Gastrointestinal parasites in captive olive baboons in a UK safari park.

From the safety inside vehicles, Knowsley Safari offers visitors a close-up encounter with captive olive baboons. As exiting vehicles may be contaminated with baboon stool, a comprehensive coprological inspection was conducted to address public health concerns. Baboon stools were obtained from vehicles, and sleeping areas, inclusive of video analysis of baboon­vehicle interactions. A purposely selected 4-day sampling period enabled comparative inspections of 2662 vehicles, with a total of 669 baboon stools examined (371 from vehicles and 298 from sleeping areas). As informed by our pilot study, front-line diagnostic methods were: QUIK-CHEK rapid diagnostic test (RDT) (Giardia and Cryptosporidium), Kato­Katz coproscopy (Trichuris) and charcoal culture (Strongyloides). Some 13.9% of vehicles were contaminated with baboon stool. Prevalence of giardiasis was 37.4% while cryptosporidiosis was <0.01%, however, an absence of faecal cysts by quality control coproscopy, alongside lower than the expected levels of Giardia-specific DNA, judged RDT results as misleading, grossly overestimating prevalence. Prevalence of trichuriasis was 48.0% and strongyloidiasis was 13.7%, a first report of Strongyloides fuelleborni in UK. We advise regular blanket administration(s) of anthelminthics to the colony, exploring pour-on formulations, thereafter, smaller-scale indicator surveys would be adequate.

ON LIVER FLUKE (FASCIOLA HEPATICA) IN CAPTIVE VICUÑAS (VICUGNA VICUGNA) AT KNOWSLEY SAFARI, PRESCOT, UNITED KINGDOM.

Knowsley Safari (KS), Prescot, United Kingdom houses a variety of captive exotic ungulates. As part of their animal welfare plan, a prospective coprological survey was undertaken for liver fluke. In June 2021, 330 fecal samples, representative of 18 exotic ungulate species, were processed by sedimentation and filtration, with examination by coproscopy. Finding fascioliasis in all five vicuña alone, with fecal egg counts ranging from one to eight eggs per gram, anthelminthic treatment was attempted twice, with three coprological reviews. While the first anthelminthic treatment (oxyclozanide) was equivocal, the second anthelminthic treatment (triclabendazole) was proven effective upon two later follow-ups. An initial malacological survey of 16 freshwater sites in KS, first found Galba truncatula at two sites in June 2021, then upon more extensive searching subsequently within the vicuña's enclosure. It appears that F. hepatica was locally acquired, being the first report of fascioliasis within captive vicuñas in the United Kingdom. To develop a better fluke-management plan, regular coprological and malacological surveillance is justified, perhaps with molecular xenomonitoring of snails, alongside prompt administration of appropriate flukicide as required.

Detecting Schistosoma mansoni infections among pre-school-aged children in southern Ghana: a diagnostic comparison of urine-CCA, real-time PCR and Kato-Katz assays.

BACKGROUND: In Ghana, pre-school-aged children (PSAC) are at risk of intestinal schistosomiasis and are living in need of praziquantel treatment. To better assess the infection burden within this vulnerable demographic group, we have provided a comparative assessment of the prevalence of Schistosoma mansoni in pre-school-aged children by urine circulating cathodic antigen (CCA) dipsticks, real-time PCR Taqman® faecal assays and Kato-Katz coproscopy. METHODS: In all, 190 pre-school-aged children were sampled from three endemic communities (viz. Tomefa, Torgahkope/Adakope, and Manheam) around Weija dam, Southern Ghana. Fresh stool and urine samples were collected from all participants for diagnosis. RESULTS: Among all the three communities, the urine-CCA assay recorded the highest prevalence values of 90.5% (95% CI 80.4-96.4), 87.9% (95% CI 76.7-95), and 81.2% (95% CI 69.9-89.6) in Tomefa, Torgahkope/Adakope, and Manheam respectively. Prevalence by real-time PCR was 50% (95% CI 35.5-64.5), 8% (95% CI 2.2-19.2) and 16.7% (95% CI 8.3-28.5), while by Kato-Katz was 55.6% (95% CI 42.5-68.1), 8.6% (95% CI 2.9-19) and 11.6% (95% CI 5.1-21.6) respectively. Children aged 1 year and over were found to be positive with the urine-CCA assay; by the ages of 3-4, over 50% were urine-CCA patent. The sensitivity and specificity of the POC-CCA dipsticks, when compared against the combined results of Kato-Katz/TaqMan results was found to be 84.1% (95% CI = 72.7-92.1) and 12.9% (95% CI = 6.6-22) respectively. CONCLUSIONS: We propose that the urine-CCA dipstick may be a useful rapid diagnostic tool to estimate the prevalence of intestinal schistosomiasis in PSAC, particularly in rapid identification of at-risk areas. However, our assessment has shown that it possible to record false positives when compared to combined Kato-Katz and qPCR results. To guide PSAC praziquantel treatment needs, we propose the urine CCA assay should be included in routine surveillance of intestinal schistosomiasis alongside other diagnostics such as Kato-Katz and urine filtration.

Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species.

With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato-Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

Consistent detection of Trypanosoma brucei but not T. congolense DNA in faeces of experimentally infected cattle.

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein-Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.

Development, validation, and pilot application of a high throughput molecular xenomonitoring assay to detect Schistosoma mansoni and other trematode species within Biomphalaria freshwater snail hosts.

Schistosomiasis is a neglected tropical disease (NTD) caused by infection with parasitic trematodes of the genus Schistosoma that can lead to debilitating morbidity and mortality. The World Health Organization recommend molecular xenomonitoring of Biomphalaria spp. freshwater snail intermediate hosts of Schistosoma mansoni to identify highly focal intestinal schistosomiasis transmission sites and monitor disease transmission, particularly in low-endemicity areas. A standardised protocol to do this, however, is needed. Here, two previously published primer sets were selected to develop and validate a multiplex molecular xenomonitoring end-point PCR assay capable of detecting S. mansoni infections within individual Biomphalaria spp. missed by cercarial shedding. The assay proved highly sensitive and highly specific in detecting and amplifying S. mansoni DNA and also proved highly sensitive in detecting and amplifying non-S. mansoni trematode DNA. The optimised assay was then used to screen Biomphalaria spp. collected from a S. mansoni-endemic area for infection and successfully detected S. mansoni infections missed by cercarial shedding as well as infections with non-S. mansoni trematodes. The continued development and use of molecular xenomonitoring assays such as this will aid in improving disease control efforts, significantly reducing disease-related morbidities experienced by those in schistosomiasis-endemic areas.

Comparative performance evaluation of blood film microscopy for the diagnosis of bovine trypanosomosis by some laboratories in North-central Nigeria.

Background: Due to its affordability in disease-affected communities and suitability for field application, microscopy has historically been considered the gold standard for field diagnosis of trypanosomosis in rural settings. Aim: This works aims to compare the performance of microscopists on bovine trypanosome microscopy by organizing the first comparative assessment on a correct reading of slides by laboratory professionals using the read slide results and a structured interviewer-administered questionnaire in North-central Nigeria. Methods: Ten participants were addressed, as they were sent a panel of two slides (Slide 1: No Trypanosome present; Slide 2: Trypanosome present) and a questionnaire. Results: All participants greater than 41 years old reported correctly the presence and absence of parasites on slides. Only 3/8 of microscopists from routine diagnostic laboratories reported correctly the presence of the parasite. Conclusion: Our study confirmed errors in reading slides. Therefore, training of microscopists besides a nationwide quality assessment is recommended.

Molecular Epidemiology and Assemblage Typing of Giardia duodenalis in School-Age Children Situated along the Southern Shoreline of Lake Malawi, Malawi.

Almost all human giardiasis infections are caused by Giardia duodenalis assemblages A and B. Differentiation between human infections with these assemblages, as well as between single-assemblage (A or B) and mixed-assemblage (A and B) infections, is therefore needed to better understand the pathological impact of infection with either, or both, assemblages. We assessed the prevalence of G. duodenalis assemblages A and B using 305 fecal samples provided by school-age children situated along the southern shoreline of Lake Malawi. Concurrently, intestinal pathology data were also collected to test for association(s) between assemblage infection status and intestinal health. Prevalence of G. duodenalis infection was 39.3% by real-time polymerase chain reaction. Of all identified infections, 32% were single G. duodenalis assemblage A and 32% were single G. duodenalis assemblage B, whereas 33% were mixed-assemblage infections. Fifteen unique G. duodenalis assemblage A and 13 unique G. duodenalis assemblage B ß-giardin haplotypes were identified. There was a positive association between single infection with G. duodenalis assemblage B and both self-reporting of abdominal pain (odds ratio [OR]: 3.05, P = 0.004) and self-reporting of diarrhea (OR: 3.1, P = 0.003). No association between single infection with assemblage A and any form of intestinal pathology was found. Additionally, there was a positive association between mixed-assemblage infections and self-reporting of abdominal pain (OR: 3.1, P = 0.002). Our study highlights the importance G. duodenalis assemblage typing and reaffirms the need for improved access to water, sanitation and hygiene infrastructure in rural areas of low- and middle-income countries.

First Molecular Identification of Bulinus africanus in Lake Malawi Implicated in Transmitting Schistosoma Parasites.

The freshwater snail genus Bulinus plays a vital role in transmitting parasites of the Schistosoma haematobium group. A hybrid schistosome between S. haematobium and S. mattheei has been recently detected using DNA-based identification methods in school children along the Lake Malawi shoreline in Mangochi District. This finding raised the need for contemporary revaluation of local interactions between schistosomes and snails, with a particular focus on snail species within the Bulinus africanus group. In 2017 and 2018, malacological surveys sampled several freshwater sites in Mangochi District. Collected snails (n = 250) were characterised using cytochrome oxidase subunit 1 gene (cox1), with DNA barcoding of the 'Folmer' region and a rapid PCR-RFLP typing assay with double digestion with HaeIII and SacI restriction enzymes. DNA cox1 sequence analysis, with phylogenetic tree construction, suggested the presence of at least three B. africanus group taxa in Lake Malawi, B. globosus, alongside first reports of B. africanus and B. angolensis, which can be differentiated by PCR-RFLP methods. In addition, a total of 30 of the 106 B. africanus group snails (28.30%) were positive to the Schistosoma-specific screen using real-time PCR methods. This study provides new insight into the recent changes in the epidemiology of urogenital schistosomiasis as likely driven by a new diversity of B. africanus group snails within the Lake.

Scaling up of tsetse control to eliminate Gambian sleeping sickness in northern Uganda.

BACKGROUND: Tsetse flies (Glossina) transmit Trypanosoma brucei gambiense which causes Gambian human African trypanosomiasis (gHAT) in Central and West Africa. Several countries use Tiny Targets, comprising insecticide-treated panels of material which attract and kill tsetse, as part of their national programmes to eliminate gHAT. We studied how the scale and arrangement of target deployment affected the efficacy of control. METHODOLOGY AND PRINCIPAL FINDINGS: Between 2012 and 2016, Tiny Targets were deployed biannually along the larger rivers of Arua, Maracha, Koboko and Yumbe districts in North West Uganda with the aim of reducing the abundance of tsetse to interrupt transmission. The extent of these deployments increased from ~250 km2 in 2012 to ~1600 km2 in 2015. The impact of Tiny Targets on tsetse populations was assessed by analysing catches of tsetse from a network of monitoring traps; sub-samples of captured tsetse were dissected to estimate their age and infection status. In addition, the condition of 780 targets (~195/district) was assessed for up to six months after deployment. In each district, mean daily catches of tsetse (G. fuscipes fuscipes) from monitoring traps declined significantly by >80% following the deployment of targets. The reduction was apparent for several kilometres on adjacent lengths of the same river but not in other rivers a kilometre or so away. Expansion of the operational area did not always produce higher levels of suppression or detectable change in the age structure or infection rates of the population, perhaps due to the failure to treat the smaller streams and/or invasion from adjacent untreated areas. The median effective life of a Tiny Target was 61 (41.8-80.2, 95% CI) days. CONCLUSIONS: Scaling-up of tsetse control reduced the population of tsetse by >80% across the intervention area. Even better control might be achievable by tackling invasion of flies from infested areas within and outside the current intervention area. This might involve deploying more targets, especially along smaller rivers, and extending the effective life of Tiny Targets.

A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

Assessing expanded community wide treatment for schistosomiasis: Baseline infection status and self-reported risk factors in three communities from the Greater Accra region, Ghana.

BACKGROUND: This paper reports on the baseline prevalence and associated risk factor findings of a pilot, longitudinal study exploring community-wide treatment of schistosomiasis and soil-transmitted helminthiasis, using albendazole plus praziquantel in the Greater Accra region of Ghana. METHOD: From three communities, at least, 658 individuals were enrolled into the study via random household selection. Prevalence and intensity of schistosomiasis and STH infection were determined from stool and urine samples with a questionnaire being administered in order to explore other morbidities and risk factors. Factor analysis of household demographic variables was undertaken to generate a socioeconomic score; this was then further categorised into tertiles. Proportional-odds cumulative logit generalised estimating equation (GEE) models were used to investigate categorical ordinal intensity of infection associations with morbidity. Separately, logistic GEE models were used to investigate risk factor associations with infection prevalence. RESULTS: Both Schistosoma haematobium and S. mansoni were prevalent in the three communities, with the prevalence of S. haematobium ranging from 3.3% (24/679; 95% CI = 1.9-4.7) to 19% (114/632; 95% CI = 15.8-22.2) and S. mansoni ranging from 30% (202/679; 95% CI = 26.5-33.5) to 78.3% (409/536; 95% CI = 74.7-81.9). The total prevalence of STH across all three sites was negligible at 1.3% (24/1847; 95% CI = 0.8-1.9) comprising mainly hookworm (10/1847). Multivariable statistical models indicated males to be 2.3 (95% CI = 1.7-3.3) times more likely to have a high intensity S. mansoni infection and 1.5 (95% CI = 1.1-2) times more likely to have a high intensity of S. haematobium infection compared to females. There was no significant difference in the likelihood of infection with S. mansoni between adults and school age children (SAC), however S. haematobium infections were found to be 2.5 (95% CI = 1.8-3.5) times more likely to occur in school age children than in adults. Multivariable statistical models (adjusted for age and sex) indicated an association between schistosomiasis and a number of self-reported morbidity indicators (notably diarrhoea and blood in stool and urine). Low socio-economic status was also associated with SCH infection (OR: 2; 95% CI = 1.3-3.2). CONCLUSION: The communities targeted by this study showed a range of Schistosoma prevalence's of infection, from hypo-endemic through to meso-endemic and hyper-endemic. The prevalence of SCH across the different age groups in the study locations highlights the large number of individuals currently being left out of the standard morbidity control method of annual treatment of the SAC.

Evidence of the absence of human African trypanosomiasis in two northern districts of Uganda: Analyses of cattle, pigs and tsetse flies for the presence of Trypanosoma brucei gambiense.

BACKGROUND: Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda. METHODS: We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples. RESULTS: In total, 39/2,088 (1.9%; 95% CI = 1.9-2.5) cattle, 25/400 (6.3%; 95% CI = 4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI = 1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense. CONCLUSION: The lack of T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.

Expanding molecular diagnostics of helminthiasis: Piloting use of the GPLN platform for surveillance of soil transmitted helminthiasis and schistosomiasis in Ghana.

The efforts to control and eradicate polio as a global health burden have been successful to the point where currently only three countries now report endemic polio, and the number of cases of polio continues to decrease. The success of the polio programme has been dependant on a well-developed network of laboratories termed the global polio laboratory network (GPLN). Here we explore collaborative opportunities with the GPLN to target two of the 18 diseases listed as a neglected tropical diseases (NTD) namely soil transmitted helminthiasis (STH) and Schistosomiasis (SCH). These were chosen based on prevalence and the use of faecal materials to identify both polio, STH and SCH. Our study screened 448 faecal samples from the Ghana GPLN using three triplex TaqMan assays to identify Ascaris lumbricoides, Necator americanus, Ancylostoma spp, Trichuris trchiura, Strongyloides stercoralis and Schistosoma spp. Our results found a combined helminth prevalence of 22%. The most common helminth infection was A. lumbricoides with a prevalence of 15% followed by N. americanus (5%), Ancylostoma spp. (2.5%), Schistosoma spp. (1.6%) and S. stercoralis (1%). These results show that it is possible to identify alternative pathogens to polio in the samples collected by the GPLN platform and to introduce new diagnostic assays to their laboratories. The diagnostic methods employed were also able to identify S. stercoralis positive samples, which are difficult to identify using parasitological methods such as Kato-Katz. This study raises the possibility of collaboration with the GPLN for the surveillance of a wider range of diseases which would both benefit the efforts to control the NTDs and also increase the scope of the GPLN as a diagnostic platform.

The COUNTDOWN Study Protocol for Expansion of Mass Drug Administration Strategies against Schistosomiasis and Soil-Transmitted Helminthiasis in Ghana.

(1) Background: Current international policy for schistosomiasis and soil-transmitted helminthiasis (STH) control emphasises mass administration of deworming drugs in school-based programmes. However, this approach is insufficient to control the transmission of these diseases, and their burden in non-school cohorts is recognised, albeit under-researched. This research will investigate the feasibility and acceptability of expanding access to praziquantel (PZQ) against schistosomiasis, and albendazole (ALB) against STH, to communities in selected transmission settings in Ghana. (2) Methods: A three-site longitudinal study will be implemented to investigate the effectiveness of expanding treatment strategies for PZQ and ALB to community members. In the context of community mass drug administration (to preschool children, school non-attending children, and adults, including pregnant women), the intervention will be assessed in a random sample of community members, at baseline with follow-up at 6, 12, and 18 months. In each community, 658 participants will be enrolled, and 314 followed up at each time point. The primary outcome measure is the prevalence of infection of Schistosoma haematobium and/or S. mansoni at study endpoint, as assessed by longitudinal surveys. Secondary outcomes are to quantify the infection of schistosomiasis and STH infections in non-treated cohorts, reductions in prevalence of STH, and intensity of schistosomiasis and STH, and treatment coverage. Nested within this study will be qualitative, cost-benefit, and cost-effectiveness evaluations that will explore accessibility, feasibility, and economic impact of expanded treatment from different complementary perspectives. (3) Discussion: Using a multidisciplinary approach, this study will generate evidence for improved availability, acceptability, affordability, and accessibility to deworming drugs against schistosomiasis and STH to individuals and communities in Ghana. This is likely to have considerable research, programmatic, and political value to contribute evidence for national programme policy development within Ghana, and, more broadly, World Health Organization policy development.

Urogenital schistosomiasis and soil-transmitted helminthiasis (STH) in Cameroon: An epidemiological update at Barombi Mbo and Barombi Kotto crater lakes assessing prospects for intensified control interventions.

BACKGROUND: The crater lakes of Barombi Mbo and Barombi Kotto are well-known transmission foci of schistosomiasis and soil-transmitted helminthiasis having had several important control initiatives previously. To collect contemporary epidemiological information, a cross-sectional survey was undertaken inclusive of: signs and symptoms of disease, individual treatment histories, local water, sanitation and hygiene (WASH)-related factors and malacological surveillance, with molecular characterisation of specimens. METHODS: At each lake, a community cross-sectional survey was undertaken using a combination of stool and urine parasitological sampling, and interview with pro-forma questionnaires. A total of 338 children and adults participated. Material from snail and parasite species were characterised by DNA methods. RESULTS: Egg-patent prevalence of urogenital schistosomiasis was 8.7% at Barombi Mbo (all light-intensity infections) and 40.1% at Barombi Kotto (21.2% heavy-intensity infections). Intestinal schistosomiasis was absent. At Barombi Kotto, significantly more women reported signs and symptoms associated with female genital schistosomiasis. While there had been extensive recent improvement in WASH-related infrastructure at Barombi Mbo, water contact risk scores were higher among schistosomiasis-infected participants (P < 0.001) and at Barombi Kotto in general (P < 0.001). Across both lakes, mean prevalence of STH was very low (6.3%) evidencing an impressive decrease of 79.0% over the last decade; neither Strongyloides stercoralis nor Ascaris lumbricoides were found. A total of 29 freshwater sampling sites were inspected for snails, 13 in Barombi Mbo and 16 in Barombi Kotto; water chemistry differed significantly (P < 0.0001) between lakes for both mean pH (7.9 v. 9.6) and mean conductivity (64.3 µS v. 202.1 µS) respectively. Only two Bulinus camerunensis found on the central island of Barombi Kotto were observed to shed schistosome cercariae, but schistosome DNA was later detected in Bulinus sampled from both lakes as well as in Indoplanorbis exustus, an invasive species from Asia. CONCLUSIONS: STH is currently at very low levels while urogenital schistosomiasis is of greatest concern at Barombi Kotto. This assessment highlights a unique opportunity for further study of the epidemiological dynamics at these crater lakes, to evaluate future intensified interventions both in terms of gaining and sustaining control at Barombi Kotto or in moving towards local interruption of transmission of both diseases at Barombi Mbo.

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring.

BACKGROUND: As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. METHODS AND FINDINGS: The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. CONCLUSION: We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.

RNA-mediated gene suppression and in vitro culture in Hymenolepis microstoma.

Hymenolepis microstoma, the mouse bile-duct tapeworm, is a classical rodent-hosted model that provides easy laboratory access to all stages of the life cycle. Recent characterisation of its genome has greatly advanced its utility for molecular research, albeit contemporary techniques such as those for assaying gene function have yet to be developed in the system. Here we present research on the development of RNA-mediated gene suppression via RNA interference (RNAi), and on in vitro culture of the enteric, adult phase of the life cycle to support this work. We demonstrate up to 80% quantitative suppression of a Hox transcript via soaking activated juvenile worms with double-stranded RNAs. However, we were unable to achieve segmentation of the worms in culture despite extensive manipulations of the culture media and supplements, preventing functional interpretation. An alternative, in vivo approach to RNAi was also tested by exposing cysticercoids prior to inoculation in mice, but fluorescent labelling showed that the RNAs did not sufficiently penetrate the cyst body and no difference in expression was found between exposed and control groups grown in vivo. Genomic and transcriptomic data revealed that H. microstoma has two orthologs each of Dicer, Drosha and Ago-1-like genes and that expression of one of the Ago-1 genes appears exclusive to germline development, suggesting that two or more independent RNA-mediated pathways are in operation. These studies demonstrate the viability of RNAi in H. microstoma and extend the utility of the model for research in the genomic era.

Spatial and temporal consistency of putative reference genes for real-time PCR in a model tapeworm.

Relative quantification of gene expression by real-time PCR relies on the use of reference genes whose expressed levels remain consistent across experimental conditions. Here we compare expression levels of commonly employed endogenous housekeeping genes against a developmental regulatory gene in the model tapeworm Hymenolepis microstoma, examining variation both spatially across regions of the adult worm and temporally through the course of larval metamorphosis. ß-Tubulin, RNA polymerase II and 60S ribosomal subunit L28 showed the most variance among candidate reference genes when comparing changes in expression along the anteroposterior gradient of development represented by the adult body, whereas expression of 18S rDNA and cyclic AMP were highly consistent and could be used reliably for relative quantification. The transcription factor Hox4, referenced to either 18S or cAMP, showed 3-fold higher expression levels in the neck region than in more mature regions of the strobila. In contrast, variance among samples representing progressive stages of larval metamorphosis were greater for candidate reference genes than for Hox4, indicating that none of the candidates can be used reliably for quantifying relative changes in gene expression during metamorphosis.