ESCMID Online Lecture Library. Molecular diagnosis of malaria. by author

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1 Molecular diagnosis of DNA malaria Aldert Bart Parasitology Medical Microbiology Academic Medical Center Amsterdam ESCMID course

2 Putative advantages of molecular diagnosis of malaria correct (unequivocal?) identification of species increased sensitivity identification of mixed infections quantitative (if realtime format is used) yet you spent 2 hours on a malaria diagnosis practical without DNA tests

3 What do we expect of a malaria test?

4 Inter-species differences of 18S rrna gene P. falciparum P. vivax P. ovale P. Malariae P. knowlesi Amplification with Plasmodium genus specific primers? Unknown amplicon

5 Nested PCR on 18S rdna Nested PCR with specific primers on amplicon from genus specific primers? P. falciparum P. vivax P. ovale P. malariae Products differ in size

6 Species specific hybridization Hybridization with specific Taqman probes Screening probe? Specific probes P. falciparum P. vivax P. ovale P. malariae Rougemont et al 2004 JCM; Shokoples et al 2009 JCM

7 Real time PCR advantages: shorter turn around time additional identification of PCR product through hybridization or melting curve

8 Technical aspects original purpose of test scale and time to result quality control false positives: contamination nested PCR assays open systems/closed systems lab set-up false negatives: inhibition DNA extraction internal control

9 positive control P. falciparum P. falciparum negative P.vivax P.malariae positive control ~2 hours amplification ~1 hour running time 5 minutes visualization Conventional PCR Realtime PCR P.m P.o ~1 hour amplification & detection P.v P.f

10 Technical aspects original purpose of test scale quality control false positives: contamination nested PCR assays open systems/closed systems lab set-up false negatives: inhibition DNA extraction internal control

11 UK NEQAS (UK QCMD quality control (UK Nat l External Quality Assessment Service) QCMD (Quality Control for Molecular Diagnostics) INSTAND e.v e.v. (Society for Promotion of Quality Assurance in the Medical Laboratories ) no international quality assurance scheme for molecular diagnosis of malaria

12 Only quantification, no species

13 Technical aspects original purpose of test scale quality control false positives: contamination nested PCR assays open systems/closed systems lab set-up false negatives: inhibition DNA extraction internal control

14 nested PCR: DNA extraction primary amplification secondary amplification closed system: tube is discarded unopened after analyses reagent preparation isolation open system: tubes opened for analysis amplification & analysis Unidirectional workflow required with different labs

15 Technical aspects original purpose of test scale quality control false positives: contamination nested PCR assays open systems/closed systems lab set-up false negatives: inhibition DNA extraction internal control

16 sample collection on filters: easy storage, but more prone to contamination and human error manual extraction: easy for small numbers, but more prone to contamination and human error automated extraction: less contamination during extraction, but only costeffective for large numbers

17 epidemiology in mosquito in man in animals primary diagnosis saliva/urine blood PCR for malaria confirmatory diagnosis mixed infections

18 Phylogenetic relationships among Plasmodium species Prugnolle F et al. PNAS 2010;107:

19 this PCR seems too sensitive for clinical purposes in an endemic population

20 epidemiology in man in mosquito primary diagnosis saliva/urine blood PCR for malaria confirmatory diagnosis mixed infections

21 Blood remains the sample material of choice

22 Only limited real-time evaluation in clinical import-malaria settings Morassin AJTMH 2002: all patients in 1 year in Toulouse An average of 2.48 PCR-based diagnoses daily 32 discrepancies: 14 (previously) under treatment 10 (semi-)immune immigrants 5 travelers under chemoprofylaxis (1 treated) 3 unknown

23 Discrepancies microscopy-qpcr: 17 positive by QPCR and ICT (treated?) 10 positive by PCR only ( false positive?)

24 Only limited evaluation in clinical import-malaria settings Shokoples JCM April 2009: confirmatory test in reference lab: 30 positive patients from Alberta 2007/2008 weekly tests

25 epidemiology in man in mosquito primary diagnosis saliva/urine blood PCR for malaria confirmatory diagnosis mixed infections

26 Clin Microbiol Infect Mar 13

27 Remarkable results from molecular methods: P. ovale consists of two major clades, as divergent as different species Incardona et al, Am. J. Trop. Med. Hyg., 72(6), 2005, pp /719 Win et al. Emerg Infect Dis Jul.

28 Remarkable results from molecular methods: P. ovale consists of two major clades, as divergent as different species Clinical relevance?

29 Remarkable results from molecular methods: P. ovale consists of two major clades, as divergent as different species Diagnostic relevance

30 Evaluation of two real time methods: Discrepancies with (mixed infections with) P. ovale

31

32 conclusions evaluation in clinical import-malaria settings Khairnar Mal. J. December 2009:

33 Approach: antigen test QBC thick smear thin smear (species) our experience PCR only in selected cases Alternative Weekly testing of all samples

34 our evaluation > 100 samples retrospectively No discrepancies except in follow-up samples (PCR positive, microscopy negative)

35 our experience PCR applied 13 times in past 3 years 1x disagreement microscopists: Pf or Pf/Pm mixed infection 6x confirmation P. ovale (2) or P. vivax (4) 4x disagreement of clinician with microscopy 3x confirmed negative 1x no Plasmodium infection, but Babesia 1x too few parasites to speciate by microscopy 1x reference request: P. malariae or P. knowlesi

36 Rougemont PCRs: generic Plasmodium POS, Duplex P. falciparum, P. vivax NEG, Duplex P. ovale, P. malariae NEG Shokoples PCR: multiplex P. falciparum, P. vivax, P. ovale, P. malariae NEG in house: monoplex P. knowlesi POS PC samples J. Clin. Microbiol. doi: /jcm

37 Conclusions molecular diagnosis of malaria still limited sequence information (specificity) time to result is >2 h, cost-effectiveness increases with increasing sample numbers published methods not sufficient for primary diagnosis, but usefull as confirmatory or secondary test in reference centers This afternoon: practical session 5: molecular diagnosis of malaria