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15Th ECCMID Copenhagen Educational Workshop EW05: Diagnosis of parasitic infections arranged with EFWISG (ESCMID Food- and Water-borne Infections Study Group) and ESGCP (ESCMID Study Group for Clinical Parasitology) Convenors: Birgitta Evengard, Stockholm, SE Kevin Kerr, Harrogate, UK Faculty: Nick Beeching, Liverpool, UK Jaco Verweij, Leiden, NL Pablo Okhuysen, Houston, US Tom van Gool, Amsterdam, NL Pierre Marty, Nice, FR Herve Pelloux, Grenoble, FR Laetitia Kortbeek, Amsterdam, NL Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care History • Started in 1995 with differentiation of microscopically identical Entamoeba histolytica and Entamoeba dispar • PCR used for differentiation and confirmation Molecular diagnostics of intestinal parasites in • Further improvement of DNA isolation methods epidemiology and patient care • Introduction of real-time PCR provides – High sensitivity Jaco J. Verweij – High specificity – Multiplex several targets – (semi) quantitative results – Reduced contamination risk • Multiplex detection of intestinal parasites in patientcare and epidemiology Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Multiplex detection of diarrhoea causing protozoa in Schistosoma multiplex real-time PCR faeces using real-time PCR Treshold 10-1 10-2 10-3 10-4 10-5 10-6 (100 fgram) Entamoeba histolytica Ct-value Giardia lamblia Cryptosporidium Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Entamoeba histolytica Laboratory diagnosis of E. histolytica infections • Amoebae • Microscopy • Antigen detection • Invasive disease – trophozoites in fresh or – detection of antigen in fresh – amoebic colitis preserved stool samples stool samples – amoebic dysentery (1876) – amoebic liver abscess – cysts in iodine stained – more sensitive formalin-ether concentrate – specific • 100.000 deaths annually – not very sensitive • PCR • The cyst is the infectious form – 3 samples required and is very resistant – Differentiation from E. dispar – detection of DNA in fresh or not possible ethanol preserved stool samples – even more sensitive – specific Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology 1 1 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care Giardia lamblia Laboratory diagnosis of Giardia infections • Flagellate • Microscopy • One of the main non-viral • Antigen detection – trophozoites in fresh or causes of diarrhoea – detection of antigen in fresh preserved stool samples or preserved stool samples (1681) • Trophozoites attach to intestinal – cysts in iodine stained epithelial cells (non-invasive) formalin-ether concentrate – more sensitive – 2 samples required • The cyst is the infectious form – not very sensitive and is very resistant cyst – 3 samples required • PCR – detection of DNA in fresh or ethanol preserved stool samples – more sensitive Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Microscopy, antigen ELISA and G. lamblia real-time PCR for the detection of G. KML Giardia lamblia infections in 10 patients with complaints of diarrhoea Validation Giardia in HGC-PCR • 30 samples from 10 patients • 362 samples microscopy, antigentest and PCR. – Microscopy positive 11/30 Microscopie Antigeentest PCR Aantal Min Ct Max Ct Median Ct – Antigen positive 13/30 - - - 254 + + + 75 20,1 38,0 26,1 - + + 9 27,3 36,7 32,0 – PCR positive 29/30 - - + 24* 30,7 42,8 35,8 362 • *10 samples of 4 patients in which Giardia infection is confirmed by microscopy and/or antigen test in a subsequent sample. • 11 samples of 2 x 3 patients from 2 families in Giardia infection was confirmed in a family member. • 3 samples of 3 patients in which only was positive 1 of 3 samples. Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Cryptosporidium parvum/hominis • Coccidia • first human case in 1976 • severe diarrhoea in AIDS patients • We estimate that 403,000 people had watery diarrhoea attributable to this outbreak. Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology 2 2 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care Microscopy and real-time PCR for the detection of a Cryptosporidium infection Laboratory diagnosis of Cryptosporidum infections in a immuno-compromised child with complaints of diarrhoea Technique to detect Cryptosporidium infection • Microscopy • Antigen detection date Microscopy (ZN) PCR Ct value – oocysts in fresh or – detection of antigen in fresh preserved stool samples or preserved stool samples 31-05-2002 - - - • modified Ziehl-Neelsen – more sensitive? 12-09-2002 - + 32.7 • auramine – specificity? • safranine 17-09-2002 - + 28.0 • monoclonal antibodies • PCR 10-02-2003 + + 26.8 – detection of DNA in fresh or – not very sensitive ethanol preserved stool 18-02-2003 + + 27.9 • specificity and samples 10-04-2003 - + 37.1 sensitivity of the Mab? – more sensitive 29-04-2003 + + 31.2 – multiple samples Morgan et al 1998 13-05-2003 + + 27.8 Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Performance of molecular diagnostic approach in In summary different populations • Three diarrhoea causing protozoa • Groningen, patients attending their general practitioner – E. histolytica; G. lamblia; Cryptosporidium • “Microscopy” is the classic diagnostic method • Zwolle, patients attending their general practitioner and peripheral • Separate alternative methods have been developed and have proved hospital to be more sensitive and specific – E. histolytica; G. lamblia; Cryptosporidium • Disadvantage: separate techniques for optimal detection of the – D. fragilis separate parasites • Antwerp, patients attending the Travel Medicine Clinic – E. histolytica; G. lamblia; Cryptosporidium – S. stercoralis • Malawi, population in which HIV infection is highly endemic – E. histolytica; G. lamblia; Cryptosporidium – E. bieneusi; Encephalitozoon Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Results Groningen, general practitioner setting Results Groningen, general practitioner setting N=950 Microscopy PCR Study groups E. histolytica 0 0 June – September 2005 Giardia lamblia (n=720) 41 67 Giardia lamblia (n=230) not done 15 Microscopy Bacteriology Cryptosporidium not done 45 n=1636 n=1184 Helminths 0 not done real time PCR Non-pathogenic protozoa 34 not done n=950 Culture Campylobacter spc. 90 Salmonella spc 29 Shigella sonnei 2 Center of Infectious Diseases Ten Hove et al. Clin Microbiol Infect. 2007 13:1001-7 Center of Infectious Diseases Ten Hove et al. Clin Microbiol Infect. 2007 13:1001-7 Department of Parasitology Department of Parasitology 3 3 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care Groningen data Prevalence(%) of Giardia per age group Prevalence(%) of Cryptosporidium per age group Microscopy Real-time PCR 30 25 25 20 20 15 15 10 10 5 5 0 Percentage 0 <5 5-14 15-29 30-49 50-64 >64 total <5 5-14 15-29 30-49 50-64 >64 total age group Center of Infectious Diseases Ten Hove et al. Clin Microbiol Infect. 2007 13:1001-7 Center of Infectious Diseases Ten Hove et al. Clin Microbiol Infect. 2007 13:1001-7 Department of Parasitology Department of Parasitology Results Zwolle, general practitioner and peripheral hospital N=397 Ct-values in time-course experiment (L. Bruijnesteijn et al. In prep) • D. fragilis Weeks after storage before isolation – Since its discovery, the pathogenicity of this organism 0 1 2 3 8 has remained controversial – No cyst stage Unpreserved sample – Preserved (SAF) faeces 1 29.3 29.1 29.2 29.0 29.0 2 28.6 27.5 28.1 28.3 27.9 3 28.7 27.0 29.1 27.7 27.3 4 30.2 28.2 30.3 30.0 29.0 5 28.4 27.2 27.0 27.2 27.0 6 30.9 29.1 30.0 30.1 30.6 7 35.9 36.4 36.5 37.1 37.1 Median 29.3 28.2 29.2 29.0 29.0 control 0 0 0 0 0 Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology Results Zwolle, general practitioner and peripheral hospital N=397 Study on the potential pathogenicity of D. fragilis (L. Bruijnesteijn et al. In prep) Microscopy PCR • Epidemiology of D. fragilis in relation with other diarrhoea (TFT set) (1 sample) causing pathogens E. histolytica/E. dispar 1 E. histolytica 1 • Development of genotyping methods and molecular Giardia lamblia 29 45 epidemiology of D. fragilis in symptomatic and asymptomatic cases. Cryptosporidium 2 3 D. fragilis 66 112 Non-pathogenic protozoa E. coli 8 E. nana 10 B. hominis 111 Helminths 1 Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology 4 4 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care Results Antwerp Travel Clinic N=2717 Strongyloides stercoralis diagnostics Microscopy PCR E. histolytica/E.dispar 152 • MICROSCOPY E. histolytica 13 – Detection of L1-larvae in stool concentrates Giardia lamblia 110 158 Cryptosporidium 13 32 – Detection of L1-larvae in L1 larve Strongyloides stercoralis 4 22 Baermann sediments – Detection of L3 larvae in stool culture L3 larve Center of Infectious Diseases (Ten Hove et al. in preparation) Center of Infectious Diseases Department
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