[Show abstract][Hide abstract] ABSTRACT: The current reference test for the detection of S. mansoni in endemic areas is stool microscopy based on one or more Kato-Katz stool smears. However, stool microscopy has several shortcomings that greatly affect the efficacy of current schistosomiasis control programs. A highly specific multiplex real-time polymerase chain reaction (PCR) targeting the Schistosoma internal transcriber-spacer-2 sequence (ITS2) was developed by our group a few years ago, but so far this PCR has been applied mostly on urine samples. Here, we performed more in-depth evaluation of the ITS2 PCR as an alternative method to standard microscopy for the detection and quantification of Schistosoma spp. in stool samples.
Microscopy and PCR were performed in a Senegalese community (n = 197) in an area with high S. mansoni transmission and co-occurrence of S. haematobium, and in Kenyan schoolchildren (n = 760) from an area with comparatively low S. mansoni transmission. Despite the differences in Schistosoma endemicity the PCR performed very similarly in both areas; 13-15% more infections were detected by PCR when comparing to microscopy of a single stool sample. Even when 2-3 stool samples were used for microscopy, PCR on one stool sample detected more infections, especially in people with light-intensity infections and in children from low-risk schools. The low prevalence of soil-transmitted helminthiasis in both populations was confirmed by an additional multiplex PCR.
The ITS2-based PCR was more sensitive than standard microscopy in detecting Schistosoma spp. This would be particularly useful for S. mansoni detection in low transmission areas, and post-control settings, and as such improve schistosomiasis control programs, epidemiological research, and quality control of microscopy. Moreover, it can be complemented with other (multiplex real-time) PCRs to detect a wider range of helminths and thus enhance effectiveness of current integrated control and elimination strategies for neglected tropical diseases.
[Show abstract][Hide abstract] ABSTRACT: In an effort to enhance accuracy of diagnosis of Schistosoma haematobium, this study explores day-to-day variability and diagnostic performance of real-time PCR for detection and quantification of Schistosoma DNA compared to other diagnostic tools in an endemic area before and after treatment.
Previously collected urine samples (N = 390) from 114 preselected proven parasitological and/or clinical S. haematobium positive Kenyan schoolchildren were analyzed by a Schistosoma internal transcribed spacer-based real-time PCR after 14 years of storage. Pre-treatment day-to-day fluctuations of PCR and microscopy over three consecutive days were measured for 24 children using intra-class correlation coefficient. A combined 'gold standard' (PCR and/or microscopy positive) was used to measure sensitivity and negative predictive value (NPV) of several diagnostic tools at baseline, two and 18 months post-treatment with praziquantel.
All 24 repeatedly tested children were PCR-positive over three days with little daily variation in median Ct-values, while 83.3% were found to be egg-positive for S. haematobium at day 1 and 75.0% at day 2 and 3 pre-treatment, signifying daily fluctuations in microscopy diagnosis. Of all 114 preselected schoolchildren, repeated microscopic measurements were required to detect 96.5% versus 100% of positive pre-treatment cases by single PCR. At two months post-treatment, microscopy and PCR detected 22.8% versus 69.3% positive children, respectively. Based on the 'gold standard', PCR showed high sensitivity (>92%) as compared to >31% sensitivity for microscopy, both pre- and post-treatment.
Detection and quantification of Schistosoma DNA in urine by real-time PCR was shown to be a powerful and specific diagnostic tool for detection of S. haematobium infections, with less day-to-day variation and higher sensitivity compared to microscopy. The superior performance of PCR before, and two and 18 months post-treatment provides a compelling argument for PCR as an accurate and reproducible tool for monitoring treatment efficacy.
[Show abstract][Hide abstract] ABSTRACT: Schistosoma haematobium eggs and Schistosoma DNA levels were measured in urine samples from 708 girls recruited from 18 randomly sampled primary schools in South Africa. Microscopic analysis of two 10-mL urine subsamples collected on three consecutive days confirmed high day-to-day variation; 103 (14.5%) girls had positive results at all six examinations, and at least one positive sample was seen in 225 (31.8%) girls. Schistosoma-specific DNA, which was measured in a 200-μL urine subsample by using real-time polymerase chain reaction, was detected in 180 (25.4%) cases, and levels of DNA corresponded significantly with average urine egg excretion. In concordance with microscopic results, polymerase chain reaction results were significantly associated with history of gynecologic symptoms and confirmed highly focal distribution of urogenital schistosomiasis. Parasite-specific DNA detection has a sensitivity comparable to single urine microscopy and could be used as a standardized high-throughput procedure to assess distribution of urogenital schistosomiasis in relatively large study populations by using small sample volumes.
The American journal of tropical medicine and hygiene 01/2014; 90(3). DOI:10.4269/ajtmh.13-0406 · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report a case of symptomatic visceral Armillifer pentastomiasis in a 23-year-old female Liberian immigrant to The Netherlands. The patient was referred to the gynecologist because of lower abdominal pain. During laparotomy, multiple adhesions were seen in the lower pelvis and a hydrosalpinx with an encapsulated Armillifer nymph, most likely Armillifer armillatus, was found. Key features of the parasite's cuticle which facilitate the diagnosis of pentastomiasis, are presented. Symptomatic pentastomiasis is uncommon, and most cases are diagnosed incidentally during surgery for other reasons, or at autopsy. With regard to increasing international migration, other imported pentastomiasis cases to Europe and North America are reviewed, and more cases are likely to be seen in the future.
Travel Medicine and Infectious Disease 10/2013; 12(2). DOI:10.1016/j.tmaid.2013.10.011 · 1.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi.
A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16-5.33) and 9.04 (95%CI 2.52-32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18-11.20); 16.98 (95%CI 3.88-74.35) and 44.91 (95%CI 5.23-385.77) for low, moderate and high load respectively).
This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.
[Show abstract][Hide abstract] ABSTRACT: Subcutaneous (upper leg) sparganosis case in the Netherlands (2008) from female patient, born in 1973 in China, lives in the Netherlands since 1991, singular nodule
slides of presentation at AMC in 2009 (Dutch)
[Show abstract][Hide abstract] ABSTRACT: A real-time PCR method targeting the small subunit of the rRNA gene was developed for the detection of Strongyloides stercoralis DNA in faecal samples, including an internal control to detect inhibition of the amplification process. The assay was performed on a range of well-defined control samples (n=145), known positive faecal samples (n=38) and faecal samples from a region in northern Ghana where S. stercoralis infections are highly endemic (n=212), and achieved 100% specificity and high sensitivity. The use of this assay could facilitate monitoring the prevalence and intensity of S. stercoralis infections during helminth intervention programs. Moreover, the use of this assay in diagnostic laboratories could make the introduction of molecular diagnostics feasible in the routine diagnosis of S. stercoralis infections, with a two-fold increase in the detection rate as compared with the commonly used Baermann sedimentation method.
Transactions of the Royal Society of Tropical Medicine and Hygiene 03/2009; 103(4):342-6. DOI:10.1016/j.trstmh.2008.12.001 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 23-year-old woman was working in a factory unpacking jars of Spanish olives when a spider bit her finger. She presented to the emergency outpatient department carrying the spider in a box. She was worried and wanted to know whether the spider, which may have originated from Spain, was poisonous. After a ten minute Internet search the spider was identified as Segestria florentina. It can inject neurotoxins causing temporary local numbness and pain. The patient was reassured and discharged without further treatment. Later, experts confirmed the species. Internet-based search engines can draw attention to unusual diagnoses which otherwise may easily be overlooked. A general search engine can also provide access to subjects that fall outside the scope of the more usual medical sources.
Nederlands tijdschrift voor geneeskunde 01/2009; 153:A116.
[Show abstract][Hide abstract] ABSTRACT: A real-time polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 2 region of the ribosomal RNA gene was developed for the detection of Isospora belli DNA in fecal samples, including an internal control to detect inhibition during the amplification process. The assay was performed on species-specific DNA controls (n = 27) and a range of positive (n = 21) and negative (n = 120) stool samples, and achieved 100% specificity and sensitivity. The simple fecal sample collection procedure, the high-throughput potential, and the possibility of quantification makes the I. belli real-time PCR assay a powerful diagnostic tool for epidemiologic studies with possibilities for extension to other helminthes and protozoa using additional molecular targets. In addition, this Isospora PCR could augment the clinical laboratory diagnosis of isosporiasis, in particular, in patients with a travel history to developing countries.
[Show abstract][Hide abstract] ABSTRACT: A multiplex real-time PCR was developed and evaluated for the simultaneous detection of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples. Using well-defined control samples (N = 150), known positive fecal samples (N = 50), and fecal samples from an area in Ghana where human infections with all 3 nematode species are endemic (N = 339), the method proved to be highly specific and sensitive. Cycle threshold (Ct) values, reflecting parasite-specific DNA load, showed significant correlation with the intensity of infection as measured by microscopy using Kato-Katz fecal smears or by species specific third-stage larval count after coproculture. The multiplex real-time PCR described combined with the simple fecal sample collection procedure and the potential for high throughput makes this approach a powerful diagnostic tool to study species-specific transmission patterns of human hookworm-like infections. Moreover, this procedure facilitates monitoring of intervention programs and allows species-specific detection of treatment failure following rounds of mass treatment.
The American journal of tropical medicine and hygiene 11/2007; 77(4):685-90. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A real-time polymerase chain reaction (PCR) method targeting the 5.8S ribosomal RNA gene was developed for the detection of Dientamoeba fragilis in stool samples. The PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n=29) and a range of stool samples (n=85), and achieved high specificity and sensitivity. D. fragilis DNA could be detected in unpreserved fecal samples up to 8 weeks after storage at 4 degrees C. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique for the routine diagnosis of intestinal D. fragilis infections.
[Show abstract][Hide abstract] ABSTRACT: A multiplex real-time polymerase chain reaction (PCR) method was developed for the simultaneous detection of Enterocytozoon bieneusi (n = 30) and Encephalitozoon spp. (n = 3) in stool samples. The multiplex PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n = 22) and a range of well-defined stool samples (n = 140), and it achieved 100% specificity and sensitivity. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique in the routine diagnosis of intestinal microsporidian infections.
[Show abstract][Hide abstract] ABSTRACT: In northern Togo and Ghana, human infection with the parasitic nematode Oesophagostomum bifurcum is of major health importance. Elsewhere, oesophagostomiasis is considered a zoonotic infection, non-human primates being the natural host. We examined 349 faecal samples of the olive baboon, mona monkey and black and white colobus monkey from two geographically distinct areas in Ghana, outside the region endemic for O. bifurcum in humans. Using both microscopy and species-specific PCR, we found a high prevalence of O. bifurcum (75-99%) in olive baboons and mona monkeys. The majority of the test-positive faecal samples contained large numbers of larvae after copro-culture (>100). No O. bifurcum was detected in the faeces of the black and white colobus monkeys. Observational studies on the behaviour of the non-human primates, focusing on defecation, food consumption and the sharing of habitat with the local human population, indicated favourable conditions for zoonotic transmission. Given that no human infection with O. bifurcum has been reported from either study area, the present findings support the hypothesis that O. bifurcum from humans in the north of Ghana, and O. bifurcum from olive baboons and/or mona monkeys are distinct.
Tropical Medicine & International Health 12/2005; 10(12):1315-20. DOI:10.1111/j.1365-3156.2005.01527.x · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa.
Tropical Medicine & International Health 07/2005; 10(6):574-80. DOI:10.1111/j.1365-3156.2005.01440.x · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In November-December 2002, stool samples from a random sample of the human population (N = 190) in the Garu area of northern Ghana were checked for intestinal helminths, using a single Kato smear and duplicate coprocultures for each subject. All 190 subjects were subsequently treated with a single, 400-mg dose of albendazole and 146 of them were successfully re-examined 21-28 days post-treatment. Prior to treatment, 75.5% of the Kato smears were found to contain 'hookworm-like' eggs (with a geometric mean egg count among the positives of 578 eggs/g faeces), and the third-stage larvae of Oesophagostomum bifurcum and hookworm were found in the cultures of stools from 34.2% and 77.4% of the subjects, respectively. Among the subjects who had positive Kato smears before treatment, albendazole treatment led to a cure 'rate' of 79.0% and an egg-reduction 'rate' of 73.5%. The results from the coprocultures indicated cure 'rates' of 98.0% for O. bifurcum but only 51.3% for hookworm. Only one subject was still positive for O. bifurcum after treatment. Among those still positive for hookworm after treatment, the larva-reduction 'rate' was 79.8%. The egg-/larva-reduction 'rates' among those with heavy infections prior to treatment were >90%, whether the data analysed came from the Kato smears or the coprocultures. It may be concluded that a single dose of albendazole is very likely to cure an O. bifurcum infection and to reduce greatly the intensity (but not the prevalence) of any hookworm infections.
Pathogens and Global Health 06/2004; 98(4):385-90. DOI:10.1179/000349804225003370 · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.
[Show abstract][Hide abstract] ABSTRACT: Since the redescription of the potentially invasive Entamoeba histolytica, separating it from the morphologically identical non-invasive Entamoeba dispar, there is a need for the reassessment of epidemiological data on amoebiasis. In this context we conducted a descriptive survey on the presence of E. histolytica and E. dispar in a rural area in northern Ghana. We found a high prevalence (39.8%) of the E. histolytica/E. dispar complex with microscopy, but E. histolytica and E. dispar-specific DNA amplification using real-time polymerase chain reaction identified only one E. histolytica case and revealed a considerably higher prevalence of E. dispar (82.8%).
Tropical Medicine & International Health 01/2004; 8(12):1153-6. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.