Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

The ongoing risk of Leishmania donovani transmission in eastern Nepal: an entomological investigation during the elimination era.

BACKGROUND: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. METHODS: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. RESULTS: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. CONCLUSIONS: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal.

Diagnosis of neglected tropical diseases among patients with persistent digestive disorders (diarrhoea and/or abdominal pain ≥14 days): Pierrea multi-country, prospective, non-experimental case-control study.

BACKGROUND: Diarrhoea still accounts for considerable mortality and morbidity worldwide. The highest burden is concentrated in tropical areas where populations lack access to clean water, adequate sanitation and hygiene. In contrast to acute diarrhoea (<14 days), the spectrum of pathogens that may give rise to persistent diarrhoea (≥14 days) and persistent abdominal pain is poorly understood. It is conceivable that pathogens causing neglected tropical diseases play a major role, but few studies investigated this issue. Clinical management and diagnostic work-up of persistent digestive disorders in the tropics therefore remain inadequate. Hence, important aspects regarding the pathogenesis, epidemiology, clinical symptomatology and treatment options for patients presenting with persistent diarrhoea and persistent abdominal pain should be investigated in multi-centric clinical studies. METHODS/DESIGN: This multi-country, prospective, non-experimental case-control study will assess persistent diarrhoea (≥14 days; in individuals aged ≥1 year) and persistent abdominal pain (≥14 days; in children/adolescents aged 1-18 years) in up to 2000 symptomatic patients and 2000 matched controls. Subjects from Côte d'Ivoire, Indonesia, Mali and Nepal will be clinically examined and interviewed using a detailed case report form. Additionally, each participant will provide a stool sample that will be examined using a suite of diagnostic methods (i.e., microscopic techniques, rapid diagnostic tests, stool culture and polymerase chain reaction) for the presence of bacterial and parasitic pathogens. Treatment will be offered to all infected participants and the clinical treatment response will be recorded. Data obtained will be utilised to develop patient-centred clinical algorithms that will be validated in primary health care centres in the four study countries in subsequent studies. DISCUSSION: Our research will deepen the understanding of the importance of persistent diarrhoea and related digestive disorders in the tropics. A diversity of intestinal pathogens will be assessed for potential associations with persistent diarrhoea and persistent abdominal pain. Different diagnostic methods will be compared, clinical symptoms investigated and diagnosis-treatment algorithms developed for validation in selected primary health care centres. The findings from this study will improve differential diagnosis and evidence-based clinical management of digestive syndromes in the tropics. TRIAL REGISTRATION: ClinicalTrials.gov; identifier: NCT02105714 .

Prevalence of Shigella species and their antimicrobial resistance patterns in Eastern Nepal.

The study was conducted to determine the prevalence of Shigella species and their antimicrobial resistance patterns in eastern Nepal. Stool samples submitted to the diagnostic laboratory of B.P. Koirala Institute of Health Sciences, Nepal, during August 2000-July 2004, were cultured for Shigella species and were confirmed by biochemical and serological tests. Of 53 Shigella species isolated, Shigella dysenteriae type 1 was the most predominant isolate (73.7%), followed by S. flexneri (23%) and S. boydii (4%). The majority (79%) of Shigella species were isolated from children aged less than five years. An overall high resistance was observed for trimethoprim-sulphamethoxazole, ampicillin, nalidixic acid, mecillinam, and ciprofloxacin. There was a statistically significant (p < 0.001) increasing trend in the prevalence of ciprofloxacin resistance in S. dysenteriae type 1. The results suggest reconsideration of the empiric use of these antimicrobial agents for shigellosis. A further study is required to evaluate additional antimicrobial agents.