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Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975-1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.

When properly used and adequately maintained, a biological safety cabinet (BSC) provides protection to the laboratory worker, the products, and the environment. Knowledge about proper use of the BS...

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.

Between July 2009 and June 2014, a total of 1,546 fecal specimens were collected from children

In this study, we developed a new metal mesh device (MMD) sensor to quantify the amount of aerosol particles. MMDs are substrate materials consisting of thin membranes with a periodic structure on smooth surfaces that can be used to detect and separate particles. The surface structure comprises square apertures of characteristic size that bestow unique optical properties. Aerosol particles, including the damaging environmental pollutant particulate matter 2.5 (PM2.5), were separated based on size and captured on MMD surfaces using a small air pump. The amount of captured particles was evaluated by band shifts in infrared (IR) transmittance spectra. Furthermore, bacterial genomic DNA attached to aerosol particles could be extracted with high yield, and extracted DNA resulted in bacterial constitution, as demonstrated by next-generation sequencing (NGS) analysis of bacterial 16S ribosomal DNA. MMD sensors were used to analyze aerosol particles from suburbs of Kenya and Japan. The results provided information on the bacterial biota of the environment in each local area, and pathogenic bacteria were identified. The results suggest that MMD sensors are useful tools for epidemiological and environmental studies, as well as quality control applications for drugs, fine chemicals, and food components.

OBJECTIVES A two-dose oral monovalent rotavirus vaccine (RV1) was introduced into the Kenyan National Immunization Program in July 2014. We assessed trends in hospitalisation for rotavirus-specific acute gastroenteritis (AGE) and strain distribution among children

In this study, a sensitive fluorescence sensor was developed for the detection of small, fluorescence-labeled particles dispersed in a solution. The prototype system comprises of a laser confocal optical system and a mechanical sample stage to detect photon bursting of fluorescence-labeled small particles in sample volumes less than 5 μL within 3 minutes. To examine the feasibility of the prototype system as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. The advantages and disadvantages of this immunosensor with respect to ELISA and RT-PCR, the current gold standards for virus detection, are discussed.

A monovalent rotavirus vaccine (RV1) was introduced into the National Immunization Program in Kenya in July 2014. We examined the impact of the vaccine on hospitalization for all-cause acute gastroenteritis (AGE) and rotavirus-specific AGE and strain distribution at a large referral hospital which serves a predominantly peri-urban population in Central Kenya. Data on rotavirus AGE and strain distribution were derived from ongoing hospital-based AGE surveillance. Hospital administrative data were used to compare trends in all-cause AGE. Pre-vaccine (July 2009-June 2014) and post-vaccine (July 2014-June 2016) periods were compared for changes in hospitalization for all-cause AGE and rotavirus AGE and strain distribution. Following the vaccine introduction, the proportion of children aged

Rotavirus gastroenteritis is an important cause of childhood morbidity and mortality in Kenya. In July 2014, Kenya introduced the rotavirus vaccine into her national immunization program. Although immunization coverage is crucial in assessing the real-world impact of this vaccine, variability in the vaccine coverage across the country is likely to occur. In view of this, we estimated the extent of coverage for the rotavirus vaccine at two socio-economically different sub-counties using the administrative data. The findings indicate disparities in vaccine coverage and access between the sub-counties and, thus, underscore the need to strengthen immunization systems to facilitate timely, accessible, and equitable vaccine delivery across the country. Both sub-counties recorded high vaccine dropout, suggestive of poor utilization of the vaccine. In this regard, increased social mobilization is needed to encourage vaccine compliance and to enhance tracking of vaccine defaulters. While efforts to improve the accuracy of the administrative coverage estimates are crucial, vaccination coverage surveys will be needed to verify the administrative coverage data and help identify specific factors relating to rotavirus vaccine coverage in the country.

長崎大学学位論文 [学位記番号]博（医歯薬）甲第963号 [学位授与年月日]平成29年3月21日, (2017-03-21)

G12 rotaviruses are globally emerging rotavirus strains causing severe childhood diarrhea. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed, of which only one G12P[4] and one G12P[6] are from Africa. In this study, we sequenced and characterized the complete genomes of three G12 strains (RVA/Human-tc/KEN/KDH633/2010/G12P[6], RVA/Human-tc/KEN/KDH651/2010/G12P[8], and RVA/Human-tc/KEN/KDH684/2010/G12P[6]) identified in three stool specimens from children with acute diarrhea in Kenya, Africa. On whole genomic analysis, all three Kenyan G12 strains were found to have a Wa-like genetic backbone: G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strains KDH633 and KDH684) and G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strain KDH651). Phylogenetic analysis showed that most genes of the three strains examined in this study were genetically related to globally circulating human G1, G9, and G12 strains. Of note is that the NSP4 genes of strains KDH633 and KDH684 appeared to be of porcine origin, suggesting the occurrence of reassortment between human and porcine strains. Furthermore, strains KDH633 and KDH684 were very closely related to each other in all the 11 gene segments, indicating derivation of the two strains from a common origin. On the other hand, strain KDH651 consistently formed distinct clusters of 10 of the 11 gene segments (VP1-2, VP4, VP6-7, and NSP1-5), indicating a distinct origin of strain KDH651 from that of strains KDH633 and KDH684. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Kenya. Our observations will provide important insights into the evolutionary dynamics of emerging G12 rotaviruses in Africa.

Pathogens handled in a Biosafety Level 3 (BSL-3) containment laboratory pose significant risks to laboratory staff and the environment. It is therefore necessary to develop competency and proficiency among laboratory workers and to promote appropriate behavior and practices that enhance safety through biosafety training. Following the installation of our BSL-3 laboratory at the Center for Microbiology Research-Kenya Medical Research Institute in 2006, a biosafety training program was developed to provide training on BSL-3 safety practices and procedures. The training program was developed based on World Health Organization specifications, with adjustments to fit our research activities and biosafety needs. The program is composed of three phases, namely initial assessment, a training phase including theory and a practicum, and a final assessment. This article reports the content of our training program.

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., Tropical Medicine and Health, 41(1), pp.27-37; 2013

Rapid detection of viruses and bacteria in clinical specimens is important for the prevention of epidemics. In this study, we developed a sensitive fluorescence sensor that can detect small, fluorescence-labeled particles dispersed in a solution. The system comprises a laser confocal optical system and a mechanical sample stage. The laser generates fluorescence fluctuations from small particles in a sample droplet on the sample stage. The optical system collects this epifluorescence data with a highly sensitive photomultiplier tube. A prototype detected particles within a few minutes in sample volumes smaller than 20 μL. To examine the feasibility of using this immunosensor as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. We evaluated the advantages and disadvantages of the immunosensor in comparison with ELISA and RT-PCR, which are considered to be the gold standards for virus detection.

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.

Identification of effective indigenous rhizobia isolates would lead to development of efficient and affordable rhizobia inoculants. These can promote nitrogen fixation in smallholder farming systems of Kenya. To realize this purpose, two experiments were conducted under greenhouse conditions using two common bean cultivars; Mwezi moja (bush type) and Mwitemania (climbing type) along with soybean cultivar SB 8. In the first experiment, the common bean cultivars were treated with rhizobia inoculants including a consortium of native isolates, commercial isolate (CIAT 899), a mixture of native isolates and CIAT 899, and a control with no inoculation. After 30 days, the crop was assessed for nodulation, shoot and root dry weights, and morphological features. In the second experiment, soybean was inoculated with a consortium of native isolates, commercial inoculant (USDA 110), and a mixture of commercial and native isolates. Remarkably, the native isolates significantly (p<0.001) increased nodulation and shoot dry weight across the two common bean varieties compared to the commercial inoculant, CIAT 899. Mixing of the native rhizobia species and commercial inoculant did not show any further increase in nodulation and shoot performance in both crops. Further field studies will ascertain the effectiveness and efficiency of the tested indigenous isolates.

This study introduces a new optical metal mesh (MM) sensor for detection of aerosol particles. The base material of the sensor corresponds to a thin metal membrane with unique optical properties and a micromesh structure. A small air pump pulls air through the MM membrane, permitting capture of aerosol particles on the micromesh surface and subsequent detection of the particles via infrared (IR) techniques. Here, MM membranes with different square apertures (side length: 4.0, 1.8, or 1.1 μm) were combined to form two-membrane stacked MM sensors. The sensors were used to collect particulate matter 2.5 (PM2.5; average diameter: ≤ 2.5 μm) as an indication of environmental pollution in the suburbs of Nairobi, Kenya. The elemental composition of each particle within captured PM2.5 was determined via wavelength-dispersive X-ray spectrometry. The results suggest that stacked MM sensors can successfully provide information about aerosol particle pollutants in the local environment.