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ESCMID Online Lecture Library © by Author Microscopy and PCR for diagnosis of parasitic infections: a tale of two amazing powerful techniques Tom van Gool MD, PhD, Aldert Bart PhD Section Clinical Parasitology, Department Medical Microbiology, Academic Medical Center, Amsterdam, ESCMID OnlineNetherlands Lecture Library © by author Academic Medical Center (AMC), Amsterdam, Netherlands Patients from routine clinical care large university hospital, Dept. ESCMIDTropical Medicine Online and general practitioners Lecture from surroundings. Library © by author Microscopy: an old, but still extremely useful diagnostic tool in clinical parasitology! Needed: 1: a microscope 2: a well trained technician 3: saline, iodine, or other (cheap) stain…. and… a wealth of information becomes available! ESCMID Online Lecture Library © by author Molecular Diagnosis Acanthamoeba detection and typing Parasitic Infections, Angiostrongylus detection (AMC, NL) Babesia detection and typing Blastocystis detection and typing Cryptosporidium detection Dientamoeba fragilis detection Entamoeba detection and typing Echinococcus detection and typing ….a large variety of Giardia detection and typing protozoa and helminths.... Leishmania detection and typing Malaria detection and typing Microsporidium detection and typing Opisthorchis detection and typing Schistosoma spp detection and typing Toxocara detection TrypanosomaESCMID cruzi detection Online and typing Lecture Library Intestinal helminths i.e. strongyloides © by author Current priorities in diagnostic approaches - Malaria - Leishmania - Intestinal parasites ESCMID Online Lecture Library © by author Malaria Caused by protozoan parasites: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi Most often severe illness, with P. falciparum often lethal. Effective treatment with early diagnosis. Al patients returning from malarious areas with fever: direct blood examination for malaria! ESCMID Online Lecture Library © by author Malaria What do we need for diagnosis in a routine clinical setting in returning travellers: ► Speed (result < 1h after having received blood sample) ► High sensitivity and specificity (for malaria) ► Good species determination: P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi ► Means to calculate parasitemia (for P. falciparum) ESCMID Online Lecture Library © by author Basic diagnostic methods for malaria in routine setting in western (hospital) laboratory Antigen test Thick smear = Thin = smear ESCMID Online Lecture Library © by author Giemsa stains Fast stains Fields stain Thick smear 2 minutes !! Diff Quick stain Thin smear ESCMID Online Lecture 1 Library minute ! Giemsa GURR Giemsa MERCK © by20 - 30author minutes stain New(er) tests for malaria diagnosis: also “microscopy”… • Quantitative Buffy Coat (QBC) • Antigen detection (RDT) • Automatic analysers (CD4000) • Polymerase Chain Reaction (PCR) ESCMID Online Lecture Library © by author QBC Quantitative Buffy Coat Inside tube: coating with acridine orange which stains nucleated cells and malaria parasites ESCMID Online Lecture Library © by author Sampling blood for QBC ESCMID Online Lecture Library © by author QBC-tube in fluorescence microscopy 5 minute reading of entire length beneath buffy coat . .. Buffy coat Concentration of parasites Sensitivity ≈ 1p/ul Most reliable: 8000 ESCMID Online Lecture patientsLibrary in 25 years: Clearly visible no failures © bynucleus author and cytoplasm Sensitivity 8 different microscopic methods among 109 postive patients with imported malaria (NL)…. Results with different techniques Thin Field's thick smear Giemsa thick smear smear Thin smear combined with: Giemsa Giemsa Giemsa Results GTS800 fields 100 fields 200 fields 400 fields 100 fields 200 fields 400 fields 5 min thick smear thick smear thick smear QBC "Gold standard" (3 min) (6 min) (12 min) (3 min) (6 min) (12 min) 100 fields 200 fields 400 fields P. falciparum 83 72 77 78 77 79 80 74 77 79 80 0 P. vivax 20 20 20 20 20 20 20 20 20 20 20 0 P. ovale 5 3 3 4 3 4 5 5 5 5 5 0 P. malariae 1 1 1 1 1 1 1 1 1 1 1 0 Sp. Unknown 0 11 6 5 6 5 3 2 4 4 3 109 All species (total) 109 107 107 108 107 109 109 102 107 109 109 109 Sensitivity 100% 98,2% 98,2% 99,1% 98,2% 100% 100% 93,6% 98,2% 100% 100% 100% Thick smear Fields Thick smear Giemsa 200 fields: 98% 100 fields (3 min.) : 98% QBC: 200 fields: (6 min.): 100% ESCMID Online Lecture Library100% © by author Thin smear 5 min.: 94% Malaria, molecular diagnosis Fascinating, challenging… But…. With experienced personnel at hand, can MD outclass microscopy and rapid tests in a routine clinical setting with returning travellers, clinically suspected of having malaria ? ESCMID Online Lecture Library © by author Speed: fast hand held PCR machine Biohazard No ESCMID Onlineparasitological Lecture Library © by authorassay Fast PCR system for clinical lab • GeneXpert takes ~1 hour from sample to result • No clinical parasitology assays (yet?) ESCMID Online Lecture Library © by author Often in use: MagnaPure 96 and LC480 ~1 hour and ~1,75 hour runtime In total ~ 2 - 3 h for result malaria diagnosis…. ESCMID Online Lecture Library MagnaPure 96 © by author LC 480 Performance of PCR’s for malaria diagnosis Non- endemic areas Bourgeois et al CMI, 2009 “In 17 studies (2002-2008) using realtime PCR methods, a total of 4963 samples were tested. In 1394 samples were positive by real-time PCR, microscopy detected parasites in only 1317 samples (94.4%)….” Endemic areas ESCMID Online Lecture Library © by author However….in western countries often also very good agreement in between classical diagnostic methods and real time PCR! Caldero, Malaria Journal, 2013, Italy 1) imported malaria - symptomatic patients 2) no follow-up included after start of therapy 3) technically skilled personnel Bourgeois et al Clinical Microbiology And Infection, 2009, France 126 + 128 + + DiscrepanciesESCMID especially withOnline regard to Lecture Library species differentiation / mixed infection…. 270 negative 270 negative © by author AMC, molecular diagnosis malaria Travel to south-east Multiplex- 4 species Asia, Genus specific PCR PCR (Shokoples P. malariae - like ? : (Rougemont 2004) 2009) P. knowlesi PCR (Link 2012) ESCMID Online Lecture Library © by author Comparison results microscopy and PCR in routine clinical setting AMC (2010-2011) Dutch origin: 126 (50%) 252 patients Foreign origin: 126 (50%) 329 samples 252 samples (= patients) 77 control samples after before start of treatment (76,6%) start of therapy (23,4%) Microscopy (QBC,ICT,Thick and Thin Smear) PCR Microscopy (QBC,ICT,Thick and Thin Smear) PCR Positive with microscopy: 31 Positive with microscopy: 34 Postive with PCR: 31 Positive with PCR: 61! Negative with microscopy: 221 Negative with PCR: 221 ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author Molecular Diagnosis Malaria: its current position …... In general: use for for special situations only: • Suspicion mixed infections • Very low parasitemia / no species differentiation possible. • Differentiation P. malariae and P. knowlesi (morphologically similar) ESCMID Online Lecture Library © byLink author et al. JCM 2012 Leishmaniasis ESCMID Online Lecture Library © by author The disease: leishmaniasis ♦ Disease caused by protozoan parasite, transmitted to humans by phlebotome sandflies ♦ 350 million people at risk of leishmaniasis worldwide Visceral disease: 500.000 cases /year (muco) cutaneous disease: 1,5 million/ year ♦ Endemic in 82 countries: 21 in “New World”, 61 “Old World” ESCMID Online Lecture Library © by author Increased numbers of patients with leishmaniasis who visited tropical and endemic European countries: Academic Medical Centre, Amsterdam: 1979 - 2012 ESCMID Online Lecture Library © by author Bart et al, Eurosurveillance 2013 unknown Pakistan Central America Belize Costa Rica Central or South America South America Bolivia Geographic origin of Brazil imported leishmaniasis Peru Surinam AMC, 2005-2012 Spain Afghanistan Italy Malta Morocco Israel Sudan ESCMID Online Lecture patients (size ~Library number) research area © by author Bart et al, Eurosurveillance 2013 Visceral Leishmaniasis (“kala azar”) • Infection with L. infantum or L. donovani • Infection of internal organs • Symptoms: non specific at beginning, fever, weakness, loss of appetite, weight loss hepatosplenomegaly • Laboratory: pancytopenia (anaemia, neutropenia, ESCMID thrombocytopenia),Online Lecture hypergammaglobulinemia Library © by author Specific diagnosis visceral leishmaniasis DiaMed-IT Leish dipstick: antibodies to rk39 antigen Microscopy Reliable result:15 min ! ≈ 30 min CDC Culture (1- 4 weeks) Bone marrow Direct Agglutination Test Molecular diagnosis: (DAT)ESCMID : 12 h Onlinepunction Lecture Library PCR and sequencing © by author (when microscopy is negative) Cutaneous Leishmaniasis: different regions, different Leishmania species! species involved… Old World L. major L. tropica L. infantum L. mexicana L. amazonensis New world L. braziliensis L. guyanensis ESCMID Online LectureL. panamensis Library © by author L. naiffi Cutaneous leishmaniasis from the “Old World”: different clinical presentations….. ** * ESCMID Online Lecture Library * ** P. P.A.M. van© Thiel by author and the “New World”…. (extended damage due to L. braziliensis) ESCMID Online Lecture Library © by author Different species, different symptomatology, but also different treatment needed…! ESCMID Online Lecture Library © by author Traditional techniques used for diagnosis of cutaneous leishmaniasis Microscopic diagnosis - fast - cheap - dis.: limited sensitivity?
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