Summary Background Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). Methods GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. Findings We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni or C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2–96·0) at the population level, compared with 51·5% (48·0–55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6–80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. Interpretation A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. Funding Bill & Melinda Gates Foundation.

Abstract. In December 2006, Rift Valley fever (RVF) was diagnosed in humans in Garissa Hospital, Kenya and an outbreak reported affecting 11 districts. Entomologic surveillance was performed in four districts to determine the epidemic/epizootic vectors of RVF virus (RVFV). Approximately 297,000 mosquitoes were collected, 164,626 identified to species, 72,058 sorted into 3,003 pools and tested for RVFV by reverse transcription-polymerase chain reaction. Seventyseven pools representing 10 species tested positive for RVFV, including Aedes mcintoshi/circumluteolus (26 pools), Aedes ochraceus (23 pools), Mansonia uniformis (15 pools); Culex poicilipes , Culex bitaeniorhynchus (3 pools each); Anopheles squamosus , Mansonia africana (2 pools each); Culex quinquefasciatus , Culex univittatus , Aedes pembaensis (1 pool each). Positive Ae. pembaensis , Cx. univittatus , and Cx. bitaeniorhynchus was a first time observation. Species composition, densities, and infection varied among districts supporting hypothesis that different mosquito species serve as epizootic/ epidemic vectors of RVFV in diverse ecologies, creating a complex epidemiologic pattern in East Africa.

BackgroundIncreased frequency of arbovirus outbreaks in East Africa necessitated the determination of distribution of risk by entomologic arbovirus surveillance. A systematic vector surveillance programme spanning 5 years and covering 11 sites representing seven of the eight provinces in Kenya and located in diverse ecological zones was carried out.MethodsMosquitoes were sampled bi-annually during the wet seasons and screened for arboviruses. Mosquitoes were identified to species, pooled by species, collection date and site and screened for arboviruses by isolation in cell culture and/or RT-PCR screening and sequencing.ResultsOver 450,000 mosquitoes in 15,890 pools were screened with 83 viruses being detected/isolated that include members of the alphavirus, flavivirus and orthobunyavirus genera many of which are known to be of significant public health importance in the East African region. These include West Nile, Ndumu, Sindbis, Bunyamwera, Pongola and Usutu viruses detected from diverse sites. Ngari virus, which was associated with hemorrhagic fever in northern Kenya in 1997/98 was isolated from a pool of Anopheles funestus sampled from Tana-delta and from Aedes mcintoshi from Garissa. Insect only flaviviruses previously undescribed in Kenya were also isolated in the coastal site of Rabai. A flavivirus most closely related to the Chaoyang virus, a new virus recently identified in China and two isolates closely related to Quang Binh virus previously unreported in Kenya were also detected.ConclusionActive transmission of arboviruses of public health significance continues in various parts of the country with possible undetermined human impact. Arbovirus activity was highest in the pastoralist dominated semi-arid to arid zones sites of the country where 49% of the viruses were isolated suggesting a role of animals as amplifiers and indicating the need for improved arbovirus disease diagnosis among pastoral communities.

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2. Fever is a symptom common to a wide variety of infectious diseases, including some of the leading causes of death in subSaharan Africa (SSA). Many etiologic studies have been performed for respiratory infections, diarrheal illness, and meningitis (1, 2). However, the incidence and etiology of undifferentiated fever are less clear (3). Most research has examined individual agents such as Plasmodium, Salmonella, and specific zoonotic or arboviral pathogens (4-6) by utilizing blood culture (7) or a complex mixture of rapid, serologic, culture, and molecular assays and algorithms to determine an etiologic agent (8).We describe our initial development and validation of a TaqMan array card (TAC) that uses quantitative reverse transcription-PCR (qRT-PCR) for the simultaneous detection of 15 viruses, 8 bacteria, and 3 protozoa of particular relevance to SSA (5, 9-13), with the intended use for outbreak investigation and acute febrile illness (AFI) surveillance. Previous TAC assays have been developed for respiratory diseases, enteric diseases, and etiologies of neonatal sepsis (14-16), and we have shown their robust and comparable performance across several countries (17). Once developed, TaqMan array cards are stable at 4°C for 2 years, can be shipped at ambient temperature, and minimize several cumbersome steps in the field, such that they are as easy to perform as individual quantitative PCR (qPCR) assays.This work was primarily a development exercise since clinical

Arthropod-borne viruses are a major constituent of emerging infectious diseases worldwide, but limited data are available on the prevalence, distribution, and risk factors for transmission in Kenya and East Africa. In this study, we used 1,091 HIV-negative blood specimens from the 2007 Kenya AIDS Indicator Survey (KAIS 2007) to test for the presence of IgG antibodies to dengue virus (DENV), chikungunya virus (CHIKV) and Rift Valley fever virus (RVFV).The KAIS 2007 was a national population-based survey conducted by the Government of Kenya to provide comprehensive information needed to address the HIV/AIDS epidemic. Antibody testing for arboviruses was performed on stored blood specimens from KAIS 2007 through a two-step sandwich IgG ELISA using either commercially available kits or CDC-developed assays. Out of the 1,091 samples tested, 210 (19.2%) were positive for IgG antibodies against at least one of the three arboviruses. DENV was the most common of the three viruses tested (12.5% positive), followed by RVFV and CHIKV (4.5% and 0.97%, respectively). For DENV and RVFV, the participant’s province of residence was significantly associated (P≤.01) with seropositivity. Seroprevalence of DENV and RVFV increased with age, while there was no correlation between province of residence/age and seropositivity for CHIKV. Females had twelve times higher odds of exposure to CHIK as opposed to DENV and RVFV where both males and females had the same odds of exposure. Lack of education was significantly associated with a higher odds of previous infection with either DENV or RVFV (p <0.01). These data show that a number of people are at risk of arbovirus infections depending on their geographic location in Kenya and transmission of these pathogens is greater than previously appreciated. This poses a public health risk, especially for DENV.