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.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.

The epidemiology of fecal carriage of nontyphoidal Salmonella among healthy children and adults in three sites in Kenya.

BACKGROUND: Despite the importance of non-Typhoidal Salmonella (NTS) disease in Africa, epidemiologic data on carriage and transmission are few. These data are important to understand the transmission of NTS in Africa and to design control strategies. METHOD: To estimate the prevalence of stool carriage of NTS in Kenya, we conducted a cross-sectional study in Kilifi, Nairobi, and Siaya, sites with a low, moderate and high incidence of invasive NTS disease, respectively. At each site, we randomly selected 100 participants in each age-group of 0-11 months, 12-59 months, 5-14 years, 15-54 years and ≥55 years. We collected stool, venous blood (for hemoglobin and malaria rapid tests), anthropometric measurements, and administered a questionnaire on Water Access Sanitation and Hygiene (WASH) practices. Stool samples were cultured on selective agar for Salmonella; suspect isolates underwent serotyping and antimicrobial susceptibility testing. RESULT: Overall, 53 (3.5%) isolates of NTS were cultured from 1497 samples. Age-adjusted prevalence was 13.1% (95%CI 8.8-17.4) in Kilifi, 0.4% (95%CI 0-1.3) in Nairobi, and 0.9% (95%CI 0-2.0) in Siaya. Prevalence was highest among those aged 15-54 years (6.2%). Of 53 isolates; 5 were S. Enteritidis, 1 was S. Typhimurium. No S. Typhi was isolated. None of the risk factors were associated with carriage of NTS. All isolates were susceptible to all antibiotics tested, including ampicillin, chloramphenicol, ciprofloxacin and co-trimoxazole. CONCLUSION: Prevalence of fecal carriage was high in Kilifi, an area of low incidence of invasive NTS disease and was low in areas of higher incidence in Nairobi and Siaya. The age-prevalence, risk factors, geographical and serotype distribution of NTS in carriage differs from invasive disease.

Seroprevalence of non-typhoidal Salmonella disease and associated factors in children in Mukuru settlement in Nairobi County, Kenya.

Non-typhoidal Salmonella (NTS) infections remain a significant public health challenge especially in sub-Saharan Africa. NTS disease is endemic in Kenya and is associated with sporadic fatal outbreaks in several regions of the country with poor resource setting. Data is limited on background exposure of NTS in the population in endemic areas and the general immune status of the community most affected by NTS. The aim of the study was to determine the proportion of children exposed to Salmonella Enteritidis or Salmonella Typhimurium O antigen among the apparently healthy children and patients and the associated host and environmental factors among children attending selected healthcare facilities in Mukuru, Nairobi County, Kenya. A cross-sectional case-control study was conducted among patients and apparently healthy participants aged 0-5 years. Blood was collected and centrifuged to obtain serum. The serum was used to test for the presence of antibodies (IgA, IgG, IgM) against NTS using ELISA. A questionnaire was administered to obtain relevant demographic, socio-economic and healthcare utilization information. A total of 382 children were recruited into the study. The NTS seroprevalence was 12.6%. Among the apparently healthy participants, mean age of those exposed to NTS was 36 months and those not exposed was 27 months. Among patients, the mean age was 39 months and those not exposed was 30 months. The seroprevalence of NTS infection among the apparently healthy was significantly associated with cooking water, washing water and age of the child. Treating water using chlorine or boiling method was identified as being protective against contracting Salmonella Typhimurium/Enteritidis. Among the patients, the proportion of exposure was significantly associated with keeping animals and the chicken count. There is a high exposure to NTS among young children below five years of age and the population has developed immunity to the disease.

Cystic echinococcosis in donkeys in eastern Africa.

Cystic echinococcosis (CE) is endemic in humans and domestic animals in eastern Africa. All the species of the Echinococcus granulosus sensu lato complex have been reported in this region except for E. equinus, possibly due to the small number of studies involving equids. This study reports the frequency of different Echinococcus species in donkeys from eastern Africa. A total of 5961 donkeys were examined during meat inspection in 3 slaughterhouses in Kenya. Identification of Echinococcus spp. was achieved through polymerase chain reaction-restriction fragment-length polymorphism and sequencing of the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 gene. The prevalence of CE was 5.7% (337/5961). The 263 genotyped cysts belonged to E. equinus (n = 163), E. granulosus sensu stricto (n = 70), E. canadensis (G6/7) (n = 26) and E. ortleppi (n = 4). One donkey harboured a metacestode of Spirometra theileri. All E. equinus cases, except 2, originated from southern Ethiopia, whereas the other species were more evenly distributed across the study area. Most of the cysts belonging to E. equinus were fertile (111/163), while those of the other species were non-fertile. This is the first report of Echinococcus spp. in donkeys from sub-Saharan Africa and the first confirmation of E. equinus in East Africa. The frequent fertility of E. equinus cysts in donkeys affirms their suitability as intermediate hosts of this species, while low frequency and cyst fertility suggest a marginal role of donkeys in the transmission of E. granulosus s. s., E. canadensis (G6/7) and E. ortleppi.

Antimicrobial Resistance Rates and Surveillance in Sub-Saharan Africa: Where Are We Now?

Introduction: Although antimicrobials have traditionally been used to treat infections and improve health outcomes, resistance to commonly used antimicrobials has posed a major challenge. An estimated 700,000 deaths occur globally every year as a result of infections caused by antimicrobial-resistant pathogens. Antimicrobial resistance (AMR) also contributes directly to the decline in the global economy. In 2019, sub-Saharan Africa (SSA) had the highest mortality rate (23.5 deaths per 100,000) attributable to AMR compared to other regions. Methods: We searched PubMed for articles relevant to AMR in pathogens in the WHO-GLASS list and in other infections of local importance in SSA. In this review, we focused on AMR rates and surveillance of AMR for these priority pathogens and some of the most encountered pathogens of public health significance. In addition, we reviewed the implementation of national action plans to mitigate against AMR in countries in SSA. Results and Discussion: The SSA region is disproportionately affected by AMR, in part owing to the prevailing high levels of poverty, which result in a high burden of infectious diseases, poor regulation of antimicrobial use, and a lack of alternatives to ineffective antimicrobials. The global action plan as a strategy for prevention and combating AMR has been adopted by most countries, but fewer countries are able to fully implement country-specific action plans, and several challenges exist in many settings. Conclusion: A concerted One Health approach will be required to ramp up implementation of action plans in the region. In addition to AMR surveillance, effective implementation of infection prevention and control, water, sanitation, and hygiene, and antimicrobial stewardship programs will be key cost-effective strategies in helping to tackle AMR.

Antimicrobial Resistance in Endemic Enteric Infections in Kenya and the Region, and Efforts Toward Addressing the Challenges.

Resistance to commonly available antimicrobials is a major threat to the fight against endemic bacterial diseases in sub-Saharan Africa, with a majority of the population unable to afford alternative effective antimicrobial options for management of these diseases. Diseases such as typhoid, cholera, and invasive nontyphoidal Salmonella are among the key enteric infections endemic in most parts of sub-Saharan Africa, especially in displaced populations and among the urban populations living in overcrowded informal settlements. Here, we explore the prevalence and the genomic epidemiology of these infections and the growing problem of multidrug resistance, including emerging resistance to the last line of treatment for these infections. Prevalence rates to commonly available antimicrobials, including ampicillin, chloramphenicol, cotrimoxazole, and tetracycline, now range between 65% and 80%, while 15%-20% of recently studied isolates show reduced susceptibility to fluoroquinolones and emerging resistance to extended-spectrum ß-lactams mediated by the CTX-M-15 gene carried on a highly mobile genetic element. The high prevalence of multidrug-resistant isolates including resistance to reserve antibiotics, calls for enhanced control and management options. It will be important for governments in the region to enhance the implementation of national action plans, as guided by the global action plan championed by the World Health Organization, to combat the threat of antimicrobial resistance. However, to yield meaningful results, these efforts will require a strong commitment and enhancement at all levels of healthcare in order. In addition, the use of World Health Organization-approved vaccines in the short to medium term and improvement of water and sanitation in the long term will reduce the burden of disease and antimicrobial resistance in the region.

Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya.

Background: Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. Methods: In a 3-year case-control study, we investigated typhoid among children aged <16 years (4670 febrile cases and 8549 age matched controls) living in an informal settlement, Nairobi, Kenya. Results: 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1 %. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype 58), with the majority of each (76% and 88%) being multidrug-resistant strains in three sublineages of the H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. Conclusions: The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between cases and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting. Funding: National Institutes of Health (R01AI099525); Wellcome Trust (106158/Z/14/Z); European Commission (TyphiNET No 845681); National Institute for Health Research (NIHR); Bill and Melinda Gates Foundation (OPP1175797).

Entamoeba species infection in patients seeking treatment for diarrhea and abdominal discomfort in Mukuru informal settlement in Nairobi, Kenya.

Entamoeba histolytica is the only pathogenic species of the Entamoeba genus and is morphologically identical to E. dispar/E. moshkovskii (Entamoeba complex) hence cannot be microscopically differentiated. The other Entamoeba spp. found in humans (E. hartmanni, E. polecki, and E. coli) can be differentiated morphologically from this Entamoeba complex. However, some of their morphologic features overlap making differential diagnosis difficult. This study aimed at determining the occurrence of Entamoeba spp. in patients seeking treatment for diarrhea and/or abdominal discomfort at two clinics in Mukuru informal settlement in Nairobi, Kenya. Faecal samples were collected from 895 patients, examined microscopically following direct wet smear and formal-ether concentration methods. Entamoeba spp. positive faecal samples were subjected to DNA extraction and species-specific nested polymerase chain reaction of the 18S ribosomal RNA (rRNA). By microscopy, Entamoeba spp. cysts or trophozoites were detected in 114/895 (12.7%, 95% Confidence Interval (CI) 10.6-15.1) faecal samples. By nested PCR, the prevalence was: E. histolytica (7.5%, 95% CI 5.9-9.4, 67/895) and E. dispar (8.2%, 95% CI 6.5-10.2, 73/895). Among the Entamoeba spp. complex positive samples, nested PCR detected E. coli and E. hartmanni DNA in 63/114 (55.3%) and 37/114 (32.5%), samples respectively. Among the E. histolytica/E. dispar PCR negative samples (32.5%), 21 (18.4%) contained cysts of either E. coli (19) or E. hartmanni (2) by nested PCR. Entamoeba spp. infections were most common among participants aged 21-30 years; however it was not significant (P = 0.7). Entamoeba spp. infections showed an inverse relationship with diarrhea being most common among participants without diarrhea (P = 0.0). The difference was significant for E. histolytica (P = 0.0) but not significant for E. dispar (P = 0.1). Only E. dispar infections were significantly associated with sex (P = 0.0). This study highlights the need for differentiation of E. histolytica from other Entamoeba spp. by molecular tools for better management of amoebiasis.

High relatedness of invasive multi-drug resistant non-typhoidal Salmonella genotypes among patients and asymptomatic carriers in endemic informal settlements in Kenya.

Invasive Non-typhoidal Salmonella (iNTS) disease is a major public health challenge, especially in Sub-Saharan Africa (SSA). In Kenya, mortality rates are high (20-25%) unless prompt treatment is instituted. The most common serotypes are Salmonella enterica serotype Typhimurium (S. Typhimurium) and Salmonella enterica serotype Enteritidis (S. Enteritidis). In a 5 year case-control study in children residing in the Mukuru informal settlement in Nairobi, Kenya, a total of 4201 blood cultures from suspected iNTS cases and 6326 fecal samples from age-matched controls were studied. From the laboratory cultures we obtained a total of 133 S. Typhimurium isolates of which 83(62.4%) came from cases (53 blood and 30 fecal) and 50(37.6%) from controls (fecal). A total of 120 S. Enteritidis consisted of 70(58.3%) from cases (43 blood and 27 fecal) and 50(41.7%) from controls (fecal). The S. Typhimurium population fell into two distinct ST19 lineages constituting 36.1%, as well as ST313 lineage I (27.8%) and ST313 lineage II (36.1%) isolates. The S. Enteritidis isolates fell into the global epidemic lineage (46.6%), the Central/Eastern African lineage (30.5%), a novel Kenyan-specific lineage (12.2%) and a phylogenetically outlier lineage (10.7%). Detailed phylogenetic analysis revealed a high level of relatedness between NTS from blood and stool originating from cases and controls, indicating a common source pool. Multidrug resistance was common throughout, with 8.5% of such isolates resistant to extended spectrum beta lactams. The high rate of asymptomatic carriage in the population is a concern for transmission to vulnerable individuals and this group could be targeted for vaccination if an iNTS vaccine becomes available.

Diversity of Taenia and Hydatigera (Cestoda: Taeniidae) in domestic dogs in Kenya.

Taenia species of domestic dogs can cause cysticercosis and coenurosis in a wide range of intermediate hosts including humans. Most taeniids of dogs are globally distributed, but some wildlife-transmitted species can be specific for certain regions. Generally, little information exists on the species composition and frequency in most regions of the world, which impairs risk assessment and control strategies. This study determined the range of taeniid species in dogs in four widely spaced areas of Kenya by genetic identification of eggs in faeces collected from the environment. Individual taeniid eggs were characterised by nested polymerase chain reaction of NADH dehydrogenase subunit 1 and cytochrome C oxidase 1 genes, restriction fragment length polymorphism and partial sequencing. Overall 79/1621 (4.9%) faecal samples contained eggs of Taenia or Hydatigera (8.0% in Turkana, 4.8% in Isiolo, 3.8% in Maasai Mara and 1.3% in Meru). Taenia hydatigena and T. multiceps were the most frequent, found in 36 and 15 samples, respectively. Other eggs found in the faeces belonged to T. serialis (sensu lato), T. madoquae (the first record in domestic dogs), T. ovis, T. saginata and Hydatigera taeniaeformis. Polymorphism of nad1 sequences revealed 22 and 8 haplotypes of T. hydatigena and T. multiceps, respectively. The results show the involvement of dogs in both domestic and sylvatic transmission cycles. In addition to the species range, this study provides data on the intraspecific diversity of T. hydatigena and T. multiceps in Kenya, which will serve as baseline information for further studies into cysticercosis and coenurosis in livestock and humans in the region.

Factors associated with occurrence of salmonellosis among children living in Mukuru slum, an urban informal settlement in Kenya.

BACKGROUND: In Kenya, typhoid fever and invasive non-typhoidal salmonellosis present a huge burden of disease, especially in poor-resource settings where clean water supply and sanitation conditions are inadequate. The epidemiology of both diseases is poorly understood in terms of severity and risk factors. The aim of the study was to determine the disease burden and spatial distribution of salmonellosis, as well as socioeconomic and environmental risk factors for these infections, in a large informal settlement near the city of Nairobi, from 2013 to 2017. METHODS: Initially, a house-to-house baseline census of 150,000 population in Mukuru informal settlement was carried out and relevant socioeconomic, demographic, and healthcare utilization information was collected using structured questionnaires. Salmonella bacteria were cultured from the blood and faeces of children < 16 years of age who reported at three outpatient facilities with fever alone or fever and diarrhea. Tests of association between specific Salmonella serotypes and risk factors were conducted using Pearson Chi-Square (χ2) test. RESULTS: A total of 16,236 children were recruited into the study. The prevalence of bloodstream infections by Non-Typhoidal Salmonella (NTS), consisting of Salmonella Typhimurium/ Enteriditis, was 1.3%; Salmonella Typhi was 1.4%, and this was highest among children < 16 years of age. Occurrence of Salmonella Typhimurium/ Enteriditis was not significantly associated with rearing any domestic animals. Rearing chicken was significantly associated with high prevalence of S. Typhi (2.1%; p = 0.011). The proportion of children infected with Salmonella Typhimurium/ Enteriditis was significantly higher in households that used water pots as water storage containers compared to using water directly from the tap (0.6%). Use of pit latrines and open defecation were significant risk factors for S. Typhi infection (1.6%; p = 0.048). The proportion of Salmonella Typhimurium/ Enteriditis among children eating street food 4 or more times per week was higher compared to 1 to 2 times/week on average (1.1%; p = 0.032). CONCLUSION: Typhoidal and NTS are important causes of illness in children in Mukuru informal settlement, especially among children less than 16 years of age. Improving Water, Sanitation and Hygiene (WASH) including boiling water, breastfeeding, hand washing practices, and avoiding animal contact in domestic settings could contribute to reducing the risk of transmission of Salmonella disease from contaminated environments.

Prevalence and Genotyping of Echinococcus Species from Livestock in Kajiado County, Kenya.

Cystic Echinococcosis (CE) is a widespread neglected zoonotic disease and is caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato. CE is more frequent in livestock-rearing areas and where people live a nomadic or seminomadic lifestyle such as in Kajiado County, Kenya. There is limited data on CE disease situation in the county of Maasailand; the present study, therefore, reports on the prevalence of CE in cattle, sheep, and goats and their relative importance in CE transmission in Kajiado County. In total, 1,486 livestock (388 cattle, 625 sheep, and 473 goats) slaughtered in two abattoirs were examined for the presence of hydatid cysts in various organs. Cyst isolates were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the NADH dehydrogenase subunit 1 gene (nad1). The overall prevalence of CE was 14.8% (220/1486), while prevalence per livestock species was 15.2% (72/473) in goats, 14.9% (93/625) in sheep, and 14.2% (55/388) in cattle. Out of the 421 cysts isolated, 389 cysts were successfully characterized to be either E. granulosus sensu stricto (s. s.), 356/389 (91.5%), E. canadensis (G6/7), 26/389 (6.7%), or E. ortleppi, 7/389 (1.8%). This record confirms predominance of E. granulosus s. s. in Maasailand and other parts of Kenya, while the importance of E. ortleppi and E. canadensis (G6/7) to the general CE burden in Maasailand might be higher than previously thought. More so, a higher infection pressure for humans by E. granulosus s. s. based on its abundance could be speculated. The study sheds significant light on CE situation in livestock in the nomadic/seminomadic society of the Maasai in Kajiado County and provides good bases to investigate human CE in the area.

Multidrug-resistant Nontyphoidal Salmonella Hotspots as Targets for Vaccine Use in Management of Infections in Endemic Settings.

BACKGROUND: Salmonella infections cause a disproportionately high number of deaths in Africa, especially among poor urban populations. The increasing level of multidrug-resistant (MDR) infections is a major cause of concern in these settings where alternative effective treatment is unavailable. Other options for management of these infections must be sought. The knowledge of hotspots in endemic settings can help to prioritize management and control measures in Kenya and the region. METHODS: Using blood cultures, we investigated children presenting with fever of unknown origin for Salmonella infections. We performed antimicrobial susceptibility testing and whole genome sequencing to further characterize Salmonella isolates. Using Global Positioning System technologies, we mapped Salmonella isolates to households of patients in the study site and determined risk factors associated with high concentration of cases in particular sites. RESULTS: A total of 281 Salmonella species (149 from blood and 132 from fecal samples) from febrile children <5 years of age were studied. These consisted of 85 Salmonella Typhimurium, 58 Salmonella Enteritidis, 32 other nontyphoidal Salmonella (NTS) serotypes, and 126 Salmonella Typhi. The prevalence of MDR invasive NTS (iNTS) was 77.2%, with 15% resistant to ceftriaxone, a drug that is last-line treatment for iNTS and other severe gram-negative infections in Kenya. Invasive NTS and S. Typhi together mapped around common water vending points and close to sewer convergence points in the highly populated village. CONCLUSIONS: These hotspots could be targeted for management and control strategies, including a combined introduction of typhoid and iNTS vaccines, aimed at reducing transmission in these endemic settings.

Molecular characterization of Echinococcus species in dogs from four regions of Kenya.

Cystic echinococcosis is endemic both in livestock and humans in many parts of Kenya. However, very little data exists on Echinococcus infections in dogs, and therefore their role in maintaining the transmission cycles and environmental contamination with eggs of Echinococcus species is unknown. The study aimed to establish the prevalence and distribution of Echinococcus granulosus sensu lato causing infection in dogs in Kenya. A total of 1621 dog faecal samples were collected from the environment in four different regions and examined microscopically for the presence of taeniid eggs. Up to 20 individual taeniid eggs per faecal sample were picked, lysed and genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the NADH dehydrogenase subunit 1 (nad1) gene. Eleven percent (178/1621) of faecal samples had taeniid eggs, while 4.4% (71/1621) contained Echinococcus spp. eggs. Area-wise, the faecal prevalence of Echinococcus spp. was 9.2% (48/524) in Turkana, 4.0% (20/500) in Maasai Mara, 0.7% (2/294) in Isiolo and 0.3% (1/303) in Meru. E. granulosus sensu stricto (s. s.) was the dominant Echinococcus taxon, followed by E. canadensis (G6/7) that was detected in 51 and 23 faecal samples, respectively. E. ortleppi was detected in only 5 faecal samples. We report for the first time the presence of E. felidis eggs in two dog faecal samples (from Maasai Mara region). Mixed infections of these taxa were also found in faecal samples, including: E. granulosus s. s. and E. canadensis (G6/7) (n = 7), E. granulosus s. s. and E. ortleppi (n = 1) and all three species (n = 1). The dog data presented here confirm the differences in diversity and abundance of Echinococcus spp. between regions of Kenya, correspond well with previously published data from livestock, and tentatively suggest a role of domestic dogs as a link between domestic and sylvatic cycles of Echinococcus spp.

Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea.

Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers). Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

Molecular characterization of group A rotaviruses in Mukuru slums Kenya: detection of novel strains circulating in children below 5 years of age.

BACKGROUND: Gastroenteritis is a public health concern due to high morbidity and mortality among children. Rotaviruses are the leading etiological agents of severe gastroenteritis in children and accounts for more than half a million deaths per year in Africa. The study aimed at investigating the rotavirus genotypes that were circulating in children aged 5 years and below in and around Mukuru slums in Nairobi County Kenya. METHODS: A purposive cross sectional sampling method was applied where 166 samples were collected from children below 5 years of age and taken to Kenya Medical Research Institute virology laboratory. Presence of rotaviruses was determined using reverse transcription polymerase chain reaction, while extraction was done using ZR Soil/Fecal RNA MicroPrep™ extraction kit. This was followed by reverse transcription and genotyping using various group A rotavirus primers. RESULTS: The G type was successfully determined in 37 (92.5%), while the P type was successfully determined in 35 (87.5%) of the 40 (24%) page positive samples. Type G1 was the most predominant of the G types (40.5%), and the incidences of G3 and G9 were 21.6 and 32.4% respectively. Mixed types G3/G9 were detected at 5.4%. Three P types existed in Mukuru slums, P[8] (60%), P[6] (22.9%), P[4] (11.4) and their relative incidence varied over the 15 months of this study. CONCLUSIONS: The G types and P types detected in this study are important causes of acute gastroenteritis in Mukuru slums Nairobi Kenya. An indication that the prevalence of certain genotypes may change over a rotavirus season is significant and mirrors observations from studies in other tropical climates. Thus monitoring of the genotypic changes among circulating viruses should be encouraged over the coming years.

Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

BACKGROUND: Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. METHODS: In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. RESULTS: The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10-7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10-5 (~3 ng/ml) and 10-4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. CONCLUSION: The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

Loop-Mediated Isothermal Amplification Test for Trypanosoma gambiense Group 1 with Stem Primers: A Molecular Xenomonitoring Test for Sleeping Sickness.

The World Health Organization has targeted Human African Trypanosomiasis (HAT) for elimination by 2020 with zero incidence by 2030. To achieve and sustain this goal, accurate and easy-to-deploy diagnostic tests for Gambian trypanosomiasis which accounts for over 98% of reported cases will play a crucial role. Most needed will be tools for surveillance of pathogen in vectors (xenomonitoring) since population screening tests are readily available. The development of new tests is expensive and takes a long time while incremental improvement of existing technologies that have potential for xenomonitoring may offer a shorter pathway to tools for HAT surveillance. We have investigated the effect of including a second set of reaction accelerating primers (stem primers) to the standard T. brucei gambiense LAMP test format. The new test format was analyzed with and without outer primers. Amplification was carried out using Rotorgene 6000 and the portable ESE Quant amplification unit capable of real-time data output. The stem LAMP formats indicated shorter time to results (~8 min), were 10-100-fold more sensitive, and indicated higher diagnostic sensitivity and accuracy compared to the standard LAMP test. It was possible to confirm the predicted product using ESE melt curves demonstrating the potential of combining LAMP and real-time technologies as possible tool for HAT molecular xenomonitoring.

Genetic Diversity of Cryptosporidium in Children in an Urban Informal Settlement of Nairobi, Kenya.

INTRODUCTION: Globally Cryptosporidium and Giardia species are the most common non-bacterial causes of diarrhoea in children and HIV infected individuals, yet data on their role in paediatric diarrhoea in Kenya remains scant. This study investigated the occurrence of Cryptosporidium species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi. METHODS: This was a prospective cross-sectional study in which faecal specimen positive for Cryptosporidium spp. by microscopy from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to the paediatric ward at the Mbagathi District Hospital were characterized. The analysis was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene for subtyping. RESULTS: C. hominis was the most common species of Cryptosporidium identified in125/151(82.8%) of the children. Other species identified were C. parvum 18/151(11.9%), while C. felis and C. meleagridis were identified in 4 and 2 children, respectively. Wide genetic variation was observed within C. hominis, with identification of 5 subtype families; Ia, Ib, Id, Ie and If and 21 subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and HIV status or patient type. CONCLUSION: C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, whereas genetic diversity was low within C. parvum with only one subtype family IIc identified.