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.

Carbapenem-resistant Acinetobacter baumannii infections: Antimicrobial resistance patterns and risk factors for acquisition in a Kenyan intensive care unit.

Objectives: Multidrug-resistant (MDR) Acinetobacter baumannii (AB), especially carbapenem-resistant (CR) strains, presents a significant challenge in intensive care units (ICUs) but surveillance data in many resource-constrained countries is inadequate. Here, we determined the prevalence of MDRAB and risk factors for infection and mortality in ICU-admitted patients. Methods: A cross-sectional study among 132 consecutive patients between July 2019 and July 2020, with infected patients followed for 30 days from sample collection to ICU discharge/death. Blood, urine, and tracheal aspirate samples were processed following the standard bacteriological procedures. Isolate identity and antimicrobial susceptibility were elucidated by VITEK 2 Compact system. Results: The prevalence of MDRAB was 22.7% (30/132), mostly from urine samples (12.1%, 16/132), and dominated by CRAB (83.3%) that were colistin-nonresistant and exhibited high multiple antibiotic resistance indices, ranging from 0.64-0.91. Risk factors for infection were occupation (adjusted odds ratio = 4.41, P = 0.016) and interhospital referral status (adjusted odds ratio = 0.14, P = 0.001). ICU mortality was 20% (6/30). Conclusion: Our findings underpin the need for strict adherence to and evaluation of infection prevention and control, and continuous surveillance of CRAB in ICU, especially among the risk groups, in the current study setting and beyond.

Survival rate, blood feeding habits and sibling species composition of Aedes simpsoni complex: Implications for arbovirus transmission risk in East Africa.

Aedes simpsoni complex has a wide distribution in Africa and comprises at least three described sub-species including the yellow fever virus (YFV) vector Ae. bromeliae. To date, the distribution and relative contributions of the sub-species and/or subpopulations including bionomic characteristics in relation to YF transmission dynamics remain poorly studied. In this study conducted in two areas with divergent ecosystems: peri-urban (coastal Rabai) and rural (Rift Valley Kerio Valley) in Kenya, survival rate was estimated by parity in Ae. simpsoni s.l. mosquitoes sampled using CO2-baited BG Sentinel traps. We then applied PCR targeting the nuclear internal transcribed spacer 2 (ITS2), region followed by sequencing and phylogenetic analytics to identify the sibling species in the Ae. simpsoni complex among parous and blood fed cohorts. Our results show that Ae. bromeliae was the most dominant sub-species in both areas, exhibiting high survival rates, human blood-feeding, and potentially, high vectorial capacity for pathogen transmission. We document for the first time the presence of Ae. lilii in Kenya and potentially yet-to-be described species in the complex displaying human feeding tendencies. We also infer a wide host feeding range on rodents, reptile, and domestic livestock besides humans especially for Ae. bromeliae. This feeding trend could likely expose humans to various zoonotic pathogens. Taken together, we highlight the utility of genotype-based analyses to generate precision surveillance data of vector populations for enhanced disease risk prediction and to guide cost-effective interventions (e.g. YF vaccinations).

Multidrug-Resistant Shigellosis among Children Aged below Five Years with Diarrhea at Banadir Hospital in Mogadishu, Somalia.

Globally, shigellosis remains the second leading cause of diarrhea-associated deaths among children under five years of age, and the infections are disproportionately higher in resource-limited settings due to overcrowding, poor sanitation, and inadequate safe drinking water. The emergence and global spread of multidrug-resistant (MDR) Shigella are exacerbating the shigellosis burden. We adopted a cross-sectional study design to determine the distribution and antimicrobial susceptibility (AST) patterns of Shigella serogroups among children aged below five years presenting with diarrhea at Banadir Hospital in Mogadishu, Somalia, from August to October 2019. Stool and rectal swab samples were collected from 180 children consecutively enrolled using a convenient sampling technique and processed following standard bacteriological methods. AST was determined using the Kirby-Bauer disc diffusion method and interpreted as per the Clinical Laboratory Standard Institute (2018) guidelines. Shigellosis prevalence was 20.6% (37/180), and S. flexneri (26/37 (70.3%)) was the predominant serogroup. All the serogroups were 100% resistant to ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT), and tetracycline (TE). Ceftriaxone (CRO) resistance was the highest among S. sonnei (66.7%) isolates. 19.2% of S. flexneri and S. sonnei (50%) serogroups were resistant to ciprofloxacin (CIP), but all S. dysenteriae type 1 isolates remained (100%) susceptible. Forty percent of CIP-susceptible S. dysenteriae type 1 were resistant to CRO. Seven MDR Shigella phenotypes were identified, dominated by those involving resistance to AMP, SXT, and TE (100%). Our findings showed a high prevalence of shigellosis with S. flexneri as the most predominant serogroup among children under five years of age in Banadir Hospital, Somalia. AMP and SXT are no longer appropriate treatments for shigellosis in children under five years in Banadir Hospital. MDR Shigella strains, including those resistant to CIP and CRO, have emerged in Somalia, posing a public health challenge. Therefore, there is an urgent need for AMR surveillance and continuous monitoring to mitigate the further spread of the MDR Shigella strains in Banadir Hospital and beyond.

Surveillance of Culicine Mosquitoes in Six Villages of Taita-Taveta County, Kenya, With Host Determinations From Blood-Fed Females.

Culicine mosquitoes are vectors of human disease-causing pathogens like filarial worms and several arthropod-borne viruses (arboviruses). Currently, there has been an increase in emerging and re-emerging vector-borne diseases along coastal Kenya, which has been of major concern in public health. This study aimed at determining culicine mosquito species abundance, diversity and their host feeding preferences in Taita-Taveta County, Coastal Kenya. Entomological sampling was done during the long-wet season (March and May) and long dry season (June to October) 2016-2018. Mosquito sampling was done using CDC light traps and Backpack aspiration for indoor and outdoor environments. All culicine mosquitoes collected were identified morphologically and categorized according to their physiological status. Blood fed culicine mosquitoes were tested for bloodmeal sources using ELISA. In total, 3,278 culicine mosquitoes were collected, of which 738 (22.5 %) were found indoors and 2,540, (77.5 %) outdoors. The mosquitoes consisted of 18 species belonging to four genera: Aedes (7), Culex (8), Mansonia (2), and Coquillettidia (1). Overall, there was high mosquito species diversity (H) in outdoors (H = 2.4339) than in indoors (H = 2.2523), whereas even distribution (EH) was higher in indoors (EH = 0.9064) than outdoors (EH = 0.8266). Majorly the bloodmeals identified were from multiple host sources with (51.6%), single hosts (41.3%), and unidentified (7.2%). This study has demonstrated a high diversity of culicine mosquitoes with relaxed feeding tendencies. These mosquitoes are contributing to mosquito biting nuisance and the likelihood of exposure of populations to diseases of public health.

Rectal gonorrhoea and chlamydia among men who have sex with men in coastal Kenya.

Background: Men who have sex with men (MSM) have a higher prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections compared to the rest of the population, often remaining undiagnosed. In Kenya, prevalence of rectal CT and NG infection and NG antimicrobial sensitivity are poorly described. Methods: MSM who reported receptive anal intercourse (RAI) were recruited from an ongoing human immunodeficiency virus acquisition and treatment study in coastal Kenya in 2016-2017. Rectal swabs were collected at two time points 6 months apart to estimate prevalence and incidence of CT/NG infection using a molecular point-of-care assay. Participants positive for CT or NG were treated according to national guidelines. NG culture and antimicrobial susceptibility testing was performed. Participant and risk behaviour characteristics were collected and association with baseline CT/NG prevalence assessed by multivariable regression analysis. Results: Prevalence of CT/NG in 104 MSM was 21.2% (CT 13.5%, NG 9.6%, dual infection 1.9%) at baseline and 25.9% in 81 MSM at follow-up (CT 14.8%, NG 14.8%, dual infection 3.7%). CT/NG incidence was estimated at 53.0 (95% CI, 34.5-81.3) per 100 person-years. Most CT/NG positive participants were asymptomatic: 95.5% at baseline and 100% at follow-up. CT/NG infection was associated with being paid for sex [adjusted odds ratio (aOR)=6.2, 95% CI (1.7-22.9)] and being in formal employment [aOR=7.5, 95% CI (1.1-49.2)]. Six NG isolates were obtained at follow-up; all were susceptible to ceftriaxone and cefixime and all were resistant to penicillin, tetracycline and ciprofloxacin. Conclusions: There is a high prevalence and incidence of asymptomatic rectal CT and NG in MSM reporting RAI in coastal Kenya. MSM who were paid for sex or had formal employment were more likely to be infected with CT/NG suggesting increased risk behaviour during transactional sex. Antimicrobial susceptibility results suggest that current antibiotic choices in Kenya are appropriate for NG treatment.