Many different intestinal parasite species can co-occur in the same population. However, classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite species in a given population. The present study investigated intestinal parasitic infections in Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of five classical microscopy techniques and real-time PCR for the detection of a broad spectrum of parasites was compared. A cross-sectional population-based survey was performed. One stool sample per participant (n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, coproculture and real-time PCR. We found that virtually all people (96%) harbored at least one helminth, and that almost half (49%) harbored three helminths or more. Remarkably, Strongyloides stercoralis infections were widespread with a prevalence of 48%, and Ancylostoma spp. prevalence was higher than that of Necator americanus (25% versus 15%), the hookworm species that is often assumed to prevail in East-Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum of parasite species. However, FEC also missed a considerable number of infections, notably S. stercoralis, Schistosoma mansoni and G. intestinalis. PCR outperformed microscopy in terms of sensitivity and range of parasite species detected. We showed intestinal parasites—especially helminths—to be omnipresent in Inhamudima, Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classical techniques such as FEC are useful for the detection of some intestinal helminth species, but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more accurately but is generally not feasible in resource-poor settings, at least not in peripheral labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot diagnosis of parasitic infections. In populations living in adverse conditions due to poverty, a wide variety of intestinal parasite infections can be observed. These infections are usually diagnosed by stool microscopy, but can be easily missed if the procedures used are inaccurate or performed in a suboptimal way. In the present study, we investigated the prevalence of intestinal parasite infections in an informal settlement in Beira, Mozambique. We also compared the diagnostic performance of five different microscopic techniques and real-time PCR for the detection of DNA of five helminth and five protozoa species. For this purpose, a single stool sample was collected from members of 63 households, evenly distributed within the settlement. Parasitic infections were found to be highly abundant in the 303 examined samples. Virtually all individuals were found to be infected with at least one helminth species and a majority of the stools showed multiple parasites. None of the classical microscopic techniques was suitable to detect all different pathogenic species. Hence, the outcome of several microscopy procedures had to be combined to get a complete picture. We found PCR to be the most accurate diagnostic approach, even in this hyper-endemic setting. However, DNA detection is a high-tech technology, generally not applicable in resource-poor settings.

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. In the developing world, over 207 million people are infected with parasitic Schistosoma worms. Schistosoma mansoni is one of the most widespread species, and its routine diagnosis is based on microscopic detection of parasite eggs in stool samples. This technique is, however, highly observer-dependent and has suboptimal sensitivity. We compared the performance of stool microscopy with the highly specific real-time polymerase chain reaction (PCR) we recently described for the detection and quantification of parasite–specific DNA. We tested stool samples collected at two different epidemiological settings: a Senegalese population (n = 197) from a high transmission area where S. mansoni and S. haematobium are co-endemic and a Kenyan school population (n = 760) selected from zones with comparatively low S. mansoni transmission. Microscopy mostly missed low intensity infections that PCR was able to detect. Consequently, the PCR may be very useful for the detection of S. mansoni in areas with low levels of infection. Furthermore, being a highly standardized diagnostic procedure, the PCR may improve schistosomiasis control programs, epidemiological research, and quality control of microscopy. Also it can be easily combined with other PCRs to detect a wider range of helminth infections in a single stool sample.

SUMMARY: For the majority of intestinal parasites, real-time PCR-based diagnosis outperforms microscopy. However, the data for Trichuris trichiura have been less convincing and most comparative studies have been performed in populations with low prevalence. This study aims to improve detection of T. trichuria DNA in human stool by evaluating four sample preparation methods. Faecal samples (n = 60) were collected at Flores island, Indonesia and examined by microscopy. Aliquots were taken and a bead-beating procedure was used both on directly frozen stool and on material preserved with 96% ethanol. PCR on frozen samples showed 40% to be positive for T. trichiura, compared with 45% positive by microscopy. The percentage positive increased when using ethanol preservation (45·0%), bead-beating (51·7%) and a combination (55·0%) and all three methods showed significantly higher DNA loads. The various procedures had a less pronounced effect on the PCR results of nine other parasite targets tested. Most prevalent were Ascaris lumbricoides (≈60%), Necator americanus (≈60%), Dientamoeba fragilis (≈50%) and Giardia lamblia (≈12%). To validate the practicality of the procedure, bead-beating was applied in a population-based survey testing 910 stool samples. Findings confirmed bead-beating before DNA extraction to be a highly efficient procedure for the detection of T. trichiura DNA in stool.

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. Schistosoma haematobium is a blood fluke that causes severe urogenital pathology and affects millions of people, mainly in sub-Sahara Africa. Current diagnosis is based on microscopic examination of urine samples, but this method is not only observer dependent, but also known for its low sensitivity and high day-to-day variability. Accurate diagnosis is important to assess community levels of infections for consideration of deworming campaigns, and to monitor treatment efficacy. We evaluated a real-time polymerase chain reaction (PCR) assay for specific detection and quantification of Schistosoma DNA in urine samples from 114 preselected S. haematobium infected schoolchildren of endemic coastal Kenya and compared the outcome to several other diagnostic methods. Three urine samples collected over three subsequent days from 24 participants were used for Analyzing day-to-day fluctuations in egg counts and Schistosoma DNA levels. Urine was also tested two and 18 months after praziquantel treatment. Compared to microscopy, we observed less day-to-day fluctuations and higher sensitivity with real-time PCR, in particular when tested two months after therapy. Real-time PCR is therefore useful for more accurate identification of S. haematobium, especially in monitoring control interventions.