Malaria Diagnosis, Treatment, and Drug Resistance

David Sullivan MD June 1, 2011dsulliva@jhsph.edu 410 502 2522 Malaria DiagnosisTreatment andDrug Resistance

Current Malaria Diagnosis• 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

Classical Malaria• Fever• Splenomegaly• AnemiaHippocrates, 5th Century BC

“Tertian” P. vivaxComparison of MalariaFever CurvesAdapted from Thayer and HewetsonJohns Hopkins Hosp Reports V 1895 p. 3-224 “Aestivo-autumnal “Quotidian” “Quartan” P. malariae P. falciparum

Classical Malaria= Fever, Splenomegaly and Anemia Hippocrates 5th century BC

Who has malaria?A 3 year young woman from Malawi presents with 3days of fever. Mother reports no headache, chills, rigorsor joint aches. The child is warm with pallor but clearlungs and no enlarged spleen of liver. The clinic lackstests for malaria.A 23 year young woman presents to student healthservice with 3 days of fever after returning from aGlobal Health Fellowship in Mali. She reports no GI orrespiratory complaints. She took Doxycyclineintermittently because of photosensitivity. She is febrilewith a palpable spleen but not ill appearing. RapidTests for Streptococcus and the Monospot arenegative.

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 featuresAdvantages DisadvantagesCheap Lack of precisionFast Over-treatment Fever and age less than 5 is one criteria (IMCI) Other criteria add in signs and symptoms If pretest probability is >50% by parasite prevalence, This is good malaria test!!!!

Diagnosis Based on MicroscopyAdvantages DisadvantagesGold standard Time consumingQuantitative Relies upon goodUseful for other microscopes, reagents,diseases and trained technicians CDC/Dr. Michael Rein CDC/Dr. Michael Rein

LAMP (loop-mediated isothermal amplification)FIND is working with Eiken and the Hospital for TropicalDiseases in London (HTDL), in the development of aLAMP assay for the detection of Plasmodium parasites.During the last year a gene target has been identified andprimers have been optimized for the detection of 2Plasmodium genus and/or P. falciparum parasites in 1 μlof blood in a 20-minute reaction. A rapid process (~15minutes) for DNA extraction from fresh and frozen bloodand from blood dried on filter paper has also beendeveloped. QuickTimeª and a QuickTimeª and a TIFF (Uncompressed) decompressorTIFF (Uncompressed) decompressor are needed to see this picture. are needed to see this picture.

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Nature Protocols 3, - 877 - 882 (2008) Published online: 24 April QuickTimeª and a 2008 |TIFF (Uncompressed) decompressor are needed to see this picture. doi:10.1038/nprot.2008.57

Loop-mediated isothermal amplification (LAMP) of genesequences and simple visual detection of productsNature Protocols 3, - 877 - 882 (2008)Published online: 24 April 2008 | doi:10.1038/nprot.2008.57 QuickTimeª and a TIFF (Uncompressed) decompressor are needed to see this picture.

Rapid Diagnostic TestsAdvantages Disadvantages• Sensitive • HRPII:• Fast – Not suitable for non Pf species• Simple to perform – Remains positive for 2 weeks after treatment• No need for special equipment or electricity • 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 II PfHRP IIIMVSFSKNKVLSAAVFASV Secretory MVSFSKNKILSAAVFASVLLLDNLLLDNNNSAFNNNLCSKNA NNSEFNNNLFSKNAKGLNSNKRLKGLNLNKRLLHETQAHVDD leader LHESQAHAGDAHHAHHVADAHHAHHAAD AHHAHHVADAHHAHHAANAHHAHHAADAHHAHHAAD AHHAANAHHAANAHHAANAHHAAAHHAHHAADAHHAHHAAY NAHHAANAHHAANAHHAANAHHAAHHAHHAADAHHAHHASD ANAHHAANAHHAANAHHAANAHHAHHAADAHHAAY AANAHHAANAHHAANAHHAADANAHHAHHAADAHHAHHASD HGFHFNLHDNNSHTLHHAKANACAHHAADAHHAAY FDDSHHDDAHHAHHAAD AHHDGAHHDDAHHDGAHHDDAHHAHHAADAHHATD DGAHHDDAHHDGAHHDDAHHAHHAHHAAD DGAHHDGAHHDGAHHNATTHHLHARHATDAHHAADAHHATD HAHHAADAHHAADAHHATDAHHAADAHHATDAHHAADAHHAADAHHATDAHHAHHAADAHHAAAHHATDAHHATDAHHAAAHHEAATHCLRH

• 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

Result Other than P. falciparum P. falciparum P. falciparum/mixed negativeControl aldolase HRP II

RDTs in Africa? Current situationProblems Special situations• Asymptomatic • Complex emergencies parasitaemia • Malaria epidemics • Low transmission settings• Expense • Military • Travellers

RDTs in Africa Future 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 TestsWHO 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 reactingantibodies

Detecting Malaria Parasites/Products: Sensitivity Thresholds HRP-2 pLDHKenya (children) Hospital for Trop. Diseases - LondonParasites/µl n= Sensit.(%) Parasites/µl n= Sensit.(%)1-10 9/23 39 <5 13/22 6011-60 17/21 81 50-500 9/11 8161-100 14/16 88 500-1500 17/18 94101-500 57/57 100 >1500 36/36 100501-1000 12/12 100 Ref: Piper et al. Am J Trop Med Hyg 60: 109-118 (1999)Ref: Beadle et al. Lancet 343: 564-568 (1994) PCR Vietnam (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)

Current Malaria Diagnosis• 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

Malaria ControlVaccines"Stimulate the Phagocytes. Drugs are a Delusion " GBShaw, The Doctors Dilemma 1906Bednets "I myself have been infected with malaria onlyonce in spite of nineteen years of service in India andthirteen subsequent malaria expeditions to warmclimates; and I attribute this to my scrupulous use of thebed net." Ronald Ross Studies on Malaria 1928.Chemotherapy "We must learn to shoot microbes with magicbullets." Paul Ehrlich in Microbe Hunters Paul de Kruif1926

Malaria Control1. Vector control & Sanitation2. Vaccines?3. Chemotherapy Curative Protective (prophylaxis)

Vector Control ObjectivesReduce vector mosquito populationRepel the vector mosquitoesForm a barrier between vector and potential host (personal protection)Reduce the lifespan of vector mosquitoesReduce the lifespan of potentially infected vector mosquitoes

Mosquito Repellents-- NEJM 2002 347:13-8

oocysts Sporozoites Antifolates-proguanil, SporozoitesGametocytes pyrimethamine Primaquine (hypnozoites of P.vivax&ovale) merozoitesArtemisinin trophozoite schizontQuinolines- chloroquine,mefloquine, quinine 48-72 hrsAntibacterials-tetra-cycline,clindamycin,fluoroquinolone(Cipro) } ring Atovoquone Azithromycin

Five Broad Groups Based on Mechanism of Action1. Quinoline: Inhibits heme crystallization2. Artemisinin: 1. Binds heme iron and or iron and generates oxygen radicals. 2. Damages SERCA Ca++ P-ATPase3. Antifolate: Inhibit DNA synthesis4. Atovaquone: Collapses mitochondrial membrane potential

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

The Numbers70 kg person X @70 mL/kg = 4.9 L of blood @ 5 L = 5X103 ml = 5X106 µL5X106 RBCs per µL of blood2.5 X 1013 RBCs4 logs parasite biomass with each asexual cycle.Artemisinin reduces byThis is the most rapidly acting antimalarial.1% parasitemia = 1 in 100 iRBCs = 2.5 X 1011 parasites

Endoperoxide bridge generates single oxygen radicalOld “where the money is” theory: artemisinin makesoxygen radicals in digestive vacuole where lots of oxygenand heme coexist.

Target proteins• Artemisinin increases lipid damage (Berman Adams 1997)• Histidine-rich protein or translationally controlled tumor protein TCTP covalent heme- art-protein adducts• Does not prevent heme crystallization even though binds heme• inhibits hemoglobin proteases in presence of heme• Binds and inhibits calcium transporter PfATPase6

COARTEM

Drug Regimens1. Curative2. Chemoprophylaxis3. Intermittent presumptive therapy (pregnancy, disease preventionin children)

P. falciparum Is 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

Oral vs. Intravenous Chemotherapy• Signs of End Organ Involvement – Pulmonary edema – Renal failure – Coma – Severe anemia-transfusion lifesaving • Unable to Tolerate Oral MedicationsIf yes to any of overabove, then IV • Parasitemia the 5%chemotherapy.

Delay Equals Bad OutcomeDelay from onset of symptoms to medical presentationDelay from presentation until consideration of malariaDelay from consideration to microscope diagnosisDelay from diagnosis until start of therapy.Delay of reassessment (ICU care;hypoglycemia; staff not asfamiliar with IV Quinidine.

Chemoprophylaxis Principles• Avoid infection.• No drug is perfectly effective; all have risks. 20% of all people taking chemoprophylaxis report some effect.• 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).• Start chloroquine one week early to build up therapeutic doses. In emergency travel, may take chloroquine on two consecutive days.

Chemoprophylaxis Principles (cont.)1. 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.• 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.• Chemoprophylaxis of children in endemic area has been shown to reduce mortality (25%) but is not widely done.

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, mefloquineIntermittent presumptive treatment (IPTpregnancy=IPTp)• Full treatment doses at intervals to reduce disease.• 2-3 intervals at scheduled visits.• sulfadoxine-pyrimethamine

Treatment During Pregnancy1. 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 transmittersChemoprophylaxis of travelers (+) prevents infection in millions of travelers/soldiers (-) no impact on endemic diseaseMass 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 OutcomeMutation Prevalence in Sensitive Prevalence in Resistant OR (95% CI) P Infections Infections % (no./total no.) %(no./total no.)pfcrt T76* 37 (40/107) 92 (56/61) 18.8 (5.9-80.3) <0.001pfmdr 1 Y86† 49 (51/104) 75 (46/61) 3.2 (1.5-6.8) <0.001pfcrt 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 chloroquine100%90%80%70%60%50%40%30%20%10% 0% <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

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Malaria Diagnosis, Treatment, and Drug Resistance

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