Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

Molecular characterization of enterotoxigenic Escherichia coli toxins and colonization factors in children under five years with acute diarrhea attending Kisii Teaching and Referral Hospital, Kenya.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infectious diarrhea in children. There are no licensed vaccines against ETEC. This study aimed at characterizing Escherichia coli for ETEC enterotoxins and colonization factors from children < 5 years with acute diarrhea and had not taken antibiotics prior to seeking medical attention at the hospital. METHODS: A total of 225 randomly selected archived E. coli strains originally isolated from 225 children with acute diarrhea were cultured. DNA was extracted and screened by multiplex polymerase chain reaction (PCR) for three ETEC toxins. All positives were then screened for 11 colonization factors by PCR. RESULTS: Out of 225 E. coli strains tested, 23 (10.2%) were ETEC. Heat-stable toxin (ST) gene was detected in 16 (69.6%). ETEC isolates with heat-stable toxin of human origin (STh) and heat-stable toxin of porcine origin (STp) distributed as 11 (68.8%) and 5 (31.2%) respectively. Heat-labile toxin gene (LT) was detected in 5 (21.7%) of the ETEC isolates. Both ST and LT toxin genes were detected in 2 (8.7%) of the ETEC isolates. CF genes were detected in 14 (60.9%) ETEC strains with a majority having CS6 6 (42.9%) gene followed by a combination of CFA/I + CS21 gene detected in 3 (21.4%). CS14, CS3, CS7 and a combination of CS5 + CS6, CS2 + CS3 genes were detected equally in 1 (7.1%) ETEC isolate each. CFA/I, CS4, CS5, CS2, CS17/19, CS1/PCFO71 and CS21 genes tested were not detected. We did not detect CF genes in 9 (39.1%) ETEC isolates. More CFs were associated with ETEC strains with ST genes. CONCLUSION: ETEC strains with ST genes were the most common and had the most associated CFs. A majority of ETEC strains had CS6 gene. In 9 (39.1%) of the evaluated ETEC isolates, we did not detect an identifiable CF.

First report of Entamoeba moshkovskii in human stool samples from symptomatic and asymptomatic participants in Kenya.

Entamoeba moshkovskii is a member of the Entamoeba complex and a colonizer of the human gut. We used nested polymerase chain reaction (PCR) to differentiate Entamoeba species in stool samples that had previously been screened by microscopy. Forty-six samples were tested, 23 of which had previously been identified as Entamoeba complex positive by microscopy. Of the 46 specimens tested, we identified nine (19.5%) as E. moshkovskii-positive. In seven of these nine E. moshkovskii-positive samples, either E. dispar or E. histolytica (or both) were also identified, suggesting that co-infections may be common. E. moshkovskii was also detected in both symptomatic and asymptomatic participants. To the best of our knowledge, this is the first report of E. moshkovskii in Kenya.

Evaluation of the performance of a multiplex reverse transcription polymerase chain reaction kit as a potential diagnostic and surveillance kit for rotavirus in Kenya.

BACKGROUND: Diarrhea is a serious concern worldwide, especially in developing countries. Rotavirus is implicated in approximately 400,000 infant deaths annually. It is highly contagious elevating the risk of outbreaks especially in enclosed settings such as daycare centers, hospitals, and boarding schools. Reliable testing methods are critical for early detection of infections, better clinical management, pathogen surveillance and evaluation of interventions such as vaccines. Enzyme immunoassays have proved to be reliable and practical in most settings; however, newer multiplex reverse transcription polymerase assays have been introduced in the Kenya market but have not been evaluated locally. METHODS: Stool samples collected from an ongoing Surveillance of Enteric Pathogens Causing diarrheal illness in Kenya (EPS) study were used to compare an established enzyme immunoassay, Premier™ Rotaclone® (Meridian Bioscience, Cincinnati, Ohio, U.S.A.), that can only detect group A rotavirus against a novel multiplex reverse transcription polymerase chain reaction kit, Seeplex® Diarrhea-V ACE Detection (Seegene, Seoul, Republic of Korea), that can detect rotavirus, astrovirus, adenovirus, and norovirus genogroups I and II. Detection frequency, sensitivity, specificity, turnaround time, and cost were compared to determine the suitability of each assay for clinical work in austere settings versus public health work in well-funded institutes in Kenya. RESULTS: The Premier™ Rotaclone® kit had a detection frequency of 11.2%, sensitivity of 77.8%, specificity of 100%, turnaround time of 93 min and an average cost per sample of 13.33 United States dollars (USD). The Seeplex® Diarrhea-V ACE Detection kit had a detection frequency of 16.0%, sensitivity of 100%, specificity of 98.1%, turnaround time of 359 min and an average cost per samples 32.74 United States dollars respectively. The detection frequency sensitivity and specificity of the Seeplex® Diarrhea-V ACE Detection kit mentioned above are for rotavirus only. CONCLUSIONS: The higher sensitivity and multiplex nature of the Seeplex® Diarrhea-V ACE Detection kit make it suitable for surveillance of enteric viruses circulating in Kenya. However, its higher cost, longer turnaround time and complexity favor well-resourced clinical labs and research applications. The Premier™ Rotaclone®, on the other hand, had a higher specificity, shorter turnaround time, and lower cost making it more attractive for clinical work in low complexity labs in austere regions of the country. It is important to continuously evaluate assay platforms' performance, operational cost, turnaround time, and usability in different settings so as to ensure quality results that are useful to the patients and public health practitioners.

A Comparison of Stool Enteropathogen Detection by Semiquantitative PCR in Adults With Acute Travelers' Diarrhea Before and 3 Weeks After Successful Antibiotic Treatment.

We evaluated stool enteropathogen detection by semiquantitative polymerase chain reaction (PCR) in 108 subjects with travelers' diarrhea before and 3 weeks after treatment. Stool samples from 21 subjects were positive for the same pathogen species at both visits. We discuss factors that should be considered when interpreting stool PCR data after treatment.

Comparison of stool collection and storage on Whatman FTA Elute cards versus frozen stool for enteropathogen detection using the TaqMan Array Card PCR assay.

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers' diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the 'reference standard'. Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively. TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.