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. 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. 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. ClinicalTrials.gov; identifier: NCT02105714 .

Background: Trypanosoma cruzi, the protozoan agent of Chagas disease, is comprised of at least 6 genetic lineages (TcI-TcVI). Their geographical distribution, clinical associations and reservoir hosts are not fully elucidated, as genotyping is hampered due to the difficulty in isolating representative populations of organisms. Lineage-specific serological techniques may address these issues. Methods: Trypanosoma cruzi lineage-specific serological assays were performed on human, canine, feline and armadillo sera from the Gran Chaco in northern Argentina, a region of ongoing transmission. Synthetic peptides representing lineage-specific epitopes of the trypomastigote small surface antigen (TSSA) were used in ELISA, and the TcII/V/VI shared epitope peptide (TSSApep-II/V/VI) was used in the Chagas Sero K-SeT rapid diagnostic test (RDT). Results: Chagas Sero K-SeT RDT, using Protein G to detect human and canine IgG, was at least as sensitive as TSSApep-II/V/VI ELISA using specific secondary antibodies. For sera from humans TSSApep-II/V/VI seroprevalence by Chagas Sero K-SeT was 273/393 (69.5%), for dogs 48/73 (65.8%) and for armadillos 1/7 (14.3%); by ELISA for cats 5/19 (26.3%). The seroprevalence for humans was similar to that for Bolivian patients, amongst whom we previously observed an association of TSSApep-II/V/VI seropositivity with severity of cardiomyopathy. In humans, prevalence of TSSApep-II/V/VI recognition was associated with locality, and with increasing and decreasing age within the Qom and Creole populations, respectively. For dogs TSSApep-II/V/VI recognition was associated with being born before community-wide insecticide spraying (P = 0.05) and with Qom household (P < 0.001). Conclusions: We show here that Chagas Sero K-SeT RDT can replace ELISA for TSSApep-II/V/VI serology of humans and dogs; for humans there were statistically significant associations between a positive Chagas Sero K-SeT RDT and being resident in Area IV, and for dogs association with Qom household or with being born before the mass spraying campaign; we also show that with cats the TcII/V/VI epitope can be detected by ELISA. We assessed the lineage distribution in an unprecedented 83% of the human T. cruzi-seropositive population. These results form the basis for more detailed studies, enabling rapid in-the-field surveillance of the distribution and clustering of these lineages among humans and mammalian reservoirs of T. cruzi infection.

The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a CLA as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania donovani complex. Without treatment, VL is fatal. Although diagnostic techniques, mainly based on the detection of anti-Leishmania antibodies are available, invasive procedures such as microscopy from spleen or bone marrow aspirates are still required for the diagnosis of seronegative VL suspects, for the detection of recurrent cases and to confirm cure after successful treatment. Previous investigations showed the potential of IgG1 as a biomarker of post-chemotherapeutic relapse for VL in rapid diagnostic tests (RDTs) sensitised with crude lysate antigen (CLA). Here we employed in silico tools to search for desired protein features in a large number of L. donovani antigens detected by human IgG1 in western blots. We then employed prediction algorithms to profile epitopes from the shortlisted proteins. We screened a panel of high-scoring peptides in a high-throughput manner using arrays, with low reagent consumption. The most reactive peptide was adapted to RDTs, showing promising results of both sensitivity and specificity. This peptide has the potential of replacing the CLAs in IgG1 RDTs. Thus we believe that in silico tools can be used to optimise wet lab experiments for a rational search of biomarkers.

Introduction. Acinetobacter baumannii infections can be extremely challenging to treat owing to the worldwide prevalence of multidrug-resistant isolates, especially against carbapenems. Colonization with carbapenem-resistant A. baumannii (CRAb) requires rapid action from an infection control perspective because the organism is known for its propensity for epidemic spread. Hypothesis/Gap Statement. There is an unmet medical need to rapidly identify CRAb to enable appropriate antimicrobial treatment and to prevent transmission. Aim. Our aim was to expand the OXA-detection abilities of the rapid immunochromatographic test (ICT) OXA-23 K-SeT (Coris BioConcept) to include OXA-40- and OXA-58-like carbapenemases, which together confer carbapenem resistance to more than 94 % of CRAb isolates worldwide. Methodology. We used hybridoma technology to generate mAbs against OXA-40 and OXA-58 and selected them for productivity and specificity against recombinant and endogenous OXA-40 and OXA-58. Combinations of the resulting mAbs were analysed in ICT format for their ability to detect recombinant rOXA-40His6 or rOXA-58His6, respectively. Subsequently, selected antibody pairs were implemented into single-OXA-40 or single-OXA-58 prototypes and the final OXA-23/40/58/NDM ICT and were evaluated on clinical Acinetobacter spp. isolates with well-defined carbapenem resistance mechanisms. Results. Five anti-OXA-40 and anti-OXA-58 mAbs were selected. Competition ELISA with combinations of these antibodies revealed that the anti-OXA-40 antibodies bind to one of two binding clusters on OXA-40, while anti-OXA-58 antibodies bind to one of four binding clusters on OXA-58. Direct binding to the corresponding antigen in an ICT format has left only three antibodies against rOXA-40His6 and rOXA-58His6, respectively for the subsequent sandwich ICT selection procedure, which revealed that the anti-OXA-40 (#5) and anti-OXA-58 (#A8) mAbs in combination with the cross-reactive mAb #C8 performed best. They were implemented into single-OXA-40 and single-OXA-58 ICT prototypes and evaluated. These single ICT prototypes demonstrated 100 % specificity and sensitivity. Based on these results, an OXA-23/40/58/NDM-ICT was developed, complemented with OXA-23 and NDM-specific detection. An evaluation with selected carbapenem-resistant Acinetobacter spp. isolates (n=34) showed 100 % specificity. Conclusion. With this easy-to-use detection assay, one can save 12–48 h in diagnostics, which helps to treat patients earlier with appropriate antibiotics and allows immediate intervention to control transmission of CRAb.

Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field. Canine leishmaniasis (CanL) is a widespread severe chronic disease in dogs, caused by the protozoan parasite Leishmania infantum. Leishmania parasites are transmitted by the bite of phlebotomine sand flies, of which the most important one in southwestern Europe is Phlebotomus perniciosus. A sand fly bite is accompanied by the inoculation of saliva, which elicits a specific antibody response in the host. Past studies have used these antibodies as markers of exposure to sand flies. Recently, a rapid sand fly exposure test was prepared using one salivary protein of P. perniciosus, the SP03B protein. This rapid test possessed a high sensitivity and specificity when screening experimentally exposed versus non-bitten dogs. However, since naturally exposed dogs possess a higher variability of antibodies against sand fly saliva, we have optimized the previously proposed rapid test and evaluated its detectability on field samples. In this study we present the optimized rSP03B sero-strip that possesses a higher specificity and an ability to detect seropositive dogs from a naturally exposed dog population in a similar fashion as standard serological methods. Furthermore, we have optimized it with whole canine blood which renders it suitable for use in field conditions.

Background: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. Methodology/Principal findings: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. Conclusions/Significance: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.