug is necessary. Also it has issues with teratogenic effects and neurological toxicity necessitates cautious use. It is the only drug for treatment of severe malaria besides quinine. Resistance to artemisinin has been reported in 2009-2010. So there is an urgent need to identify newer drugs for treatment of malaria.

Slide

48 • WHO issued new guidelines on malaria treatment and

requested pharmaceutical companies to end the marketing and sale of "single-drug" artemisinin malaria medicines, in order to prevent malaria parasites from developing resistance to this drug (1-20-06 Press release).

• The artemisinin component– Reliably efficacious, rapidly effective

• Partner drug(s)– Ensures high cure rate– Very well tolerated– Reduces gametocyte carriage– Reduces de-novo resistance to partner– Prophylactic activity

Artemisinin Combination Therapy (ACT)

WHO has recommended the use of ACT in areas of high malaria transmission and where drug resistance to antimalarial drugs is prevalent. ACT achieves its antimalarial effect through an initial rapid reduction in parasite biomass attributable to the short-acting but highly potent artemisinin drug, with the subsequent removal of remaining parasites by a less-effective but slower eliminated partner drug. The use of the partner drugs ensures high cure rate although now with resistance to artemisinin, it has started to fall in some regions, the treatment is well tolerated, reduces gametocyte carriage, reduces de novo resistance to partner drug.

Slide

49

49

ACT Partner drugs, CQ resistance

Global report on antimalarial drug efficacy and drug

resistance: 2000–2010. WHO

This slide shows the partner drugs in different parts of the world depending on the resistance patterns. Various drugs are used as partner drugs such as lumefantrine, amodiaquine, mefloquine, sulfadoxine and pyrimethamine piperaquine. The bottom right map shows the regions where P.vivax is resistant to CQ.

Slide

50


• Treatment of depends on many factors including disease severity, the species of malaria parasite causing the infection, and the part of the world in which the infection was acquired. – Planning, Case history very important

• Think about the probability that the organism is resistant to certain antimalarial drugs.

• Additional factors such as age, weight, and pregnancy status may limit the available options for malaria treatment.

• Guidelines for U.S. Travelers– http://www.cdc.gov/malaria/diagnosis_treatment/treatment.html

So, how do you correctly treat a patient?

Interactive world map for malaria prevalence/risk:

http://apps.who.int/tools/geoserver/www/ith/index.html

CDC list of malaria countries and risk:

http://wwwnc.cdc.gov/travel/yellowbook/2010/chapter-

2/malaria-risk-information-and-prophylaxis.aspx

It can be very confusing to consider the preventive medication to take if one is visiting a certain part of the world. The points to consider are the region where you are traveling so that the treatment can be started ahead of time. If you get infected , you need to provide detailed information of the places you have traveled. Also, age, weight and pregnancy status could limit the choice of drugs. Detailed information for US travelers is available on the CDC website.

Slide

51


• Reduce the transmission of the disease by mosquito control

• Mosquito nets treated with long-lasting insecticide, a very cost-effective method

• Indoor residual spraying of insecticides.

• Repellents• Drugs- all groups- kill

parasites• Vaccines -block infection, kill

parasite• Pregnant women

Prevention

The prevention of malaria is very simple in theory but difficult in practice due to many different factors. It is primarily focused on avoiding getting bitten by mosquitoes, residual indoor spray to kill vectors, early diagnosis and treatment prophylaxis while traveling. Vaccines are still under development

Slide

52


• Intermittent preventive treatment (IPT)-antimalarial treatment given at regular intervals during pregnancy,

– Two to three doses of sulfadoxine-pyrimethamine (SP) administered to a pregnant woman through antenatal care services. This protects pregnant women from possible death and anemia and also prevents malaria-related low birthweight in infants

• Insecticide-treated bed nets (ITN), indoor residual spraying

• Febrile malaria case management.

• Malaria prevention is particularly challenging in HIV-infected women, who might constitute up to 40% of the antenatal population in southern Africa.

– HIV-infected pregnant women are more likely to fail antimalarial treatment and require three or more doses of IPTp

Prevention and Control of Malaria During Pregnancy

Malaria has a significant effect on pregnancy. The intermittent therapy for malaria in pregnancy help reduce anemia and adverse mother and child outcomes. The drugs used for this are sulfadoxine and pyrimethamine. Other measures include Insecticide treated nets and indoor residual spraying, febrile malaria case management. Malaria prevention is particularly challenging in HIV positive women who constitute upto 40% of the antenatal population in Africa. This is because they are more llikely to fail treatment of malaria and need three or more doses of IPTp.

Slide

53


• Pre-erythrocytic stages- directed against sporozoites and/or liver stages (prevent blood-stage infection)

• Against asexual blood stages are designed to reduce clinical severity

• Against mosquito stages are designed to halt development in the mosquito (transmission-blocking vaccines)-

• Major problem: antigens are constantly changing and developing a vaccine against these varying antigens is very difficult

Vaccines

Now lets talk a little about the vaccines for malaria. The vaccine for malaria can be one that can act at the pre-erythrocytic stages. These are designed to prevent the invasion of the RBC and liver cells i.e. they prevent blood stage infection. The other type of vaccine can be against the asexual blood stages and they are designed to reduce the clinical severity. A third type of vaccine is one that is designed to block the development of the parasite in the mosquito. These are the transmission blocking vaccines. The major problem in developing vaccine is that the antigens constantly changing.

Slide

54

54

This slide taken form the presentation by David Kaslow, delivered at the Annual Meeting of the American Society of Tropical Medicine and Hygiene (ASTMH) in 2012 shows the different vaccine candidates for malaria in their development path. The candidate that is currently in Phase III trials is the RTS,S vaccine. Multilateral Initiative on Malaria Pan-African Conference, Durban, South Africa : Results from a large-scale Phase III trial, presented in Durban in October 2013, show that the most clinically advanced malaria vaccine candidate, RTS,S, continued to protect young children and infants from clinical malaria up to 18 months after vaccination. Based on these data, GSK intends to submit, in 2014, a regulatory application to the European Medicines Agency (EMA). The World Health Organization (WHO) has indicated that a policy recommendation for the RTS,S malaria vaccine candidate is possible as early as 2015 if it is granted a positive scientific opinion by EMA. These latest results demonstrated that over 18 months of follow-up, RTS,S was shown to almost halve the number of malaria cases in young children (aged 5-17 months at first vaccination) and to reduce by around a quarter the malaria cases in infants (aged 6-12 weeks at first vaccination).

Slide

55


• Requires an integrated approach– Vectors– Proper antimalarial drugs– Access to prompt, medical treatment

• Factors contributing to disease– Reservoir-prevalence in humans– Vector-suitability of local anopheline as hosts-breeding

preferences, flight/resting behavior, abundance, temp., rainfall, humidity,

– New hosts-nonimmune hosts– Local climatic conditions

• Local geographical and hydrogeographical conditions, human activities that can determine availability of and accessibility to mosquito breeding areas

Control: Mechanisms and Challenges

These are some of the issues with control mechanisms and challenges to control of malaria. The control of malaria requires an integrated approach comprising of vector control, proper anti-malarial drugs and prompt access to medical treatment. The vector control itself is quite complex because it will depend on the vector habits and habitats, breeding locations, preferences for resting and feeding on humans, abundance, temperature, rainfall and humidity. The anti0malarial drugs are fast becoming less effective due to drug resistance. The endemic regions are mostly the poor developing regions of the world in the tropical and temperate regions where lack of resources is compounded by political strife and inadequate public health and healthcare infrastructure.

Slide

56


• Transgenic mosquitoes– Mosquitoes refractory to mouse

malaria in lab experiments- transgenic malaria-resistant mosquitoes have a selective advantage over nontransgenic mosquitoes?

– Sterile male mosquitoes released into a given environment

– Male mosquitoes carrying lethal genes causing death of newborns very young

– Disadvantages: Could resistant mosquitoes be useful in

the wild? They must survive better than non-resistant mosquitoes even when not exposed to malaria.

Not shown to work in Plasmodium falciparum

Genetic engineering and Malaria

Blocking transmission of sporozoites through the gut wall and into the salivary glands.

One of the approaches for the control of malaria that has been experimented with is the use of transgenic mosquitoes. One of the strategies has been the generation of transgenic mosquitoes that are refractory to mouse malaria. The studies showed that transgenic mosquitoes did not do any better than the non-transgenic mosquitoes when fed with non-malaria infected blood. This is not really advantageous as the transgenic mosquitoes do not show any fitness advantage over the non-transgenic mosquitoes. For the strategy to be effective, the transgenic mosquitoes would need to fare better than the non-transgenic mosquitoes. Another strategy is to release sterile males into a given environment. A third strategy is to release males carrying a lethal gene causing death of newborns very young. The challenges to using transgenic malaria resistant mosquitoes are that they must survive better than the non resistant mosquitoes even when not exposed to malaria. Also, the strategy does not work in P.falciparum.

Slide

57


• Zoonotic disease maintained by the interaction of tick vectors, transport hosts, and animal reservoirs.

• More than 100 species have been reported, only a few have been identified as causing human infections.

• Babesia microti and Babesia divergens have been identified in most human cases

• Ticks of the family Ixodidae transmit B. microti(and Lyme disease).

• Transplacentally or perinatally acquired (congenital) babesiosis have been reported.

• Blood transfusions-emerging problem

Babesia spp.

The next parasite in this lecture is Babesia spp. Babesiosis is a zoonotic infection that is transmitted by ticks and is maintained in the wild by numerous animal reservoirs and has different transport hosts. There are more than a 100 species that have been reported but only a few have been known to cause human disease. The most significant species causing human infections are Babesia microti and Babesia divergens. The ticks of the Ixodidae family are responsible for transmission to humans. These are the same that are also responsible for the transmission of Lyme disease. Babesiosis is an infection that can be transmitted by a pregnant woman to her unborn child and is an emerging problem with blood transfusions.

Slide

58


Life Cycle: Babesia

The Babesia microti life cycle involves two hosts, which include a rodent, primarily the white-footed mouse, Peromyscus leucopus, and a tick in the genus Ixodes. During a blood meal, a Babesia-infected tick introduces sporozoites into the mouse host . Sporozoites enter erythrocytes and undergo asexual reproduction (budding) . In the blood, some parasites differentiate into male and female gametes, although these cannot be distinguished by light microscopy . The definitive host is the tick. Once ingested by an appropriate tick , gametes unite and undergo a sporogonic cycle resulting in sporozoites . Transovarial transmission (also known as vertical, or hereditary, transmission) has been documented for "large" Babesia species but not for the "small" Babesia, such as B. microti . Humans enter the cycle when bitten by infected ticks. During a blood meal, a Babesia-infected tick introduces sporozoites into the human host . Sporozoites enter erythrocytes and undergo asexual replication (budding) . Multiplication of the blood-stage parasites is responsible for the clinical manifestations of the disease. Humans usually are dead-end hosts. However, human-to-human transmission is well recognized to occur via contaminated blood transfusions .

Slide

59


• Worldwide, but little is known about the prevalence of Babesia in malaria-endemic countries, where misidentification as Plasmodiumprobably occurs.

• Endemic areas are regions of tick habitat, including the forest regions of the Northeastern U.S. and temperate regions of Europe.

• In Europe, most reported cases are due to B. divergens and occur in splenectomized patients.

• In the U.S., B. microti is the agent most frequently identified (Northeast mostly, less common in Midwest), and can occur in nonsplenectomized individuals.

• High number of asymptomatic infections• The first U.S. case of babesiosis was reported on Nantucket Island in

1966. Between 1968 and 1993, more than 450 cases of Babesia infections were confirmed in the United States. – An increasing trend over the past 30 years may be the result of restocking of

the deer population, curtailment of hunting, and an increase in outdoor recreational activities.

Geographic Distribution

Babesia is found worldwide but its prevalence in malaria endemic regions is unknown as they can be mistaken as malarial parasites. The forest regions of North America and Europe that harbor the tick vector are endemic for babesiosis. In Europe, most reported cases are due to B. divergens and they occur in splenectomized patients. In the US, B.microti is the most frequent agent identified mostly in Northeast and less commonly in Midwest. And it can occur in non-splenectomized individuals. There is high number of asymptomatic infections. The first US case was reported in Nantucket in 1966

Slide

60


• Fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia.

• Symptoms typically occur after an incubation period of 1 to 4 weeks, and can last several weeks. The disease is more severe in patients who are immunosuppressed, splenectomized, and/or elderly.

• Infections caused by B. divergens tend to be more severe (frequently fatal if not appropriately treated) than those due to B. microti, where clinical recovery usually occurs.

Laboratory Diagnosis:• Microscopic examination of thick and thin blood smears

stained with Giemsa.• Isolation of the organisms by inoculation of patient

blood into hamsters or gerbils• Treatment: Clindamycin plus quinine or atovaquone

plus azithromycin

Disease, dx, tx

Lets look at the disease, diagnosis and treatment of Babesiosis. The clinical diseases is characterized by fever, chills, sweating, myalgia, enlargement of liver and spleen and hemolytic anemia. The symptoms occur after 1-4 weeks and they can last for several weeks. The disease is more severe in patients who are immunosuppressed or splenectomized or the elderly. Infections cause by B.divergens tend to be more severe and could be fatal in the absence of timely treatment. On the other hand B.microti infections usually recover clinically. The laboratory diagnosis is by examination of thick and thin blood smears stained by Giemsa. The organism is isolated by innoculation of hamsters or gerbils with the patients blood. The treatment of Babesiosis is with Clindamycin with quinine or atovaquone with azithromycin

Slide

61

61

• Babesiosis is the most frequent transfusion-transmitted infection with approximately 162 cases reported since 1980 and 12 associated fatalities in the period 2005–2008

• Currently, the blood supply is not screened for Babesia

• Donors are deferred if they have a fever at the time of donation or report a history of Babesiainfection, but this practice alone is unable to prevent asymptomatic individuals with low levels of parasitemia from serving as donors.

Babesia and blood transfusion: U.S.

Babesia is now recognized as one of the frequent transfusion transmitted infections. There have been about 162 cases that were transfusion transmitted since 1980 and 12 fatalities associated with it in the 2005-2008 period. The current diagnostic methods are inadequate for the screening of donated blood. Also, individuals with very low parasitemia are very difficult to detect. The table on the right from a 2011 paper lists the aetiological Babesia species by the number of reported cases. As we can see, the most frequent is B.microti. Below is one of the health advisory that was issued by the NY City DOH about transfusion associated Babesiosis in NYC.

Slide

62

Figure 1. Different forms of Babesia divergens in human RBCs as seen on a Giemsa-stained smear from in vitro cultured parasites (ring, dividing figure eights, Maltese cross parasites, and multiply infected RBCs).

Lobo CA, Cursino-Santos JR, Alhassan A, Rodrigues M (2013) Babesia: An Emerging Infectious Threat in Transfusion Medicine.

PLoS Pathog 9(7): e1003387. doi:10.1371/journal.ppat.1003387

http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003387

This slide shows the different forms of Babesia divergens in human RBCs on a Giemsa stained smear. You can see the different forms of the parasites like ring forms, dividing figure eights, Maltese cross forms as well as multiply infected RBCs.

Slide

63


• Avoid tick exposures– Tick infested areas (high tick season

between May and September)– Cover skin with light clothing, searching

for ticks after being outdoors and removing discovered ticks from the skin.

– Applying bug repellent with DEET

• Eradication?– Preventative measures seem to be more

recommended over vector control.– Due to the relatively low prevalence of

the disease and the presence of several reservoirs, Babesiosis is currently not a candidate for vaccine prevention.

Prevention

For prevention of Babesiosis, one should avoid the tick infested areas in seasons of high activity like summer between May and September, wear light colored clothing to easily identify ticks and removing ticks that are discovered on the skin. Eradication efforts would be a long-term project, which would significantly reduce the prevalence of both Babesiosis and Lyme disease.

Date post:	01-Oct-2018
Category:	Documents
Upload:	phungthuy
View:	215 times
Download:	0 times

Video: ...eta.health.usf.edu/publichealth/PHC4031/S14/Block3/Week15_Protozoa... · falciparum and...

Documents