Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Effect of Time Since Death on Multipathogen Molecular Test Results of Postmortem Specimens Collected Using Minimally Invasive Tissue Sampling Techniques.

BACKGROUND: We used postmortem minimally invasive tissue sampling (MITS) to assess the effect of time since death on molecular detection of pathogens among respiratory illness-associated deaths. METHODS: Samples were collected from 20 deceased children (aged 1-59 months) hospitalized with respiratory illness from May 2018 through February 2019. Serial lung and/or liver and blood samples were collected using MITS starting soon after death and every 6 hours thereafter for up to 72 hours. Bodies were stored in the mortuary refrigerator for the duration of the study. All specimens were analyzed using customized multipathogen TaqMan® array cards (TACs). RESULTS: We identified a median of 3 pathogens in each child's lung tissue (range, 1-8; n = 20), 3 pathogens in each child's liver tissue (range, 1-4; n = 5), and 2 pathogens in each child's blood specimen (range, 0-4; n = 5). Pathogens were not consistently detected across all collection time points; there was no association between postmortem interval and the number of pathogens detected (P = .43) and no change in TAC cycle threshold value over time for pathogens detected in lung tissue. Human ribonucleoprotein values indicated that specimens collected were suitable for testing throughout the study period. CONCLUSIONS: Results suggest that lung, liver, and blood specimens can be collected using MITS procedures up to 4 days after death in adequately preserved bodies. However, inconsistent pathogen detection in samples needs careful consideration before drawing definitive conclusions on the etiologic causes of death.

Knowledge and attitude of Kenyan healthcare workers towards pandemic influenza disease and vaccination: 9 years after the last influenza pandemic.

BACKGROUND: Healthcare workers (HCWs) are at high risk of exposure and transmission of infectious respiratory pathogens like influenza. Despite the potential benefits, safety and efficacy of influenza vaccination, vaccines are still underutilized in Africa, including among HCWs. METHOD: From May-June 2018, we conducted a cross-sectional, self-administered, written survey among HCWs from seven counties in Kenya and assessed their knowledge attitudes and perceptions towards pandemic influenza disease and vaccination. Using regression models, we assessed factors that were associated with the HCW's knowledge of pandemic influenza and vaccination. RESULTS: A total of 2,035 HCWs, representing 49% of the targeted respondents from 35 health facilities, completed the question. Sixty eight percent of the HCWs had ever heard of pandemic influenza, and 80.0% of these were willing to receive pandemic influenza vaccine if it was available. On average, Kenyan HCWs correctly answered 55.0% (95% CI 54.0-55.9) of the questions about pandemic influenza and vaccination. Physicians (65.6%, 95% CI 62.5-68.7) and pharmacists (61.7%, 95% CI 57.9-65.5) scored higher compared to nurses (53.1%, 95% CI 51.7-54.5). HCWs with 5 or more years of work experience (55.8, 95% CI 54.5-57.0) had marginally higher knowledge scores compared to those with less experience (53.9%, 95% CI 52.5-55.3). Most participants who were willing to receive pandemic influenza vaccine did so to protect their relatives (88.7%) or patients (85.9%). CONCLUSION: Our findings suggest moderate knowledge of pandemic influenza and vaccination by HCWs in Kenya, which varied by cadre and years of work experience. These findings highlight the need for continued in-service health education to increase the HCW's awareness and knowledge of pandemic influenza to increase acceptance of influenza vaccination in the case of a pandemic.

Postmortem Study of Cause of Death Among Children Hospitalized With Respiratory Illness in Kenya.

BACKGROUND: In resource-limited settings, acute respiratory infections continue to be the leading cause of death in young children. We conducted postmortem investigations in children <5 years hospitalized with a clinical diagnosis of respiratory disease at Kenya's largest referral hospital. METHODS: We collected respiratory and other tissues postmortem to examine pathologic processes using histology, molecular and immunohistochemistry assays. Nasopharyngeal, trachea, bronchi and lung specimens were tested using 21-target respiratory pathogen real-time reverse transcription polymerase chain reaction assays deployed on Taqman Array Cards. Expert panels reviewed all findings to determine causes of death and associated pathogens. RESULTS: From 2014 to 2015, we investigated 64 pediatric deaths (median age 7 months). Pneumonia was determined as cause of death in 70% (42/52) of cases where death was associated with an infectious disease process. The main etiologies of pneumonia deaths were respiratory syncytial virus (RSV) (n = 7, 19%), Pneumocystis jirovecii (n = 7, 19%), influenza A (n = 5, 14%) and Streptococcus pneumoniae (n = 5, 14%)-10% of cases had multi-pathogen involvement. Among the other 10 deaths associated with a nonpneumonia infectious process, 4 did not have an etiology assigned, the others were associated with miliary tuberculosis (2), cerebral thrombosis due to HIV (1), Enterobacteriaceae (1), rotavirus (1), and 1 case of respiratory infection with severe hypokalemia associated with RSV. CONCLUSIONS: In spite of well-established vaccination programs in Kenya, some deaths were still vaccine preventable. Accelerated development of RSV monoclonal antibodies and vaccines, introduction of seasonal influenza vaccination, and maintenance or improved uptake of existing vaccines can contribute to further reductions in childhood mortality.

Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport.

Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport-associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of Campylobacter bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of Eucampyvirinae CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log10 PFU mL-1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze-dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (p = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log10 PFU mL-1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log10 PFU mL-1). We propose that lyophilization offers a convenient method to preserve and transport Campylobacter phages, with minimal titer reduction after the drying process.

The Epidemiology and Burden of Influenza B/Victoria and B/Yamagata Lineages in Kenya, 2012-2016.

BACKGROUND: The impact of influenza B virus circulation in Sub-Saharan Africa is not well described. METHODS: We analyzed data from acute respiratory illness (ARI) in Kenya. We assessed clinical features and age-specific hospitalization and outpatient visit rates by person-years for influenza B/Victoria and B/Yamagata and the extent to which circulating influenza B lineages in Kenya matched the vaccine strain component of the corresponding season (based on Northern Hemisphere [October-March] and Southern Hemisphere [April-September] vaccine availability). RESULTS: From 2012 to 2016, influenza B represented 31% of all influenza-associated ARIs detected (annual range, 13-61%). Rates of influenza B hospitalization and outpatient visits were higher for <5 vs ≥5 years. Among <5 years, B/Victoria was associated with pneumonia hospitalization (64% vs 44%; P = .010) and in-hospital mortality (6% vs 0%; P = .042) compared with B/Yamagata, although the mean annual hospitalization rate for B/Victoria was comparable to that estimated for B/Yamagata. The 2 lineages co-circulated, and there were mismatches with available trivalent influenza vaccines in 2/9 seasons assessed. CONCLUSIONS: Influenza B causes substantial burden in Kenya, particularly among children aged <5 years, in whom B/Victoria may be associated with increased severity. Our findings suggest a benefit from including both lineages when considering influenza vaccination in Kenya.

Factors Influencing Acceptance of Post-Mortem Examination of Children at a Tertiary Care Hospital in Nairobi, Kenya.

BACKGROUND: Clinical autopsies are not often part of routine care, despite their role in clarifying cause of death. In fact, autopsy rates across the world have declined and are especially low in sub-Saharan Africa. OBJECTIVES: We set out to identify factors associated with acceptance of pediatric autopsies among parents of deceased children less than five years old, and examined local preferences for minimally invasive tissue sampling (MITS) procedures during post-mortem (PM) examinations. METHODS: From December 2016 to September 2017, we contacted 113 parents/next of kin who had been previously approached to consent to a PM examination of their deceased child as part of a Kenyan study on cause of death. Interviews occurred up to three years after the death of their child. FINDINGS: Seventy-three percent (83/113) of eligible study participants were enrolled, of whom 62/83 (75%) had previously consented to PM examination of their child. Those who previously consented to PM had higher levels of education, were more likely employed, and had more knowledge about certain aspects of autopsies than non-consenters. The majority (97%) of PM consenters did so because they wanted to know the cause of death of their child, and up to a third believed autopsy studies helped advance medical knowledge. Reasons for non-consent to PM examination included: parents felt there was no need for further examination (29%) or they were satisfied with the clinical diagnosis (24%). Overall, only 40% of study participants would have preferred MITS procedures to conventional autopsy. However, 81% of autopsy non-consenters would have accepted PM examination if it only involved MITS techniques. CONCLUSION: There is potential to increase autopsy rates by strengthening reasons for acceptance and addressing modifiable reasons for refusals. Although MITS procedures have the potential to improve autopsy acceptance rates, they were not significantly preferred over conventional autopsies in our study population.

Comparison of Minimally Invasive Tissue Sampling With Conventional Autopsy to Detect Pulmonary Pathology Among Respiratory Deaths in a Resource-Limited Setting.

OBJECTIVES: We compared minimally invasive tissue sampling (MITS) with conventional autopsy (CA) in detection of respiratory pathology/pathogens among Kenyan children younger than 5 years who were hospitalized with respiratory disease and died during hospitalization. METHODS: Pulmonary MITS guided by anatomic landmarks was followed by CA. Lung tissues were triaged for histology and molecular testing using TaqMan Array Cards (TACs). MITS and CA results were compared for adequacy and concordance. RESULTS: Adequate pulmonary tissue was obtained by MITS from 54 (84%) of 64 respiratory deaths. Comparing MITS to CA, full histologic diagnostic concordance was present in 23 (36%) cases and partial concordance in 19 (30%), an overall 66% concordance rate. Pathogen detection using TACs had full concordance in 27 (42%) and partial concordance in 24 (38%) cases investigated, an overall 80% concordance rate. CONCLUSIONS: MITS is a viable alternative to CA in respiratory deaths in resource-limited settings, especially if combined with ancillary tests to optimize diagnostic accuracy.

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya.

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 × 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in ≥ 90% reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.

Feasibility of Using Mobile ECG Recording Technology to Detect Atrial Fibrillation in Low-Resource Settings.

BACKGROUND: Screening for atrial fibrillation (AF), a major risk factor for stroke that is on the rise in Africa, is becoming increasingly critical. OBJECTIVES: This study sought to examine the feasibility of using mobile electrocardiogram (ECG) recording technology to detect AF. METHODS: In this prospective observational study, we used a mobile ECG recorder to screen 50 African adults (66% women; mean age 54.3 ± 20.5 years) attending Kijabe Hospital (Kijabe, Kenya). Five hospital health providers involved in this study's data collection process also completed a self-administered survey to obtain information on their access to the Internet and mobile devices, both factors necessary to implement ECG mobile technology. Outcome measures included feasibility (completion of the study and recruitment of the patients on the planned study time frame) and the yield of the screening by the mobile ECG technology (ability to detect previously undiagnosed AF). RESULTS: Patients were recruited in a 2-week period as planned; only 1 of the 51 patients approached refused to participate (98% acceptance rate). All of the 50 patients who agreed to participate completed the test and produced readable ECGs (100% study completion rate). ECG tracings of 4 of the 50 patients who completed the study showed AF (8% AF yield), and none had been previously diagnosed with AF. When asked about continuous access to Internet and personal mobile devices, almost all of the health care providers surveyed answered affirmatively. CONCLUSIONS: Using mobile ECG technology in screening for AF in low-resource settings is feasible, and can detect a significant proportion of AF cases that will otherwise go undiagnosed. Further study is needed to examine the cost-effectiveness of this approach for detection of AF and its effect on reducing the risk of stroke in developing countries.