Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia.

AIM: This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance. METHODS: Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/µL and, tenfold serially diluting it up to 10 pg/µL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples. RESULTS: Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7-100.0) and 98.0% (90.7-99.0), respectively. The area under the ROC curve was 1.00 (1.00-1.00). CONCLUSIONS: The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.

Antibiotic-Resistant Vibrio cholerae O1 and Its SXT Elements Associated with Two Cholera Epidemics in Kenya in 2007 to 2010 and 2015 to 2016.

Multidrug-resistant Vibrio cholerae O1 strains have long been observed in Africa, and strains exhibiting new resistance phenotypes have emerged during recent epidemics in Kenya. This study aimed to determine the epidemiological aspects, drug resistance patterns, and genetic elements of V. cholerae O1 strains isolated from two cholera epidemics in Kenya between 2007 and 2010 and between 2015 and 2016. A total of 228 V. cholerae O1 strains, including 226 clinical strains isolated from 13 counties in Kenya during the 2007-2010 and 2015-2016 cholera epidemics and two environmental isolates (from shallow well water and spring water isolates) isolated from Pokot and Kwale Counties, respectively, in 2010 were subjected to biotyping, serotyping, and antimicrobial susceptibility testing, including the detection of antibiotic resistance genes and mobile genetic elements. All V. cholerae isolates were identified as El Tor biotypes and susceptible to ceftriaxone, gentamicin, and ciprofloxacin. The majority of isolates were resistant to trimethoprim-sulfamethoxazole (94.6%), streptomycin (92.8%), and nalidixic acid (64.5%), while lower resistance was observed against ampicillin (3.6%), amoxicillin (4.2%), chloramphenicol (3.0%), and doxycycline (1.8%). Concurrently, the integrating conjugative (SXT) element was found in 95.5% of the V. cholerae isolates; conversely, class 1, 2, and 3 integrons were absent. Additionally, 64.5% of the isolates exhibited multidrug resistance patterns. Antibiotic-resistant gene clusters suggest that environmental bacteria may act as cassette reservoirs that favor resistant pathogens. On the other hand, the 2015-2016 epidemic strains were found susceptible to most antibiotics except nalidixic acid. This revealed the replacement of multidrug-resistant strains exhibiting new resistance phenotypes that emerged after Kenya's 2007-2010 epidemic. IMPORTANCE Kenya is a country where cholera is endemic; it has experienced three substantial epidemics over the past few decades, but there are limited data on the drug resistance patterns of V. cholerae at the national level. To the best of our knowledge, this is the first study to investigate the antimicrobial susceptibility profiles of V. cholerae O1 strains isolated from two consecutive epidemics and to examine their associated antimicrobial genetic determinants. Our study results revealed two distinct antibiotic resistance trends in two separate epidemics, particularly trends for multidrug-associated mobile genetic elements and chromosomal mutation-oriented resistant strains from the 2007-2010 epidemic. In contrast, only nalidixic acid-associated chromosomal mutated strains were isolated from the 2015-2016 epidemic. This study also found similar patterns of antibiotic resistance in environmental and clinical strains. Continuous monitoring is needed to control emerging multidrug-resistant isolates in the future.

Case Report: Bacillus pumilus-Caused Bacteremia in a Patient with Food Poisoning.

Bacillus pumilus has rarely been reported as a cause of human infections. We report a case of a B. pumilus causing food poisoning in an adult male. A 51-year-old Japanese man complained of severe abdominal cramps, fever with chills, diarrhea, dizziness, and loss of appetite after eating reheated rice with stewed minced meat purchased from a Kenyan restaurant. Bacillus pumilus was isolated from blood culture and was identified using a biochemical test and 16S rRNA gene sequencing analysis. The patient was treated with probiotics and ciprofloxacin and recovered after 3 days. To our knowledge, this is the first report describing the potential role of B. pumilus as a foodborne pathogen in Kenya and highlights the importance of good hygiene and food preparation practices.

Biosafety and biosecurity capacity building: insights from implementation of the NUITM-KEMRI biosafety training model.

The NUITM-KEMRI biosafety training program was developed for capacity building of new biosafety level three (BSL-3) laboratory users. The training program comprehensively covers biosafety and biosecurity theory and practice. Its training curriculum is based on the WHO biosafety guidelines, local biosafety standards, and ongoing biosafety level three research activities in the facility, also taking into consideration the emerging public health issues. The program's training approach enhances the participant's biosafety and biosecurity knowledge and builds their skills through the hands-on practice sessions and mentorship training. Subsequently, the trainees are able to integrate acquired knowledge and good practices into their routine laboratory procedures. This article describes implementation of the NUITM-KEMRI biosafety training program.

Burden of Rotavirus and Enteric Bacterial Pathogens among Children under 5 Years of Age Hospitalized with Diarrhea in Suburban and Rural Areas in Kenya.

This cross-sectional descriptive study aimed to investigate the incidence of rotavirus and enteric bacterial infections among children up to 5 years old with diarrhea living in suburban and rural areas of Kenya. Between August 2011 and December 2013, a total of 1,060 diarrheal fecal specimens were obtained from 722 children at Kiambu County Hospital (KCH), located in a suburban area, and from 338 children from Mbita District Hospital (MDH), located in a rural part of western Kenya. Of the 1,060 isolates, group A rotavirus was detected in 29.6% (214/722) and 11.2% (38/338) fecal specimens from KCH and MDH, respectively. Diarrheagenic Escherichia coli (DEC) was found to be the most frequently isolated bacterial pathogens in both study areas (32.8% at KCH and 44.1% at MDH). Two different mixed infection patterns (virus/bacteria and bacteria/bacteria) were observed among patients. A significantly higher infection rate of rotavirus (17.6%, p = 0.001) and DEC (10.5%, p = 0.007) were observed during the dry season. Our study found that in both suburban and rural settings in Kenya, rotavirus and DEC are the principal cause of pediatric diarrhea and exhibit higher incidence during the dry season.

An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1.

In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment.

Prevalence, seasonal variation, and antibiotic resistance pattern of enteric bacterial pathogens among hospitalized diarrheic children in suburban regions of central Kenya.

BACKGROUND: The epidemiology of enteric pathogens has not been well studied in Kenya because of wide disparities in health status across the country. Therefore, the present study describes the prevalence of enteropathogenic bacteria, their seasonal variation, and antibiotic resistance profiles among hospitalized diarrheic children in a suburban region of central Kenya. METHODS: Fecal samples were collected between July 2009 and December 2013 from a total of 1410 children younger than 5 years, hospitalized with acute diarrhea in Kiambu County Hospital, Kenya. Conventional culture, biochemical, and molecular methods were conducted to identify causative bacterial pathogens and their virulence factors. Antimicrobial susceptibility tests were performed using E-test strips and VITEK-2 advanced expert system (AES) to evaluate the drug-resistance pattern of the isolates. RESULTS: Of the 1410 isolates, bacterial infections were identified in 474 cases. Diarrheagenic Escherichia coli (DEC) was the most frequently isolated pathogen (86.5%). Other pathogens such as Aeromonas (5.5%), Shigella (4%), Salmonella (3.4%), Providencia (3.2%), Vibrio spp. (1.1%), Yersinia enterocolitica (1.1%), and Plesiomonas shigelloides (0.2%) were also identified. Mixed bacterial infection was observed among 11.1% of the cases. The highest infection rate was found during the dry season (59.3%, p = 0.04). Most of the DEC was found to be multidrug resistant to trimethoprim/sulfamethoxazole 97.6%, amoxicillin 97.6%, erythromycin 96.9%, ampicillin 96.6%, and streptomycin 89%. CONCLUSIONS: This study suggests that DEC is the leading diarrhea-causing bacterial pathogen circulating in central Kenya, and seasonality has a significant effect on its transmission. Proper antibiotic prescription and susceptibility testing is important to guide appropriate antimicrobial therapy.

First Report of a Foodborne Providencia alcalifaciens Outbreak in Kenya.

Providencia alcalifaciens is an emerging bacterial pathogen known to cause acute gastroenteritis in children and travelers. In July 2013, P. alcalifaciens was isolated from four children appearing for diarrhea at Kiambu District Hospital (KDH) in Kenya. This study describes the outbreak investigation, which aimed to identify the source and mechanisms of infection. We identified seven primary and four secondary cases. Among primary cases were four mothers who had children and experienced mild diarrhea after eating mashed potatoes. The mothers reported feeding children after visiting the toilet and washing their hands without soap. P. alcalifaciens was detected from all secondary cases, and the isolates were found to be clonal by random amplified polymorphic DNA (RAPD) fingerprinting. Our study suggests that the outbreak was caused by P. alcalifaciens, although no fluid accumulation was observed in rabbit ileal loops. The vehicle of the outbreak was believed to be the mashed potato dish, but the source of P. alcalifaciens could not be confirmed. We found that lack of hygiene, inadequate food storage, and improper hand washing before food preparation was the likely cause of the current outbreak. This is the first report of a foodborne infection caused by P. alcalifaciens in Kenya.

The NUITM-KEMRI P3 Laboratory in Kenya: Establishment, Features, Operation and Maintenance.

A biocontainment facility is a core component in any research setting due to the services it renders towards comprehensive biosafety observance. The NUITM-KEMRI P3 facility was set up in 2007 and has been actively in use since 2010 by researchers from this and other institutions. A number of hazardous agents have been handled in the laboratory among them MDR-TB and yellow fever viruses. The laboratory has the general physical and operational features of a P3 laboratory in addition to a number of unique features, among them the water-air filtration system, the eco-mode operation feature and automation of the pressure system that make the facility more efficient. It is equipped with biosafety and emergency response equipments alongside common laboratory equipments, maintained regularly using daily, monthly and yearly routines. Security and safety is strictly observed within the facility, enhanced by restricted entry, strict documentation and use of safety symbols. Training is also engrained within the operation of the laboratory and is undertaken and evaluated annually. Though the laboratory is in the process of obtaining accreditation, it is fully certified courtesy of the manufactures' and constructed within specified standards.