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 of Parasitology Department of Parasitology
Results Antwerp Travel Clinic N=2717 Malawi, population in which HIV infection is highly (Ten Hove et al. in preparation) endemic (N=182)
Microscopy PCR Microscopy Antigen PCR Schistosoma mansoni 14 - E. histolytica 0 - 0 Hookworm 13 - Giardia lamblia 6 14 43 Ascaris lumbricoides 8 - Cryptosporidium 18 18 38 Trichuris trichiura 14 - Taenia spec. 1 - Microsporidium 12 - 36 S. haematobium 1 - S. stercoralis 2 - not done Enterobius vermicularis 1 - S. mansoni 1 - not done
Hookworm 1 - not done Cyclospora 4 - Ascaris 1 - not done Isospora belli 2 - Dientamoeba fragilis 76 -
Center of Infectious Diseases (Ten Hove et al. in preparation) Center of Infectious Diseases (Van Lieshout et al. in preparation) Department of Parasitology Department of Parasitology
Detection of Microsporidian DNA with PCR in faeces Microsporidia (Katzwinkel et al, 1996)
• Semi-nested multiplex PCR 1 1 1 1
• ITS rRNA gene 1 2 3 4 Eb Ei neg M
• Specific for E. bieneusi
• Specific for other Microsporidia (ie E. intestinalis)
Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology
5 5 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care
Available (multiplex) real-time PCRs
Targets with internal control Status E. histolytica; E. dispar Published E. histolytica; G. lamblia; Cryptosporidium Published E. bieneusi; Encephalitozoon Published D. fragilis Published C. cayetanensis Published I. belli In press N. americanus; A. duodenalis; O. bifurcum Published S. mansoni; S. haematobium Published Schistosoma genus Submitted S. stercoralis In preparation O. felineus In preparation A. lumbricoides In preparation Leishmania In use Toxoplasma In use Plasmodium species Published
Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology
E. bieneusi and Encephalitozoon spp. real-time PCR Most (cost) effective way to use these assays results in fecal samples from different patient groups
• Is a different approach needed from the approach that is Underlying condition Number PCR result Ct-value used in choosing conventional diagnostics?
HIV infection 16 E. bieneusi 15.8-36.5 • Can we choose test based on complaints? Kidney transplantation 6 E. bieneusi 18.9-24.8 Hypogammaglobulinemia 1 E. bieneusi 21.6 • Is it useful to detect every parasite in each population? Adoption (1 & 4 years old) 2 E. bieneusi 29.8; 34.8 Traveler 1 E. bieneusi 23.1 Unknown 4 E. bieneusi 17.9-21.6
HIV infection 2 Encephalitozoon spp. 22.4; 24.7 Unknown 1 Encephalitozoon spp. 34.0
Patients from 1996-2005 N=33
Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology
E. bieneusi and Encephalitozoon spp. real-time PCR E.bieneusi PCR in hospital population Ethiopia results in fecal samples 2005-2006
Underlying condition Number PCR result Ct-value
Kidney transplantation 1 E. bieneusi 20.0 Liver transplantation 1 E. bieneusi 39.0 Diabetes 1 E. bieneusi 21.6 Colitis ulcerosa 21-09-2006 E. bieneusi 21.9 09-10-2006 E. bieneusi 35.1 18-10-2006 E. bieneusi 38.1 12-01-2007 negative
HIV infection 1 Encephalitozoon spp. 31.0 Kidney transplantation 1 Encephalitozoon spp. 32.9 Diabetes 1 Encephalitozoon spp. 38.9
Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology
6 6 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care
Encephalitozoon in hospital population Ethiopia E.bieneusi PCR in hospital population LUMC
Patient with hypogammaglobulinemia
Center of Infectious Diseases Center of Infectious Diseases Department of Parasitology Department of Parasitology
Encephalitozoon in hospital population LUMC E. bieneusi in hospitalized diarrhoea patients - Malawi
40
Ct (median) of all samples: 30 37.2 34.6 28.0
20
10 % positives (Ct % positives < 35)
0 controls (n = 73) acute (n = 119) chronic (n = 141) diarrhoea groups
Center of Infectious Diseases Center of Infectious Diseases Van Lieshout et al. unpublished data Department of Parasitology Department of Parasitology
E. bieneusi in hospitalized diarrhoea patients - Malawi Polymorphic sites in the ITS sequence
Nucleotide position in ITS sequence 92 102 112 121 133 139 146 153 157 173 177 179 180 183 191 223 235 236 239 251 253 Genotype 50 Ct (median) of all samples: A C C A C A C C C G G A G C C C C A C C C C 37.1 36.8 35.9 28.0 40 B ------T - - - - T - - C T - - A G - T - A - - A ------T - A D - - - - G - - A - - G ------30 K - - - - G ------UG2145 - - - - G ------G - T - - PE6624 - - - - G - - - - - G ------20 SP1 - - - - G ------T T - SP2 - - - - G ------T - - T T -
% positives (Ct % positives < 35) 10 SP3 - - - - G ------T - - - T - SP4 - - - - G ------T - - T - - SP5 - - - - G ------T - - 0 SP6 - - - - G - - A ------HIV negative (n = 46) CD4 >200 (n = 47) CD4 100-200 (n = 41) CD4<100 (n = 138) SP7 - - - - G ------T - - HIV positive CD4 counts SP8 T - - A G - - - A - - A - - - - G G T - A SP9 - A - - G T ------SP10 - - C - - G - - A A - A T T T - - - T - - Center of Infectious Diseases Van Lieshout et al. unpublished data Center of Infectious Diseases Klaartje Spee unpublished data Department of Parasitology Department of Parasitology
7 7 Jaco J. Verweij Molecular diagnostics of intestinal parasites in epidemiology and patient care
Phylogenetic tree based on E. bieneusi ITS sequences Genotypes found in specific patient groups
I Malawi Children HIV+ N=9 D, K, SP2, SP5
II Malawi Children HIV- N=4 K, SP1, SP2, SP6
III Malawi Adults HIV+ N=21 K, PE6624, UG2145, SP2, SP3, SP4, SP6, SP7,
IV LUMC Adults HIV+ N=7 B(4x), D, K, SP8
V LUMC Adults transplant pat. N=5 C
V LUMC Adults HIV-/? N=7 A, K, SP9, SP10
Center of Infectious Diseases Ten Hove in preparation Center of Infectious Diseases Ten Hove manuscript in prep Department of Parasitology Department of Parasitology
8 8 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Cyclospora: An Emerging Foodborne pathogen
Pablo C. Okhuysen, MD The University of Texas Health Science Center –Medical School at Houston, Texas, USA
3/25/2008 1
Outline
Characteristics of Cyclospora that n distinguish it from other Apicomplexan parasites Life cycle and sporulation requirements n Epidemiology and outbreaks n Clinical Manifestations n Diagnosis and Treatment n
3/25/2008 2
Cyclospora
First human cases described in 1977 n Previously referred to as cyanobacterium- n like bodies, fungal spores, blue-green algae, “large”cryptosporidia Confirmed as belonging to the Cyclospora n genus in 1993 Closely related to Eimeria, distantly related n to Isospora, Sarcocystis and Toxoplasma
3/25/2008 3
9 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Host Specificity
Cyclospora-like organisms have been identified n Cyclospora in mice, rats, chicken, ducks, dogs, primates and others
n Coprophagy vs. zoonosis with Cyclospora other than C. cayetanensis
n Unknown if animals can serve as a reservoir for C. cayetanensis Attempts to infect animals have been n unsuccessful except for the albino mouse ID50 for humans unknown, challenge studies n with 200 - 49,000 oocysts unsuccessful Alfano-Sobsey et.al. Emerg Infect Dis.2004 10;726-8
3/25/2008 4
Cyclospora in Primates
Study of 511 stool specimens in 11 distinct n primates in 10 locations in Kenya Oocysts typed for 18S rRNA analysis: n
n Vervet monkeys (43/102, 42%) C. cercopotheci n C. cercopotheci
n Yellow and olive baboons (19/206, 9%) C. papionis n C. papionis
n Black and white colobus monkeys (19/76, 25%) C. colobi n C. colobi No seasonality, high host specificity in primates n Eberhard ML et.al. J Parasitol. 2001;87:1394-7
3/25/2008 5
Characteristics
n Oocysts measure 8-10 um in diameter
n Contain two sporocysts, each one with four sporozoites Fresh Day 5
n Unsporulated phase: outer membrane, outer fibrillar coat, cell wall, and membrane
n Sporulated sporozoites show a membrane bound nucleus and micronemes Day 10 Rupture
http://www.dpd.cdc.gob
3/25/2008 6
10 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Life Cycle
Sporulation n
n Outside the host
n Optimal at 27 to 32 °C for 8 to 11 days Oocysts survive: n
n Freezing, 2% formalin n 2% potassium dichromate and chlorination
3/25/2008 7
FoodNet Active Surveillance 2006 16 14 Cases / 12 10 100,00 8 population 6 1.9 0.9 4 2 0
HUS Shigella Listeria Salmonella Cyclospora Campylobacter Cryptosporidium 3/25/2008 http://www.cdc.gov/foodnet/ 8
Effects of Gamma Irradiation
At doses of 0.4 kGy excystation occurs but n not infectious to mice
Dubey et.al. Int J Parasitol. 1998;28:36 9-75
3/25/2008 9
11 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Epidemiology
Occurs worldwide in clusters and n sporadically High prevalence in Nepal, Peru, Egypt, Haiti n An infrequent cause of travelers’diarrhea n Increased frequency in HIV co-infection n and other immunosuppressive conditions Outbreaks have been related to: n Contaminated water storage tanks n Imported raspberries, mixed vegetables, basil n
3/25/2008 10
Epidemiology
Produce is contaminated when sprayed or n washed with untreated water Developing countries: 0.1 to 0.5% prevalence n Raspberry farm workers in Guatemala: n n 6/182 (3%) Highest risk in children 8 mo to 5 years of age n n Cryptosporidium mean age 2 years n Cyclospora mean age 5 years Highest incidence in Spring and Summer and n rainy seasons Bern et. al. Emerg Infect Dis 1999;5:766-74
3/25/2008 11
Cryptosporidium vs. Cyclospora Guatemalan Children Characteristic Cyclo Crypto
Episodes / year (all) 0.20 0.22
Incidence Peaks 1 yr Constant 0.42 0.21
Isolation and Declines Remains Diarrhea with time constant
Association House Animals No latrine
3/25/2008 Bern et. al. Emerg Infect Dis 1999;5:766-74 12
12 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Molecular Epidemiology
Small subunit rRNA allows for genotyping n Intervening transcribed spacer (ITS) n sequencing shows a high variability Useful for linking cases to a single source n Parasites from patients can contain multiple n ITS1 genotypes
n Variability within the genome of a single clone n Multiple clones from a single source “clonal polyparisitism” Adam et.al. J Clin Microbiol, 2000;38: 2339-43 Oliver et. al. Int J Parasitol, 2001;31:1475-78 3/25/2008 13
Clinical Manifestations
In indigenous populations n n Asymptomatic infection is common even in some immunocompromised patients Incubation period n One to 11 days n One to 11 days Symptoms n Flu-like illness may precede diarrhea n Flu-like illness may precede diarrhea Watery diarrhea (average of 6 stools/day) n Watery diarrhea (average of 6 stools/day) Upper GI symptoms common, nausea, abdominal pain n Upper GI symptoms common, nausea, abdominal pain Fatigue, myalgia, cramps, flatus, n Fatigue, myalgia, cramps, flatus, Fever in 25% of cases n Fever in 25% of cases
3/25/2008 14
Clinical Manifestations
Duration of illness 2 - 7 weeks n Cyclic or relapsing n Dehydration, weight loss n Clinically cannot be differentiated from n other causes of diarrhea such as Cryptosporidium, Giardia Case reports of reactive arthritis, Guillain n Barre
3/25/2008 15
13 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Clinical Symptoms Outbreak Wedding Reception 1998 Diarrhea 57 (100) Bloating 36 (63) n n Weight loss 53 (93) Myalgia 36 (63) n n Fatigue 52 (91) Chills 29 (51) n n Anorexia 51 (90) Sub. Fever 22 (39) n n Weakness 46 (81) Vomiting 21 (37) n n Abd. Pain 43 (75) Headache 21 (37) n n Duration >3 wk (60%) Recurring Symptoms n n in 81% of cases
Fleming, C Arch Intern Med. 1998;158:1121-1125
3/25/2008 16
HIV related
n Longer duration of illness CD4 <200 cells/mL n CD4 <200 cells/mL
n Can involve the biliary tree and cause cholecystitis
n Responds to therapy but relapses are common
n Secondary prophylaxis indicated in HIV
De Gorgolas MJ et.al. Ann Intern Med. 2001;134:166
3/25/2008 17
Histopathology
Can be detected in jejunal aspirates or in n biopsy specimens In tissues, Cyclospora is identified in n supranuclear cytoplasmic space Inflammation of submucosa n Deposition of electron dense myelin - like n deposits
Connor, BA et.al. Clin Infect Dis. (1999);28:1216-22 3/25/2008 18
14 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Diagnosis
Microscopic exam n
n Twice the size of cryptosporidia (8-10 um)
n Visualized in fresh mount n Acid fast staining with Ziehl-Neelsen or Kinyoun
n Safranin and lacto phenol cotton blue stain oocysts uniformly Shedding mirrors clinical illness n In prolonged cases, xylose absorption can be n abnormal
http://www.dpd.cdc.gov 3/25/2008 19
Autofluorescence
Cyclospora oocysts exhibit intense blue n color when excited with UV light using the filter set at 330-365 nm Less intense green fluorescence with blue n excitation filter at 450-490 nm Can be done on a wet mount without dyes n and age does not affect this property
http://www.dpd.cdc.gov
3/25/2008 20
Treatment
Trimethroprim / sulfamethoxasole n Two prospective, double blind, placebo n controlled studies have been done using160/800 mg Recommended dose is twice daily for 7 days n In HIV infection, tmp/smx used q.i.d and then n q.i.d chronic suppression three times a week Case reports of failures when using n trimethroprim alone Hogue CW Lancet. 1995;345:691-3 Slim et. al. J Travel Med. 1997;4;:44-45
3/25/2008 21
15 Pablo Okhuysen Cyclospora – an emerging food-borne parasite
Treatment
Ciprofloxacin n In a study in Haiti, found to be similar to n tmp/smx in efficacy Treatment failures reported n Nitazoxanide n Case reports of efficacy n An alternative in sulfa allergic patients or n failures with other antibiotics
Zimmer S et. al. Clin Infect Dis. 2007;44:467-70 Verdier et. al Ann Intern Med. 2000;885-6 Diaz3/25/2008 E Am J Trop Med. 2003;68:384-5 22
16 Pierre Marty Cutaneous leishmaniasis
Rapid Diagnosis of Cutaneous Leishmaniasis
Pierre Marty Equipe de Recherche sur les Leishmanioses (ERLEISH) Faculté de Médecine Université de Nice-Sophia Antipolis and Centre Hospitalier Universitaire de Nice France
Geographical distribution
88 countries, Prevalence: 12 millions, Incidence: 2 millions (CL 1.5 M, VL 0.5 M)
7 countries = 90% CL: Afghanistan, Algeria, Saudi Arabia, Syria, Iran, Brazil, Peru 5 countries = 90% CL: India, Bangladesh, Nepal, Sudan, Brazil
Visceral Leishmaniases
Clinical presentation
Leishmania infantum Morocco Leishmania donovani, Sudan
17 Pierre Marty Cutaneous leishmaniasis
Amastigotes (May Grünwald Giemsa stain) M.E. Bougnoux
Cutaneous leishmaniases The difficulty Great variety of clinical presentations Great polymorphism related to species and hosts !
Leishmania major Kairouan, Tunisia Leishmania infantum Leishmania braziliensis Campania, Italy Bolivia (P. Desjeu x)
Cutaneous leishmaniases The difficulty Similar clinical spectrum to leishmaniasis
-leprosy -skin cancers -mycobacteriosis -cutaneous mycoses -sarcoidosis -insect bite granuloma … etc
common in leishmaniasis-endemic areas
18 Pierre Marty Cutaneous leishmaniasis
Cutaneous Leishmaniases
Leishmania infantum Nice area, France
Cutaneous Leishmaniases
Leishmania major Biskra area, Algeria
Cutaneous Leishmaniases
Leishmania aethiopica Ethiopia
19 Pierre Marty Cutaneous leishmaniasis
Cutaneous Leishmaniases
Leishmania mexicana Mexico
Cutaneous Leishmaniases
Leishmania infantum Nice area France
Cutaneous Leishmaniases
Leishmania killicki Tunisia
20 Pierre Marty Cutaneous leishmaniasis
Cutaneous Leishmaniases
Leishmania infantum Imperia Italy
Cutaneous Leishmaniases
Leishmania infantum Antibes area France
Cutaneous Leishmaniases
Leishmania infantum Imperia Italy
21 Pierre Marty Cutaneous leishmaniasis
Cutaneous Leishmaniases
Leishmania donovani PKDL
India (WHO)
Classical diagnosis methods of Cutaneous Leishmaniasis
Skin biopsy or Skin aspiration Microscopic examination
-Histopathological study (fixed lesion biopsy)
-May Grünwald Giemsa smears stained (not fixed biopsy or aspiration) Culture on Nicolle-Novy-McNeal or Schneider medium (biopsy and aspiration) zymodeme identification
Cutaneous biopsy: numerous amastigote forms MGG x 400
22 Pierre Marty Cutaneous leishmaniasis
Cutaneous biopsy: numerous amastigote forms MGG x 1000
Cutaneous biopsy: numerous promastigote forms culture
Modern diagnosis methods of Cutaneous Leishmaniasis
Molecular diagnosis PCR on skin biopsy or aspiration fresh paraffin-embedded more sensitive more rapid identification but expensive Serodiagnosis western blotting rapid tests
23 Pierre Marty Cutaneous leishmaniasis
Molecular diagnosis of Cutaneous Leishmaniasis
-PCR based methods
-Particularly useful in cases with low parasite load
-Specificity 100%
-Sensitivity improved by 20-70% compared with conventional parasitological diagnosis (time consuming, clinical expertise)
Reithinger and Dujardin, J. Clin. Microbiol., 2007, 45:21-25
Western blot in cutaneous leishmaniasis WB profiles of some patients in different groups:
A: with active lesion(s)
34 kDa B: with cured lesion(s) 31 kDa
23 kDa C: without history of 21 kDa leishmaniosis 18 kDa 14 kDa T: visceral leishmaniasis (L. AAAAAA BBBBBBBBCCT infantum) Le Fichoux et al, Poster, WordLeish 2, 2001, Hersonissos, Greece
Serological rapid diagnosis tests in cutaneous leishmaniasis
Marty et al, Med Trop, 2007, 67:79-85
24 Pierre Marty Cutaneous leishmaniasis
DiaMed-IT LEISH
Dipstick using K39 recombinant antigen, an individual test with all required accessories packed in an aluminium foil and which could be defined as a bedside test
ID-PaGIA Leishmaniasis
ID-PaGIA Leishmaniasis
antibody test
A Particle Gel ImmunoAssay based on an agglutination principle using particles coated with K39rAg. Card with 6 individual microtubes containing gel, useful to test large series
Global Results
Positive Negative Positive Negative IT-Leish IT-Leish ID-PaGIA ID-PaGIA
VL HIV- 98% 2% 98% 2% n = 46 VL HIV+ 53% 47% 70% 30% Patent n = 30 CL Leishmaniasis 49% 51% 56% 44% n = 39
Asymptomatic 5% 95% 11% 89% Contacts n = 87 Healthy controls n = 100 0% 100% 1% 99% Disease Without
25 Pierre Marty Cutaneous leishmaniasis
Serological rapid diagnosis tests in cutaneous leishmaniasis (CL)
- 50% of the sera of CL were positive in both tests
For the diagnosis of CL, we underline the usefulness of the predictive value of a negative result
Conclusion
Classical diagnosis: sensitivity related to microscopic experience but time-consuming PCR diagnosis: great threshold sensitivity, necessity of a specialized lab but expensive Rapid serological tests: great negative predictive value, easy to perform, cheap but moderate sensitivity
26 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
How far have multiplex-PCR and quality assessment of PCR come ?
H. Pelloux, Parasitology-Mycology Teaching hospital, Grenoble, France and UMR 5163 UJF-CNRS. On behalf of the ESGCP
Introduction -Large topic -Major improvements in the diagnosis of infectious diseases, particularly Parasitology, in the last decades -Quality assessment is based on both quality controls (QC) and standard quality management procedures (ISO… ) -Recent developments of PCR (qualitative, quantitative, multiplex… ) have modified the diagnosis of many parasitic diseases
Introduction
-QC for PCR are added to QC for other techniques used for the diagnosis of parasitic diseases which still remain useful -Necessary in human medicine but also in veterinary medicine, study of the environment… -Multiplex PCRs are the most sophisticated PCRs, applied to various pathogens
27 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
Introduction -References in PubMed : « Quality control Parasitology » : > 130 refs since the 90’s (much less specifically on QC for PCR) « Multiplex PCR Parasitology » : > 100 refs since the 90’s -Impossible to analyse all of them -Focus mainly on one parasitic disease as an example : Toxoplasmosis
QC PCR Toxo -Only few papers in the literature focusing on QC for PCR for Toxoplasma
Guy et al, 1996 Pelloux et al, 1998 Costa et al, 2001 Bastien et al, 2007 Kaiser et al, 2007
These papers describe different QC that have been built for PCR Toxo
QC PCR Toxo Guy E.C. et al, Eur. J. Clin. Microbiol. Infect. Dis. 1996
-Five European centers -Amniotic fluid spiked with Toxo DNA -Both false negative and false positive results -Heterogeneity of techniques -Need for external assurance scheme
28 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
QC PCR Toxo E.C. Guy et al, Eur. J. Clin. Microbiol. Infect. Dis. (1996) 836-839
QC PCR Toxo E.C. Guy et al, Eur. J. Clin. Microbiol. Infect. Dis. (1996) 836-839
QC PCR Toxo Pelloux H. et al, FEMS Microbiol. Lett. 1998
-Collaborative European study -15 centers -12 aliquots of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma -Heterogeneous PCRs and results -Need for an external quality assurance scheme
29 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
QC PCR Toxo
QC PCR Toxo
QC PCR Toxo Costa J.M. et al, Bone Marrow Transplant. 2001
-4 European laboratories -Tachyzoites of the S3 strain of Toxoplasma diluted in EDTA blood -Diversity of the techniques -Diversity of the results -Need for quality controls with reference samples
30 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
QC PCR toxo Bone Marrow Transplantation, (2001) 28, 527-528
QC PCR toxo Bastien P. et al, Clin. Microbiol. Infect. 2007 -Analysis of the french QC for PCR Toxo 2002-2004 -QC still organised -23 laboratories in France -Amniotic fluid containing different concentrations of Toxoplasma, naturally infected -Results better than in the previous published studies -Heterogeneity of techniques -This external quality assurance scheme allows to improve laboratory practice and communication among labs
QC PCR Toxo Bastien et al, Clin. Microbiol. Infect. 2007, 13:430-433
31 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
QC PCR Toxo Bastien et al, Clin. Microbiol. Infect. 2007, 13:430-433
QC PCR Toxo Kaiser K. et al, Clinica Chimica Acta, 2007 -Proficiency panel designed to assess the performance of techniques for the detection of Toxoplasma in amniotic fluid -Lyophilised samples of dilution of Toxoplasma in amniotic fluids (RH strain) -33 labs in 17 countries -Both false positive and false negative results -Need for improvement of molecular detection of Toxoplasma
QC PCR Toxo K. Kaiser et al, Clinica Chimica Acta 375 (2007) 99-103
32 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
QC PCR Toxo K. Kaiser et al, Clinica Chimica Acta 375 (2007) 99-103
QC PCR Toxo Conclusion : -External quality control schemes now exist (national or international level) -Numerous issues concerning the QC : Toxo strain, sample, DNA or parasite, storage, number of aliquots… -Heterogeneity of PCRs (in house++) -Need for a strong validation of each of them
- These validation schemes are even more important when the techniques used are more sophisticated
- For instance : PCRs with internal control are in fact a kind of multiplex PCR…
33 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
Multiplex PCR Multiplex PCR - Most sophisticated PCR (qualitative or quantitative) - Appeared in the early 90’s - More than 100 papers for the keyword « multiplex PCR parasitology » in PubMed - Main application : simultaneous detection of different strains or species or genders in one sample - But not only…
Multiplex PCR
- Exemples of co-detections that have been described - Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum - 4 species of Plasmodium - Neospora caninum, Toxoplasma gondii - Entamoeba histolytica, Entamoeba dispar
Multiplex PCR
- Chloroquine resistant or not strains of Plasmodium falciparum - Strains or species of leishmania - Enterocytozoon bieneusi, Encephalitozoon spp. -Viruses, Toxoplasma gondii - … and others A large number of parasites concerned by multiplex PCR
34 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
Multiplex PCR One example : Toxoplasma gondii Usefulness of multiplex PCR for T. gondii (chronological) - Co-detection with EBV in CSF of AIDS patients - Recognition of T. gondii stage-specific genes - Co-detection with viruses in posterior uveitis - Differentiate Neospora caninum and T. gondii in dog - Typing strains of T. gondii (congenital, immunocompromised, environnement) - Co-detection with viruses in congenital infections and bacteria, viruses in granulomatous lymphadenitis
Multiplex PCR Dabil et al, 2001 Validation of a diagnostic multiplex PCR assay for infectious posterior uveitis
→ To valide multiplex PCR on vitreous biopsy in posterior uveitis
→ CMV, HSV, VZV, Toxoplasma gondii → Comparison multiplex/monoplex on 21vitreous specimen
Multiplex PCR Results : - Monoplex : detection of fewer than 10 genomes of VZV and 100 genomes of HSV, CMV, T. gondii - Multiplex : loss of sensitivity (< 1 log, monoplex positive 18/21 samples, multiplex : 15/18) - No false positive Conclusion : - Multiplex avoids serial testing (expensive, time consuming, limited volume of sample) - But : declining sensitivity and complexity of annealing reactions
35 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
Multiplex PCR Ajzenberg D. et al, 2005 Multiplex PCR for typing strains of Toxoplasma gondii - Designed for multilocus strain typing of T. gondii. Length polymorphism of 5 microsatelllites markers - Height T. gondii strain previously sequenced at five MS markers were used as controls - Multiplex PCR easy to use (1 PCR needed to perform multilocus typing with five markers), rapid (1 day), adapted to large series
Multiplex PCR Conclusion : Multiplex PCR in Parasitology - Often « in house » - Not commercialised - Should be carefully validated (see QC… ) - Saves time but in each all the targets are used even if not medically justified (« screening ») - Future ?
Multiplex PCR
Perspectives : - In the field of Mycology and Bacteriology : one multiplex PCR commercialised for severe septis - Cost/effectiveness under evaluation
36 Herve Pelloux How far have multiplex-PCR and quality assessment of PCR come?
Conclusion :
- QC and multiplex PCR are major improvements in the field of Parasitology in the last decade - QCs are now available - Multiplex PCRs are emerging. cost/effectiveness is a major issue
37 38 Laetitia Kortbeek Intestinal parasites
National Institute for Public Health and the Environment
Rapid diagnosis of parasitic infections Intestinal parasites Titia Kortbeek
Centre for Infectious Disease Control Netherlands , Bilthoven
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Rapid diagnosis of parasitic infections
Rapid:
• Is it necessary to have a quick answer?
- Depending on request of doctor or patient
For intestinal parasites: duration of symptoms usually long;
(The patient is not sitting in the waitingroom )
• Efficiency laboratory:
- Staining Methods are time consuming
- Training technicians
- Motivation (most samples are negative)
- Level knowledge sender
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Intestinal parasites
• Which techniques are available - Preference microbiologist - Available equipment
• Which parasites do you want to detect: - All - Only Giardia and Crypto
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•Hospital setting or general practitioner - Academic -periferal hospital
•Patients: YOPI - Very Young, very Old, Pregnant and Immunocompromised
•Other available information: - Season - Duration of symptoms
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Intestinal parasites
• Valid request from clinicians?
• Algoritm for diagnostic request in gastro-enteritis patients of general practitioners
• Sometimes specific information important - E.g. contact with animals
• Clinical symptoms (fever, blood with stool, mucus) cannot be used to exclude an diagnosis or select patients
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40 Laetitia Kortbeek Intestinal parasites
Duration of Diagnostic Age Season symptoms request
1-3 days Rota (52%); NLV (13%) 0-4 yr Dec-May 4-7 days Rota (22%); Adeno (22%); NLV, SLV, Crypto (7%) >7 days NLV (18%); Rota, Adeno (7%)
1-3 days Rota (27%) ; NLV (13%); Camp., Adeno (7%) June-Nov 4-7 days Salm (17%);Camp.(13%); Rota,Adeno, Crypto (4 %) >7 days Giardia (9 %); Crypto (6%), Adeno (6%)
1-3 days Camp.(16%); NLV (14%) ;Rota (7%); Astro (6%); Salm.(4%); 4-7 days >5 yr Dec-May Camp.(18%);Rota, Crypto (5 %) ; Astro , Salm. (4%) >7 days Camp., Giardia (4%)
1-3 days Camp. (21%); Salm. (7%) 4-7 days June-Nov Camp. (20%); Giardia (5 %) >7 days Giardia (10 %); Camp. (6%),
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Intestinal parasites
Available rapid tests for intestinal parasites:
Methods: • ELISA antigen detection • Dipstick • Cassette • Direct fluorescence antibody assay
Parasites: protozoa: • Cryptosporidium • Giardia • E. histolytica/dispar
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41 Laetitia Kortbeek Intestinal parasites
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www.indmedica.com/.../003_001_crypt_figd_sm.jpg
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Intestinal parasites Important criteria: • Reliability • Sensibility and specificity • Easy to use • Costs • Hands on time • Literature comparing results in same setting • NVP and PPV are depending of prevalence
What is the golden standard?
• Microscopy or PCR? • Multiple sampling?
TFT: van Gool & Mank
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42 Laetitia Kortbeek Intestinal parasites
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Giardia
Data Dr. Theo Mank, Haarlem , the Netherlands Setting: diarrhea patients general practitioners the Netherlands
Comparing - Rida Quick - ImmunocardStat, - X/Pect - Triage
•Sensitivity : >93->95 % •Specificity : >98 %
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43 Laetitia Kortbeek Intestinal parasites
Garcia et al 1996 : evaluation 9 commercial kits Setting: human feces in 10% formaline
•DFA Techlab • Giardia/Crypto IF; Crypto IF •DFA Meridian Merifluor Crypto, •EIA Alexon ProspecT • Giardia; Crypto •EIA Meridian • Premier Giardia; Premier Crypto •Techlab CELISA •EIA Trend Giardia •EIA Cambridge Giardia
Reference test: DFA Meridian Merifluor Crypto/Giardia
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Conclusions:
DFA Giardia and DFA Crypto: •Sensitivity 100 % •Specificity 100 %
EIA Giardia •Sensitivity 94-99 % •Specificity 96-100 %
EIACrypto •Sensitivity 94-99 % •Specificity 96-100 %
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Oster et all ; Eur J Clin Microbiol Infect Dis (2006) 25: 112–115
Evaluation of the immunochromatographic CORIS Giardia- Strip test Compared to a commercial ELISA-coproantigen test (ProSpect Giardia- ELISA-microplate assay; Remel, Lenexa, KS, USA)
CORIS Giardia-Strip test
• Sensitivity 58% • Specificity 99 %
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44 Laetitia Kortbeek Intestinal parasites
Schuurman ea. 2007
Comparing microscopy, real time PCR and ImmunocardStat! • Setting: gastroenteritis patients Groningen (Northern Netherlands)
Microscopy TFT: SAF preserved • iodinestained,wet-mount preparation: suspect: chlorazol-black stain Unpreserved: • Ridleyconcentration : iodinestained,wet-mount preparation
microscopy, Sensitivity 99% Specificity 97% real-time PCR Sensitivity 100%, Specificity 92% ImmunocardStat! Sensitivity 98% Specificity 100% Nationa l Instit ute for Pub lic Health 19 and t he Env ironment
T. Weizel et al 2006 Clin Microbiol Infect 2006; 12: 656–659 • Setting: outpatient clinic of the Institute of Tropical Medicine; diarrhoea and travelling
Sensitivity Giardia: Ridascreen Giardia 82%, Rida Quick Giardia 80% Rida Quick Combi 80% Giardia-Strip 44%
Sensitivity Cryptosporidium: Rida Quick Cryptosporidium 88% Ridascreen Cryptosporidium 82% Rida Quick Combi 82% Cryptosporidium-Strip 75%
The specificity of all tests was ‡ 98%.
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Weitzel continued
• No cross-reactions with other intestinal parasites • 68 (of 220 samples): other intestinal parasites
Conclusion: - the copro-antigen assays : less time-consuming - easier to perform - but were less sensitive
• “Thus, these tests might be a useful addition to, but not a substitute for microscopical methods in the diagnosis of travel-associated giardiasis and cryptosporidiosis.”
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45 Laetitia Kortbeek Intestinal parasites
Cryptosporidium
Magi Par. Res. 2005 Diagnostics Cryptosporidium: PCR, Kinyoun acid-fast stain, ImmunoCard STAT! Setting: 127 diarrhoea immunocompetent patients in hospital Italy
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Significance of positive antigen test- negative microscopy and/or PCR? • Recent recovery • False positive
Conclusion:
..for immunocompetent persons (and hence with few oocysts, if positive), microscopy test with Kinyoun stain currently seems to be the best approach in the hands of trained microscopists examining a large number of microscopic fields. Besides, this method is cheap even when used for a small numberof samples.
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Manufacturer's Recall of Rapid Cartridge Assay Kits on the Basis of False-Positive Cryptosporidium Antigen Tests --- Colorado, 2004 Recall of ImmunoCardSTAT • MMWR Morb Mortal Wkly Rep. 2004 Mar 12;53(9):198. Centers for Disease Control and Prevention (CDC).
• The Colorado Department of Public Health and Environment (CDPHE) has determined that a fourfold increase in the number of reported cryptosporidiosis cases in Colorado during January--February 2004 might be attributed primarily to false-positive test results. Since January 1, 2004, a total of 13 in-state cases and one out-of-state case were reported to CDPHE. During the previous 7 years, an average of three cases were reported during January--February. In eight of 14 patients, rapid testing was performed by using the ImmunoCard STAT!® Cryptosporidium/Giardia Rapid Assay (Meridian Bioscience, Inc., Cincinnati, Ohio). This assay is a solid-phase qualitative immunochromatographic assay designated to detect and distinguish between Giardia intestinalis (lamblia) and Cryptosporidium parvum in aqueous extracts of human fecal specimens. Seven of these samples were tested by using lot no. 081093 (expires August 11, 2004). Of the seven samples that tested positive initially for Cryptosporidium with this lot number, four were retested by using other, more specific tests. One patient sample was positive by direct microscopy, one was negative by direct microscopy, and two were negative by direct fluorescent-antibody testing, suggesting that results for three of the four samples were false positive. The results of testing for Giardia intestinalis (lamblia) with these kits are unclear. Several other states have noted increases in the number of reported cryptosporidiosis cases that also might be associated with use of these rapid assays.
• Meridian Bioscience, Inc., has voluntarily recalled two lots (lot no. 081077 [expires July 11, 2004] and lot no. 081093) from laboratories. CDC recommends reconfirmation of positive test results obtained with ImmunoCard STAT!® rapid assays from these lots.
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46 Laetitia Kortbeek Intestinal parasites
Regnath et al; Eur J Clin Microbiol Infect Dis (2006) 25:807–809
Setting: diagnostic laboratory in Germany; 738 stoolsamples Microscopy: • Concentration and iodine- wetmounted preparation • Dried unstained slide: Crypto • EIA Ridascreen Giardia and Ridascreen Cryptosporidium
Pre-screened positive specimens • RIDAQuick Giardia, • RIDAQuick Cryptosporidium • RIDAQuick Cryptosporidium/Giardia Combi (R-Biopharm)
Conclusion: easy to use; rapid to perform ; sensitive and specific
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© Meddia, Amsterdam
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Visser et all; Int. Journal of Medical Microbiology 296 (2006) 397–403
Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: Performance and clinical implications in a non-endemic setting
Unpreserved faecal samples; Microscopic examination : suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites
Reference test: real-time PCR.
416 patients: results real time PCR:
In 283 patients (68%) DNA of E. histolytica or E. dispar positive 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%) 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples (controls)
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47 Laetitia Kortbeek Intestinal parasites
Visser continued Entamoeba test Techlab: monoclonal ab against E.histolytica and E. dispar E. histolytica II Techlab : only monoclonal ab against E.histolytica
E. histolytica/E. dispar carrier
Entamoeba test Techlab sensitivity 59% specificity 98%
E. histolytica II Techlab sensitivity 71% specificity 100%
Serology : sensitivity : 83.3% and specificity 95.2%. Serology non endemic country: sensitivity 90% and specificity 98.8%.
In comparison to real-time PCR the performances of Entamoeba testTM and E. histolytica IITM lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting.
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E. histolytica/dispar in the Netherlands
• In GE study GP patients (1996-1998): - 9 /857 cases and 4 /574 controls De Wit et all Am. J. Epidemiol. 2001 154: 666-674.
• In population based study (1998): - 1 /703 cases and 0 /673 controls
De Wit et all Clin Infect Dis 2001; 33 (3) 280-88
• Recent study in Groningen: 930 cases GP:
• no cases of Entamoeba (R. ten Hove et all (2007) Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR Clinical Microbiology and Infection 13 (10) , 1001–1007)
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Is it necessary to type Entamoeba histolytica/dispar?
WHO guidelines World Health Organization (WHO). Amoebiasis. WklyEpidemiol Rec 1997;/72:/97-9.
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Conclusions
• Most rapid tests are easy to perform and fast • Sensitivity and specificity overall is OK - Be aware that sometimes batches can go wrong
• Applicability depends on setting of the lab and type of patients
• E.histolytica /E.dispar in non endemic patients: send these rare positive samples to specialized centers
• Importance of other parasites - Travel related - Dientamoeba
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Thank you !!
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