Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters.

Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements. It further increases the multiplexing capacity of commercial dPCR devices. For example, we present a stepwise increase in multiplexing degrees from a monochrome two-plex assay in one detection channel to a six-plex REM-dPCR assay in a three-color dPCR device for KRAS/BRAF single nucleotide polymorphism (SNP) target sequences. The guidelines for the REM-dPCR design are presented, and the process from duplex to six-plex assay establishment, taking into account the target sequence-dependent effects on assay performance, is discussed. Furthermore, the assay-specific, sensitive and precise quantification of different fractions of KRAS mutant and wild-type DNA sequences in different ratios is demonstrated. To increase the device capacitance and the degree of multiplexing, the REM-dPCR uses the advantage of n target-independent reporter molecules in combination with target sequence-specific mediator probes. Different reporter types are labelled with fluorophores of different signal intensities but not necessarily different emission spectra. This leads to the generation of n independent single-positive populations in the dataspace, created by k detection channels, whereby n > k and n ≥ 2. By usage of target-independent but population-specific reporter types, a fixed set of six optimized signalling molecules could be defined. This reporter set enables the robust generation and precise differentiation of multiple fluorescence signals in dPCRs and can be transferred to new target panels. The set which enables stable signal generation and differentiation in a specified device would allow easy transfer to new target panels.

Knowledge and Practices on the Prevention and Management of Diarrhea in Children Under-2 Years Among Women Dwelling in Urban Slums of Karachi, Pakistan.

BACKGROUND: Diarrhea is the second leading cause of death especially among children. The age-proportionate mortality of diarrheal disease in infants under 2 years is 72%, among children under 5 years of age. Children living in urban slums are more prone to develop diarrhea. Although the disease can be prevented by many simple cost-effective interventions, i.e. proper sanitation and hygiene, appropriate feeding, and timely vaccination, poverty and lack of basic life amenities often potentiate diarrhea mortality. Gadap town is the largest town of Karachi with a deprived health system. This study aims to assess pediatric diarrhea prevalence and related knowledge-practice gaps in the slums of Gadap Town, Karachi, Pakistan. METHOD: A community-based cross-sectional study was conducted from November 2016 to May 2017 among mothers of children under 2 years, who were residents of Gadap Town, Karachi, Pakistan. The participants were approached by a multistage sampling method. A validated dichotomous questionnaire, piloted on 40 participants, translated into local language Urdu was used for data collection and the data was analyzed by SPSS® version 20.0. RESULTS: 51.8% (n = 199) of participants were aged between 25 and 34 years. Among all participants, 68% (n = 261) had primary level education or less, compared to 4.7% (n = 18) of women who had graduate-level education. The mean number of children per woman was 2.52 ± 1.62. Self-reported pediatric diarrhea incidence was 72.1% (n = 277). More than half (55.2% n = 149) of participants reported frequent diarrhea episodes during the 2nd year of their child's life. In this survey, we found the knowledge of women regarding diarrhea management and how to reduce diarrhea morbidity to be inadequate (p > 0.05). However, many women reported appropriate practices which can significantly reduce diarrhea morbidity (p < 0.05). CONCLUSION: While the knowledge among women on preventive measures for pediatric diarrhea was insufficient, the translation of the right knowledge into appropriate practices showed promising outcomes for reducing diarrhea morbidity. An integrated approach for improving feeding, sanitation, and hygiene practices along with continuous health education could curtail the burden of diarrhea among infants living in urban slums.

Distribution of bovine Fasciola gigantica (Cobbold, 1885) in the district des Savanes, northern Côte d'Ivoire.

Fascioliasis, caused by an infection with liver flukes of the genus Fasciola, is an important disease of livestock in most parts of the world. However, little is known about the distribution of fascioliasis in sub-Saharan Africa. We report results of a cross-sectional study conducted in 2014 in the district des Savanes in the northern part of Côte d'Ivoire. We obtained 275 livers from bovine suspected with fascioliasis and 51 unsuspected livers from 24 slaughterhouses. Livers were dissected using a standard operating procedure and all Fasciola gigantica flukes were removed from the tissues of the liver and the biliary ducts. We found F. gigantica in 125 livers from bovines suspected with fascioliasis (45.5%) in 10 departments of the district des Savanes. Among the unsuspected livers, five were positive for F. gigantica (9.8%). The distribution of fascioliasis showed considerable spatial heterogeneity, both at regional (ranging from 18.0% to 52.3%) and departmental level (ranging from 14.3% to 64.0%). Poro region was the most affected (52.3%) with a relatively homogeneous distribution. The departments most affected by fascioliasis were M'Bengué (64.0%), Sinématiali (62.1%) and Ferkessédougou (52.9%). Our study confirms that fascioliasis is an important veterinary disease in the northern part of Côte d'Ivoire, and hence, high-risk areas need to be targeted for prevention and control measures.

A Call for Systems Epidemiology to Tackle the Complexity of Schistosomiasis, Its Control, and Its Elimination.

Ever since the first known written report of schistosomiasis in the mid-19th century, researchers have aimed to increase knowledge of the parasites, their hosts, and the mechanisms contributing to infection and disease. This knowledge generation has been paramount for the development of improved intervention strategies. Yet, despite a broad knowledge base of direct risk factors for schistosomiasis, there remains a paucity of information related to more complex, interconnected, and often hidden drivers of transmission that hamper intervention successes and sustainability. Such complex, multidirectional, non-linear, and synergistic interdependencies are best understood by looking at the integrated system as a whole. A research approach able to address this complexity and find previously neglected causal mechanisms for transmission, which include a wide variety of influencing factors, is needed. Systems epidemiology, as a holistic research approach, can integrate knowledge from classical epidemiology, with that of biology, ecology, social sciences, and other disciplines, and link this with informal, tacit knowledge from experts and affected populations. It can help to uncover wider-reaching but difficult-to-identify processes that directly or indirectly influence exposure, infection, transmission, and disease development, as well as how these interrelate and impact one another. Drawing on systems epidemiology to address persisting disease hotspots, failed intervention programmes, and systematically neglected population groups in mass drug administration programmes and research studies, can help overcome barriers in the progress towards schistosomiasis elimination. Generating a comprehensive view of the schistosomiasis system as a whole should thus be a priority research agenda towards the strategic goal of morbidity control and transmission elimination.

Perceptions, knowledge, attitudes and practices for the prevention and control of lymphatic filariasis in Conakry, Republic of Guinea.

Little is known about the perceptions, attitudes and practices of lymphatic filariasis in Conakry, Republic of Guinea. Yet, such knowledge is important for an optimal design and implementation of setting-specific prevention and control measures. We conducted a cross-sectional study using a mixed methods approach. Qualitative data related to people's general experience with lymphatic filariasis, their perception of the causes of the disease, the onset of elephantiasis, care-seeking behaviour and the socioeconomic impact of lymphatic filariasis were collected through in-depth interviews with 85 respondents. Quantitative data related to strategies for prevention and the knowledge of the causes of the disease were collected by interviewing 429 people. A total of 514 individuals (313 females and 201 males), aged 10-84 years, participated. Most participants were well aware of lymphatic filariasis and they recognized the disease mainly by its disfiguring manifestation, collectively termed "elephantiasis" or "leg-swelling disease". Morbidity patterns due to filarial infection showed an increase with age (from 30 to 50 years) independent of sex. Most patients with lymphatic filariasis abandoned their jobs (73.9%) or sought other work (21.7%). The main perceived causes of acquiring lymphatic filariasis were of supernatural origin (as stated by 8.7% of patients and 5.7% of healthy subjects), while mosquito bites were mentioned by fewer participants (4.3% of patients and 4.2% of healthy subjects). A number of other causes were reported that relate to both medical and non-medical conceptions. The study also identified socioeconomic impairments and stigmatization due to elephantiasis. Taken together, community perception of lymphatic filariasis in Conakry is influenced by sociocultural conceptions. Appropriate health education campaigns aimed at enhancing community understanding of the transmission of lymphatic filariasis are required to increase the success of mass drug administration implemented for the elimination of this disease. There is a need for a morbidity management programme to alleviate lymphatic filariasis-related physical and emotional burden in Conakry.

Validation of a Point-of-Care Circulating Cathodic Antigen Urine Cassette Test for Schistosoma mansoni Diagnosis in the Sahel, and Potential Cross-Reaction in Pregnancy.

On the shores of Lake Chad, schistosomiasis among mobile pastoralists was investigated in a field laboratory. Point-of-care circulating cathodic antigen (POC-CCA) cassette test, reagent strip, and filtration were conducted on urine samples. Fresh stool samples were subjected to the Kato-Katz technique, and fixed samples were examined with an ether-concentration method at a reference laboratory. POC-CCA urine cassette tests revealed a Schistosoma mansoni prevalence of 6.9%, compared with only 0.5% by stool microscopy. Three pregnant women with otherwise negative urine and stool testing had positive POC-CCA. This observation raises concern of cross-reactivity in pregnancy. Hence, two pregnant women in Switzerland with no history of schistosomiasis were subjected to POC-CCA and one tested positive. Our data suggest that POC-CCA can be performed under extreme Sahelian conditions (e.g., temperatures > 40°C), and it is more sensitive than stool microscopy for S. mansoni diagnosis. However, potential cross-reactivity in pregnancy needs further investigation.

All that is blood is not schistosomiasis: experiences with reagent strip testing for urogenital schistosomiasis with special consideration to very-low prevalence settings.

BACKGROUND: Reagent strip testing for microhaematuria has long been used for community diagnosis of Schistosoma haematobium. Sensitivities and specificities are reasonable, and hence, microhaematuria can serve as a proxy for S. haematobium infection. However, assessment of test performance in the context of the underlying S. haematobium prevalence is rare and test parameters other than sensitivity and specificity have been neglected. METHODS: Data about the association between microhaematuria and urine filtration results from three studies were compared and put into context with findings from a recent Cochrane review. Data were stratified by S. haematobium prevalence to identify prevalence-related differences in test performance. Kappa agreement and regression models were employed to compare data for different S. haematobium prevalence categories. RESULTS: We found a "background" prevalence of microhaematuria (13 %, on average) which does not seem to be associated with schistosomiasis in most settings, irrespective of the prevalence of S. haematobium. This background level of microhaematuria might be due to cases missed with urine filtration, or alternative causes apart from S. haematobium. Especially in very-low prevalence settings, positive results for microhaematuria likely give an inaccurate picture of the extent of S. haematobium, whereas negative results are a sound indicator for the absence of infection. CONCLUSIONS: Reagent strip testing for microhaematuria remains a good proxy for urogenital schistosomiasis, but implications of test results and scope of application differ depending on the setting in which reagent strips are employed. In very-low prevalence settings, microhaematuria is an unstable proxy for urogenital schistosomiasis and treatment decision should not be based on reagent strip test results alone. Our findings underscore the need for highly accurate diagnostic tools for settings targeted for elimination of urogenital schistosomiasis.

Access to, and use of, water by populations living in a schistosomiasis and fascioliasis co-endemic area of northern Côte d'Ivoire.

Water is an essential element of life, but it can also be a source of disease. Apart from direct consumption of unsafe water, direct contact and indirect consumption puts people at risk of many different types of pathogens. Employing a mixed methods approach, consisting of questionnaires and direct observations, we assessed access to, and use of, different water sources by the participants of the district des Savanes in northern Côte d'Ivoire. The use of water sources was put in relation to the potential risk of acquiring schistosomiasis and fascioliasis. Overall, 489 people aged 8 to 82 years participated. While all participants had access to safe water, 63% were in direct contact with unimproved water and 31% directly consumed unsafe water. More than a third of the people who otherwise reported using only improved water for all activities came in contact with unimproved water through crossing open water when going to their workplace, school or other destinations. Self-reported blood in urine - a marker for Schistosoma haematobium with reasonable sensitivity and specificity - was reported by 6% (n=30), self-reported blood in stool - an unspecific marker for Schistosoma mansoni - was reported by 7% (n=35), while blood co-occurring in both urine and stool was reported by another 10% (n=48) of participants. Accessing unimproved water for any activity (including crossing) was associated with higher odds of reporting blood in urine and/or blood in stool (odds ratio: 1.90; 95% confidence interval: 1.07-3.36). Our results have important rami-fications for intervention programmes targeting neglected tropical diseases, and emphasize the need for a wider supply of safe water to rural populations, since the water supply at the workplace needs to be considered as well next to the water supply at home. Crossing of open water sources is an important risk factor for sustained transmission of schistosomiasis.

Effect of deworming on physical fitness of school-aged children in Yunnan, China: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: There is considerable debate on the health impacts of soil-transmitted helminth infections. We assessed effects of deworming on physical fitness and strength of children in an area in Yunnan, People's Republic of China, where soil-transmitted helminthiasis is highly endemic. METHODOLOGY: The double-blind, randomized, placebo-controlled trial was conducted between October 2011 and May 2012. Children, aged 9-12 years, were treated with either triple-dose albendazole or placebo, and monitored for 6 months post-treatment. The Kato-Katz and Baermann techniques were used for the diagnosis of soil-transmitted helminth infections. Physical fitness was assessed with a 20-m shuttle run test, where the maximum aerobic capacity within 1 min of exhaustive exercise (VO2 max estimate) and the number of 20-m laps completed were recorded. Physical strength was determined with grip strength and standing broad jump tests. Body height and weight, the sum of skinfolds, and hemoglobin levels were recorded as secondary outcomes. PRINCIPAL FINDINGS: Children receiving triple-dose albendazole scored slightly higher in the primary and secondary outcomes than placebo recipients, but the difference lacked statistical significance. Trichuris trichiura-infected children had 1.6 ml kg(-1) min(-1) (P = 0.02) less increase in their VO2 max estimate and completed 4.6 (P = 0.04) fewer 20-m laps than at baseline compared to non-infected peers. Similar trends were detected in the VO2 max estimate and grip strength of children infected with hookworm and Ascaris lumbricoides, respectively. In addition, the increase in the VO2 max estimate from baseline was consistently higher in children with low-intensity T. trichiura and hookworm infections than in their peers with high-intensity infections of all soil-transmitted helminths (range: 1.9-2.1 ml kg(-1) min(-1); all P<0.05). CONCLUSIONS/SIGNIFICANCE: We found no strong evidence for significant improvements in physical fitness and anthropometric indicators due to deworming over a 6-month follow-up period. However, the negative effect of T. trichiura infections on physical fitness warrants further investigation.

Dynamics of Schistosoma haematobium egg output and associated infection parameters following treatment with praziquantel in school-aged children.

BACKGROUND: Praziquantel is the drug of choice in preventive chemotherapy targeting schistosomiasis. Increasing large-scale administration of praziquantel requires monitoring of drug efficacy to detect early signs of development of resistance. Standard protocols for drug efficacy monitoring are necessary. Here, we determined the optimal time point for praziquantel efficacy assessment against Schistosoma haematobium and studied the dynamics of infection parameters following treatment. METHODS: Ninety school-aged children from south Côte d'Ivoire with a parasitologically confirmed S. haematobium infection were treated with a single oral dose of praziquantel (40 mg/kg) and followed up for 62 days post-treatment. Urine samples were collected on 23 schooldays during this period and were subjected to visual examination (macrohaematuria), urine filtration and microscopy (S. haematobium eggs) and reagent strip testing (microhaematuria, proteinuria and leukocyturia). RESULTS: Observed cure and egg reduction rates were highly dependent on the time point post-treatment. Egg reduction rates were high (>97%) in weeks 3-9 post-treatment. Cure rates were highest in weeks 6 (92.9%) and 9 (95.0%) post-treatment. The prevalence of infection-associated parameters decreased after treatment, reaching a minimum of 2.4% in weeks 5 (proteinuria) and 7 (leukocyturia) post-treatment, and 16.3% at the end of week 8 (microhaematuria). Macrohaematuria disappeared between weeks 3 and 6 post-treatment. CONCLUSIONS: For monitoring praziquantel efficacy against S. haematobium, we recommend that the cure rate is assessed at week 6 post-treatment. The egg reduction rate can be evaluated earlier, from day 14 post-treatment onwards. Reagent strips are a useful additional tool for evaluating treatment outcomes in areas with high endemicity, preferably at weeks 5 and 6 post-treatment. The delayed decrease of microhaematuria confirms that lesions in the urinary tract persist longer than egg excretion post-treatment.

An in-depth analysis of a piece of shit: distribution of Schistosoma mansoni and hookworm eggs in human stool.

BACKGROUND: An accurate diagnosis of helminth infection is important to improve patient management. However, there is considerable intra- and inter-specimen variation of helminth egg counts in human feces. Homogenization of stool samples has been suggested to improve diagnostic accuracy, but there are no detailed investigations. Rapid disintegration of hookworm eggs constitutes another problem in epidemiological surveys. We studied the spatial distribution of Schistosoma mansoni and hookworm eggs in stool samples, the effect of homogenization, and determined egg counts over time in stool samples stored under different conditions. METHODOLOGY: Whole-stool samples were collected from 222 individuals in a rural part of south Côte d'Ivoire. Samples were cut into four pieces and helminth egg locations from the front to the back and from the center to the surface were analyzed. Some samples were homogenized and fecal egg counts (FECs) compared before and after homogenization. The effect of stool storing methods on FECs was investigated over time, comparing stool storage on ice, covering stool samples with a water-soaked tissue, or keeping stool samples in the shade. PRINCIPAL FINDINGS: We found no clear spatial pattern of S. mansoni and hookworm eggs in fecal samples. Homogenization decreased S. mansoni FECs (p = 0.026), while no effect was observed for hookworm and other soil-transmitted helminths. Hookworm FECs decreased over time. Storing stool samples on ice or covered with a moist tissue slowed down hookworm egg decay (p<0.005). CONCLUSIONS/SIGNIFICANCE: Our findings have important implications for helminth diagnosis at the individual patient level and for epidemiological surveys, anthelmintic drug efficacy studies and monitoring of control programs. Specifically, homogenization of fecal samples is recommended for an accurate detection of S. mansoni eggs, while keeping collected stool samples cool and moist delayed the disintegration of hookworm eggs.

Effect of schistosomiasis and soil-transmitted helminth infections on physical fitness of school children in Côte d'Ivoire.

BACKGROUND: Schistosomiasis and soil-transmitted helminthiasis are important public health problems in sub-Saharan Africa causing malnutrition, anemia, and retardation of physical and cognitive development. However, the effect of these diseases on physical fitness remains to be determined. METHODOLOGY: We investigated the relationship between schistosomiasis, soil-transmitted helminthiasis and physical performance of children, controlling for potential confounding of Plasmodium spp. infections and environmental parameters (i.e., ambient air temperature and humidity). A cross-sectional survey was carried out among 156 school children aged 7-15 years from Côte d'Ivoire. Each child had two stool and two urine samples examined for helminth eggs by microscopy. Additionally, children underwent a clinical examination, were tested for Plasmodium spp. infection with a rapid diagnostic test, and performed a maximal multistage 20 m shuttle run test to assess their maximal oxygen uptake (VO(2) max) as a proxy for physical fitness. PRINCIPAL FINDINGS: The prevalence of Schistosoma haematobium, Plasmodium spp., Schistosoma mansoni, hookworm and Ascaris lumbricoides infections was 85.3%, 71.2%, 53.8%, 13.5% and 1.3%, respectively. Children with single, dual, triple, quadruple and quintuple species infections showed VO(2) max of 52.7, 53.1, 52.2, 52.6 and 55.6 ml kg(-1) min(-1), respectively. The VO(2) max of children with no parasite infections was 53.5 ml kg(-1) min(-1). No statistically significant difference was detected between any groups. Multivariable analysis revealed that VO(2) max was influenced by sex (reference: female, coef. = 4.02, p<0.001) and age (years, coef. = -1.23, p<0.001), but not by helminth infection and intensity, Plasmodium spp. infection, and environmental parameters. CONCLUSION/SIGNIFICANCE: School-aged children in Côte d'Ivoire showed good physical fitness, irrespective of their helminth infection status. Future studies on children's physical fitness in settings where helminthiasis and malaria co-exist should include pre- and post-intervention evaluations and the measurement of hemoglobin and hematocrit levels and nutritional parameters as potential co-factors to determine whether interventions further improve upon fitness.

Geospatial health: the first five years.

Geospatial Health is an international, peer-reviewed scientific journal produced by the Global Network for Geospatial Health (GnosisGIS). This network was founded in 2000 and the inaugural issue of its official journal was published in November 2006 with the aim to cover all aspects of geographical information system (GIS) applications, remote sensing and other spatial analytic tools focusing on human and veterinary health. The University of Naples Federico II is the publisher, producing two issues per year, both as hard copy and an open-access online version. The journal is referenced in major databases, including CABI, ISI Web of Knowledge and PubMed. In 2008, it was assigned its first impact factor (1.47), which has now reached 1.71. Geospatial Health is managed by an editor-in-chief and two associate editors, supported by five regional editors and a 23-member strong editorial board. This overview takes stock of the first five years of publishing: 133 contributions have been published so far, primarily original research (79.7%), followed by reviews (7.5%), announcements (6.0%), editorials and meeting reports (3.0% each) and a preface in the first issue. A content analysis of all the original research articles and reviews reveals that three quarters of the publications focus on human health with the remainder dealing with veterinary health. Two thirds of the papers come from Africa, Asia and Europe with similar numbers of contributions from each continent. Studies of more than 35 different diseases, injuries and risk factors have been presented. Malaria and schistosomiasis were identified as the two most important diseases (11.2% each). Almost half the contributions were based on GIS, one third on spatial analysis, often using advanced Bayesian geostatistics (13.8%), and one quarter on remote sensing. The 120 original research articles, reviews and editorials were produced by 505 authors based at institutions and universities in 52 countries. Importantly, a considerable proportion of the authors come from countries with a low or medium human development index (29.3%). In view of the increasing number of submissions, we are considering to publish more than two issues per year in the future. Finally, our vision is to open-up a new section predominantly based on visual presentations, including brief video clips, as discussed in a symposium at the 60th annual meeting of the American Society of Tropical Medicine and Hygiene in December 2011.