David Sullivan MDJune 1, 2011

dsulliva@jhsph.edu410 502 2522

MalariaDiagnosis

Treatment and Drug Resistance

• Blood smear examination: still the “Gold Standard” for diagnosing malaria; but it’s time consuming; requires technical experience; difficult to speciate.

• Rapid Diagnostic Testing, improves the turn around time, and enhances the accuracy of diagnosing P. falciparum especially in non-specialized labs.

• So far, blood-based dipsticks are in use, such as Parasight-F and NOW ICT(FDA approved) for detecting HRP2 antigen of P. falciparum and panspecies aldolase and OptiMAL for detecting species-specific Plasmodium LDH of malaria.

• Despite lab based tests, in Africa greater than 60-70% of patients identified by Clinical Diagnosis = Fever and age less than 5 years living in endemic area = malaria drug

Current Malaria Diagnosis

Classical Malaria

• Fever• Splenomegaly• Anemia

Hippocrates, 5th Century BC

144120967248240096

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P.malariae temp

Hours

“Quartan” P. malariae

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P. vivax temp

Hours

“Tertian” P. vivax

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P. falciparum

Hours

“Aestivo-autumnal “Quotidian”P. falciparum

Comparison of Malaria Fever Curves

Adapted from Thayer and HewetsonJohns Hopkins Hosp Reports V 1895 p. 3-224

Classical Malaria= Fever, Splenomegaly and Anemia

Hippocrates 5th century BC

Who has malaria?A 3 year young woman from Malawi presents with 3 days of fever. Mother reports no headache, chills, rigors or joint aches. The child is warm with pallor but clear lungs and no enlarged spleen of liver. The clinic lacks tests for malaria.

A 23 year young woman presents to student health service with 3 days of fever after returning from a Global Health Fellowship in Mali. She reports no GI or respiratory complaints. She took Doxycycline intermittently because of photosensitivity. She is febrile with a palpable spleen but not ill appearing. Rapid Tests for Streptococcus and the Monospot are negative.

Best positive predictors in endemic area presentationsHepatosplenomegaly and pallor. Headache was sensitive but not specific in adults

In returning travelers positive predictors were fever, splenomegly, jaundice and thrombocytopeniaAbsence of headache was decent negative predictor.

FIGURE 1.Severe macular whitening (solid arrow) completely surrounding the foveola of a Malawian child with cerebral malaria. Papilledema is present as well as a white-centered hemorrhage temporal to the macula and cotton wool spots above superior temporal arcade. The open arrow indicates glare (photographs provided by Nicholas A. V. Beare).

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 790–797

FIGURE 2.Macular whitening around inferior fovea and temporal macula (solid black arrow). White-centered hemorrhages are temporal to the disc and on the superior macula. Peripheral whitening is outside the vascular arcades (solid white arrow). Open arrow indicates glare.

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 790–797

FIGURE 3.White retinal vessels in an area of confluent peripheral retinal whitening.

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 790–797

Vessel changes in same child as in Figure 1, including examples of tramlining (solid arrow) and orange vessel (open arrow)

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 790–797

FIGURE 5.Large number of retinal hemorrhages in a child with cerebral malaria.

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 790–797

Diagnosis based on clinical features

Advantages

Cheap

Fast

Disadvantages

Lack of precision

Over-treatment

Fever and age less than 5 is one criteria (IMCI)Other criteria add in signs and symptomsIf pretest probability is >50% by parasite prevalence,This is good malaria test!!!!

Diagnosis Based on Microscopy

Advantages

Gold standard

Quantitative

Useful for other diseases

Disadvantages

Time consuming

Relies upon good microscopes, reagents, and trained technicians

CDC/Dr. Michael ReinCDC/Dr. Michael Rein

LAMP (loop-mediated isothermal amplification)

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FIND is working with Eiken and the Hospital for Tropical Diseases in London (HTDL), in the development of a LAMP assay for the detection of Plasmodium parasites. During the last year a gene target has been identified and primers have been optimized for the detection of 2 Plasmodium genus and/or P. falciparum parasites in 1 μl of blood in a 20-minute reaction. A rapid process (~15 minutes) for DNA extraction from fresh and frozen blood and from blood dried on filter paper has also been developed.

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Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Nature Protocols 3, - 877 - 882 (2008)Published online: 24 April 2008 | doi:10.1038/nprot.2008.57

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Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Nature Protocols 3, - 877 - 882 (2008)Published online: 24 April 2008 | doi:10.1038/nprot.2008.57

Rapid Diagnostic Tests

Advantages• Sensitive

• Fast

• Simple to perform

• No need for special equipment or electricity

Disadvantages• HRPII:

– Not suitable for non Pf species

– Remains positive for 2 weeks after treatment

• Not quantitative

• Expensive (US$0.60-2.50 per test)

Why Target HRP II for Detection?

• Multiple His-Ala repeating regions for antibody epitopes

• Present in infected RBC cytoplasm and parasite digestive vacuole

• Secreted in plasma• Directly related to parasitemia, parasite biomass, and parasite developmental stage

PfHRP IIMVSFSKNKVLSAAVFASVLLLDNNNSAFNNNLCSKNAKGLNLNKRLLHETQAHVDDAHHAHHVADAHHAHHAADAHHAHHAADAHHAHHAADAHHAHHAADAHHAHHAAYAHHAHHAADAHHAHHASDAHHAADAHHAAYAHHAHHAADAHHAHHASDAHHAADAHHAAYAHHAHHAADAHHAADAHHATDAHHAHHAADARHATDAHHAADAHHATDAHHAADAHHAADAHHATDAHHAADAHHATDAHHAADAHHAADAHHATDAHHAHHAADAHHAAAHHATDAHHATDAHHAAAHHEAATHCLRH

PfHRP IIIMVSFSKNKILSAAVFASVLLLDNNNSEFNNNLFSKNAKGLNSNKRLLHESQAHAGDAHHAHHVADAHHAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAANAHHAADANHGFHFNLHDNNSHTLHHAKANACFDDSHHDDAHHDGAHHDDAHHDGAHHDDAHHDGAHHDDAHHDGAHHDDAHHDGAHHDGAHHDGAHHNATTHHLHH

Secretory leader

• Aldolase and lactate dehydrogenase enzymes

• Abundant production by parasites

• Aldolase has over 90% identity at amino acid level

• LDH has less and enables species specific monoclonal antibodies

• LDH is basis for Optimal test

• Aldolase is in ICT test as non HRP II band

• Both aldolase and LDH have short half life and go away within 1-2 days of treatment

• HRP II can linger for more than a week

Control

HRP II

aldolase

Result

negative

P. falciparum/mixed

P. falciparum

Other than P. falciparum

RDTs in Africa?Current situation

Problems

• Asymptomatic parasitaemia

• Expense

Special situations

• Complex emergencies

• Malaria epidemics

• Low transmission settings

• Military

• Travellers

RDTs in AfricaFuture options

• Changing cost-benefit– Rising drug costs

• Possible uses– Confirmation of treatment failure (pLDH)– Severe disease in peripheral settings

• BUT…– Will RDT diagnosis change clinical practice?

• Need for operational studies

Malaria Rapid Diagnostic Tests

WHO site: http://www.wpro.who.int/sites/rdtResults of validation of rapid testshttp://www.wpro.who.int/sites/rdt/who_rdt_evaluation/call_for_testing.htm

Contains• Explanation of RDT• Use of RDT• Guidelines on purchasing an RDT including an

important table that compares good manufacturing practice on known suppliers

• Collections of published reviews and trials• Collection of publications and committee documents• Useful links pertaining to malaria diagnosis

http://www.wpro.who.int/sites/rdt/links.htm

False positives from rhuematoid factors or cross reacting antibodies

Detecting Malaria Parasites/Products: Sensitivity Thresholds

PCRVietnam (Serial dilution)Detection limits for:

- P. falciparum: 0.02-0.08 parasites/µl-P. vivax: 0.8-2.6 parasites/µl

Ref: Vu thi Ty Hang et al. Trans R Soc Trop Med Hyg 89: 44-47 (1995)

HRP-2Kenya (children)Parasites/µl n = Sensit.(%)1-10 9/23 3911-60 17/21 8161-100 14/16 88101-500 57/57 100501-1000 12/12 100Ref: Beadle et al. Lancet 343: 564-568 (1994)

pLDHHospital for Trop. Diseases - LondonParasites/µl n = Sensit.(%)<5 13/22 6050-500 9/11 81500-1500 17/18 94>1500 36/36 100Ref: Piper et al. Am J Trop Med Hyg 60: 109-118 (1999)

• Blood smear examination: still the “Gold Standard” for diagnosing malaria; but it’s time consuming; requires technical experience; difficult to speciate.

• Rapid Diagnostic Testing, improves the turn around time, and enhances the accuracy of diagnosing P. falciparum especially in non-specialized labs.

• So far, blood-based dipsticks are in use, such as Parasight-F and NOW ICT(FDA approved) for detecting HRP2 antigen of P. falciparum and panspecies aldolase and OptiMAL for detecting species-specific Plasmodium LDH of malaria.

• Despite lab based tests, in Africa greater than 60-70% of patients identified by Clinical Diagnosis = Fever and age less than 5 years living in endemic area = malaria drug

Current Malaria Diagnosis

Vaccines"Stimulate the Phagocytes. Drugs are a Delusion " GB Shaw, The Doctor's Dilemma 1906

Bednets"I myself have been infected with

malaria only once in spite of nineteen years' of service in India and thirteen subsequent 'malaria expeditions' to warm climates; and I attribute this to my scrupulous use of the bed net." Ronald Ross Studies on Malaria 1928.Chemotherapy

"We must learn to shoot microbes with magic bullets." Paul Ehrlich in Microbe Hunters Paul de Kruif 1926

Malaria Control

Malaria Control

1. Vector control & Sanitation2. Vaccines?3. Chemotherapy

Curative Protective (prophylaxis)

Vector Control Objectives

Reduce vector mosquito populationRepel the vector mosquitoes Form a barrier between vector and potential host (personal protection)

Reduce the lifespan of vector mosquitoes

Reduce the lifespan of potentially infected vector mosquitoes

Mosquito Repellents-- NEJM 2002 347:13-8

ring

trophozoiteschizont

SporozoitesGametocytes

48-72 hrs

merozoites

oocysts

Quinolines- chloroquine,mefloquine, quinineAntibacterials-tetra-cycline,clindamycin, fluoroquinolone(Cipro)

}Artemisinin

AtovoquoneAzithromycin

Primaquine(hypnozoites ofP.vivax&ovale)

Antifolates-proguanil,pyrimethamine

Sporozoites

Five Broad Groups Based on Mechanism of Action

1.Quinoline: Inhibits heme crystallization

2. Artemisinin: 1. Binds heme iron and or iron and generates oxygen radicals. 2. Damages SERCA Ca++ P-ATPase

3. Antifolate: Inhibit DNA synthesis

4. Atovaquone: Collapses mitochondrial membrane potential

5. Antibacterial: ribosome inhibition-tetracycline, clindamycin and erythromycin, DNA gyrase inhibition-fluoroquinolones

Plasmodium metabolic pathways-Gardner Nature 2002 419:498-511

Plasmodium Specialized Organelles

• Digestive vacuole physiology or the Plasmodium iron problem

• Specialized lysosome for degrading hemoglobin and making malarial pigment

• Mitochondria-lack of active TCA cycle with 99% energy from glycolysis

• Apicoplast-chloroplast origin, makes heme and fatty acids and has prokaryote ribosomes

70 kg person X @70 mL/kg = 4.9 L of blood @ 5 L = 5X103 ml = 5X106 µL

5X106 RBCs per µL of blood

2.5 X 1013 RBCs

1% parasitemia = 1 in 100 iRBCs = 2.5 X 1011 parasites

Artemisinin reduces by 4 logs parasite biomass with each asexual cycle. This is the most rapidly acting antimalarial.

The Numbers

Endoperoxide bridge generates single oxygen radical

Old “where the money is” theory: artemisinin makes oxygen radicals in digestive vacuole where lots of oxygen and heme coexist.

Target proteins

1. Artemisinin increases lipid damage (Berman Adams 1997)2. Histidine-rich protein or translationally controlled tumor

protein TCTP covalent heme-art-protein adducts3. Does not prevent heme crystallization even though binds heme4. inhibits hemoglobin proteases in presence of heme5. Binds and inhibits calcium transporter PfATPase6

COARTEM

1. Curative

2. Chemoprophylaxis

3. Intermittent presumptive therapy (pregnancy, disease prevention in children)

Drug Regimens

P. falciparumIs a

Medical emergency

Initial Management Decisions

• Oral vs. Intravenous chemotherapy– Severity of disease

• Correct Drug– Resistance ?

• Correct dose– mg/kg of salt or base

• Response to therapy– Parasite clearance time– Fever clearance time

• Check the glucose• Hypoglycemia from parasite or drugs

If yes to any of the above, then IV chemotherapy.

Oral vs. Intravenous Chemotherapy

• Signs of End Organ Involvement– Pulmonary edema– Renal failure– Coma– Severe anemia-transfusion lifesaving

• Unable to Tolerate Oral Medications

• Parasitemia over 5%

Delay Equals Bad Outcome

Delay from onset of symptoms to medical presentation

Delay from presentation until consideration of malaria

Delay from consideration to microscope diagnosis

Delay from diagnosis until start of therapy.

Delay of reassessment (ICU care;hypoglycemia; staff not as familiar with IV Quinidine.

1. Avoid infection.

2. No drug is perfectly effective; all have risks. 20% of all people taking chemoprophylaxis report some effect.

3. Prophylactic drugs like chloroquine, mefloquine, and tetracycline are not designed to eliminate hypnozoites of P. vivax or P. ovale. Primaquine can be given for radical cure if exposure is substantial (>6 months).

4. Start chloroquine one week early to build up therapeutic doses. In emergency travel, may take chloroquine on two consecutive days.

Chemoprophylaxis Principles

5. Continue drugs for 4 weeks (mefloquine 2 weeks) after leaving malarious area so that schizontocidal concentrations are present when merozoites emerge from liver. Mefloquine and Maloprim are started early to watch for side effects. Alternative is to brings drugs along for standby treatment of fevers for individuals who are pregnant, young children or people with allergies.

6. Chloroquine, quinine, pyrimethamine. and proguanil are safe during pregnancy. Sulfonamides are safe at term. Mefloquine is still under evaluation, but probably safe. Malorone is category C, probably safe but some minimal animal toxicity with atovoquone.

7. Chemoprophylaxis of children in endemic area has been shown to reduce mortality (25%) but is not widely done.

Chemoprophylaxis Principles (cont.)

Pregnancy and DrugsChemoprophylaxis• continuous provision of antenatal chemotherapy to prevent parasitemia. Chemoprophylaxis during first pregnancy does not reduce immunity in subsequent pregnancies.

• Chloroquine,amodiaquine, proguanil, mefloquine

Intermittent presumptive treatment (IPTpregnancy=IPTp)

• Full treatment doses at intervals to reduce disease.

• 2-3 intervals at scheduled visits.• sulfadoxine-pyrimethamine

Treatment During Pregnancy

1. IV quinine2. Chloroquine3. SP4. Artesunate combinations5. Quinine and clindamycin6. Amodiaquine

Prospects for control: The Drug ApproachDiagnosis and treatment of active clinical cases

(+)Reduces burden of transmitters(-) Misses asymptomatic transmitters

Chemoprophylaxis of travelers(+) prevents infection in millions of

travelers/soldiers(-) no impact on endemic disease

Mass drug administration(+) proven efficacy in elimination in

Italy Russia, China with temperate malaria.

(-) drug resistance selection, side effects of drugMass screening (diagnostic) and treatment

(+) picks up asymptomatic reservoirs of transmitters

(+) decreases drug resistance selection(-) requires sensitive point of care

diagnostic for low density parasitiemia

What Is Drug Resistance?

• The ability of a parasite strain to

survive and/or multiply despite

administration & absorption of a drug

given in doses equal to or higher than

those usually recommended but within the

limit of tolerance of the subject

( WHO 1986)

Why is it that antimalarial drug treatments do not

always work?• Wrong Diagnosis• Incorrect choice of drugs• Sub-optimal regimen (dose, schedule, duration)

• Non-compliance• Sub-optimal absorption (nausea, diarrhea, vomiting, malabsorption)

• Idiosyncratic pharmacokinetics• Poor quality drugs• Resistance of the pathogen to the drug

Spread of Chloroquine Resistance Worldwide

The positions of all of the different mutations identified from geographically diverse isolates from the Eastern and Western hemispheres are indicated by filled circles.The K (lysine) T (threonine) change at position 76 (indicated by the arrow) is critical to CQ resistance in P. falciparum.

Association of Pretreatment pfcrt T76 with Treatment Outcome

Mutation Prevalence in Sensitive Infections% (no./total no.)

Prevalence in Resistant Infections

%(no./total no.)

OR (95% CI) P

pfcrt T76* 37 (40/107) 92 (56/61) 18.8 (5.9-80.3)

<0.001

pfmdr 1 Y86† 49 (51/104) 75 (46/61) 3.2 (1.5-6.8)

<0.001

pfcrt T76 & pfmdr1 Y86

22 (22/102) 73 (43/59) 9.8 (4.4-22.1)

<0.001

*pfcrt T76 is associated with chloroquine treatment failure; †pfmdr 1 Y86 is independently associated with treatment failure; No additive effect or interaction with T76.

Adapted from Djimde et al. A Molecular Marker for Chloroquine-Resistant Falciparum Malaria. N Engl J Med 2001;344:257-63.

Age-related ability to clear chloroquine-resistant parasites when treated with

chloroquine

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

<1 1 2 3 4 5 6 7 8 9 10 11 12 13+

Age

Djimde et al 2003 Am J Trop Med Hyg 69:558-563