Identification of enteroviruses along Lake Victoria shoreline - a potential indicator of sewage pollution.

E n t e r i c v i r u s e s a r e m a i n l y t r a n s m i t t e d b y t h e f a e c a l - o r a l r o u t e a n d h a v e b e e n l i n k e d t o s e v e r a l d i s e a s e s i n c l u d i n g g a s t r o e n t e r i t i s a n d r e s p i r a t o r y i n f e c t i o n s . T h e i r p r e s e n c e i n s u r f a c e w a t e r s h a s b e e n exacerbated by p o l l u t i o n f r o m a v a r i e t y o f p o i n t s o u r c e s s u c h a s s e w a g e d i s c h a r g e . W e s t u d i e d t h e occurrence o f e n t e r o v i r u s e s i n w a t e r s a m p l e s f r o m L a k e V i c t o r i a i n K e n y a t o i n v e s t i g a t e i f t h e r e w a s a l i n k b e t w e e n s e w a g e p o l l u t i o n a n d d e t e c t i o n o f e n t e r o v i r u s e s ( E V s ) t o b u i l d a b a s e l i n e f o r a n enteric viruses monitoring platform for this region. We analysed 216 samples collected over 6 months from six different locations along the Homa Bay Pier. The six sampling locations comprised of three sites (P3, P5, P6) located <500 m from a local sewage treatment plant and pit latrines while three other sites (P1, P2, P4) were located at approximately 0.5 to 3 Km. EVs were concentrated using glass wool adsorption elution protocol and identified using the nested reverse transcription-polymerase chain reaction. The odds ratio was performed to determine whether the location of the sources of sewage pollution near the lake was associated with the EVs contamination. Five out of 108 (5 %) samples collected from the sites (P3, P5 and P6 were EV positive, while 2 % (2/108) of samples from P1, P2 and P4 were EV positive. The presence of the EVs was associated with the distance from the possible sources of faecal contamination (odds ratio 20.28 and 4.86, confidence interval 2.42, and 0.95) for pit latrines and the sewage treatment plant respectively. The result from this study indicates that sewage discharge at the shoreline of Lake Victoria may have been the source of EVs contamination. Data from this study could significantly contribute to informing risk management on sewage pollution in Lake Victoria and it is important to continue monitoring this lake for potentially pathogenic enteric viruses.

Quantification and Trends of Rotavirus and Enterovirus in Untreated Sewage Using Reverse Transcription Droplet Digital PCR.

The quantification and trends in concentrations for naturally occurring rotaviruses (RV) and enteroviruses (EV) in untreated sewage in various wastewater systems have not often been compared. There is now greater interest in monitoring the infections in the community including live vaccine efficacy by evaluating untreated sewage. The goals of this study were to 1) survey the concentrations of naturally occurring RV and EV in untreated sewage using a reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) and 2) investigate the use of a new adsorption elution (bag-mediated filtration system (BMFS) using ViroCap filters) against more traditional polyethylene glycol (PEG) precipitation for virus concentration. Sewage samples were collected from lagoons in Kenya and Michigan (MI), the United States (USA) and from wastewater treatment plants (WWTPs) in the USA. RVs were detected at geometric mean concentrations in various locations, California (CA) 1.31 × 105 genome copies/L (gc/L), Kenya (KE) 2.71 × 104 gc/L and Virginia (VA) 1.48 × 105 gc/L, and EVs geometric means were 3.72 × 106 gc/L (CA), 1.18 × 104 gc/L (Kenya), and 6.18 × 103 gc/L (VA). The mean RV concentrations using BMFS-ViroCap in split samples compared to PEG precipitation methods demonstrated that the levels were only 9% (#s BMFS/PEG) in the Michigan lagoons which was significantly different (p < 0.01). This suggests that RV concentrations in Kenya are around 1.69 × 106 gc/L. Overall, there was no difference in concentrations for the other sampling locations across the methods of virus recovery (i.e., PEG precipitation and HA filters) using one-way ANOVA (F = 1.7, p = 0.2739) or Tukey-Kramer pairwise comparisons (p > 0.05). This study provides useful data on RV and EV concentrations in untreated sewage in Kenya and the USA. It also highlights on the usefulness of the RT-ddPCR for absolute quantification of RV and EV in sewage samples. The BMFS using ViroCap filters while less efficient compared to the more traditional PEG precipitation method was able to recover RVs and EVs in untreated sewage and may be useful in poor resource settings while underestimating viruses by 1 to 1.5 logs.

Correction: Global Occurrence and Emission of Rotaviruses to Surface Waters. Pathogens 2015, 4, 229-255.

The authors wish to make the following corrections to their paper [1].[...].

Global occurrence and emission of rotaviruses to surface waters.

Group A rotaviruses (RV) are the major cause of acute gastroenteritis in infants and young children globally. Waterborne transmission of RV and the presence of RV in water sources are of major public health importance. In this paper, we present the Global Waterborne Pathogen model for RV (GloWPa-Rota model) to estimate the global distribution of RV emissions to surface water. To our knowledge, this is the first model to do so. We review the literature to estimate three RV specific variables for the model: incidence, excretion rate and removal during wastewater treatment. We estimate total global RV emissions to be 2 × 1018 viral particles/grid/year, of which 87% is produced by the urban population. Hotspot regions with high RV emissions are urban areas in densely populated parts of the world, such as Bangladesh and Nigeria, while low emissions are found in rural areas in North Russia and the Australian desert. Even for industrialized regions with high population density and without tertiary treatment, such as the UK, substantial emissions are estimated. Modeling exercises like the one presented in this paper provide unique opportunities to further study these emissions to surface water, their sources and scenarios for improved management.

Rotavirus G and P types circulating in the eastern region of Kenya: predominance of G9 and emergence of G12 genotypes.

BACKGROUND: The World Health Organization has recommended that rotavirus (RV) vaccines be included in all national immunization programs as part of a strategy to control RV-associated diarrheal diseases. Hospital-based surveillance of RV infection is therefore crucial in monitoring the impact pre- and post-vaccine introduction and also to document changes in genotype distribution. This study sought to determine the RV genotypes circulating in the eastern region of Kenya before introduction of the RV vaccine. METHODS: During September 2009 to August 2011, 500 stool samples were collected from children <5 years of age admitted for acute diarrhea in hospitals in the eastern region of Kenya and analyzed for the presence of group A RV using an enzyme immunoassay. G and P genotypes were determined using hemi-nested reverse transcriptase polymerase chain reaction. RESULTS: One hundred and eighty nine out of 500 (38%) samples analyzed were positive for rotavirus. The following G types were detected: G9 (50.9%), G1 (26.8%), G8 (12.1%), G12 (3.1%), G2 (0.6%), mixed G (1.3%) and 5.1% were G nontypeable. P types detected included: P[8] (63.7%), P[4] (12.1%), P[6] (4.5%), mixed P (7.6%) and 12.1% were P nontypeable. The most dominant strain was G9P[8] (35%), followed by G1P[8] (26.8%), G8P[4] (9.6%), G12P[6] (2.5%), G9P[6] (1.9%), G9P[4] (1.3%), G8P[8] (1.3%), and G2P[4] (0.6%). CONCLUSIONS: The present study demonstrates the recurring changing genotypes of RV circulating in Kenya, with genotypes G9, G1 and G8 being the dominant strains circulating in the eastern region of Kenya between 2009 and 2011. Additionally, G12 genotype was detected for the first time in Kenya.

Human caliciviruses detected in HIV-seropositive children in Kenya.

The human caliciviruses (HuCVs) are important causes of gastroenteritis worldwide. Norovirus (NoV) and sapovirus (SaV) have been detected in HIV-seropositive children but the genetic diversity of HuCVs circulating in these individuals is largely unknown. In this study the prevalence and genotype diversity of HuCVs circulating in Kenyan HIV-positive children, with or without diarrhea, from the year 1999 to 2000 was investigated. The overall prevalence of HuCVs was 19% with NoV predominating at 17% (18/105) and SaV present in 5.7% (6/105) of specimens. Human CVs were detected in both symptomatic (24%) and asymptomatic (16%) children. Co-infections with other enteric viruses were detected in 21.6% of children with diarrhea but only in 4.4% of children without diarrhea. Remarkable genetic diversity was observed with 12 genotypes (7 NoV, 5 SaV) being identified in 20 HuCV-infected children. NoV genogroup II (GII) strains predominated with GII.2 and GII.4 each representing 27% of the NoV-positive strains. The GII.4 strain was most closely related to the nonepidemic GII.4 Kaiso 2003 variant. Other NoV genotypes detected were GI.3, GII.6, GII.12, GII.14, and GII.17. Five different SaV genotypes (GI.2, GI.6, GII.1, GII.2, and GII.4) were characterized from six specimens. Diarrheal symptoms were not associated with any specific HuCV genotype. Overall the HuCV genotype distribution detected in this study reflects those in other studies worldwide. The strains detected are closely related to genotypes that have circulated on several continents since the year 2000.

Ovarian tissue cryopreservation by vitrification in olive baboons (papio anubis): a pilot study.

BACKGROUND: Ovarian tissue cryopreservation by vitrification is an attractive technique for fertility preservation in women. However, this technique has not been optimized. The aim of this study was to evaluate the baboon as a model for the preclinical study of ovarian tissue cryopreservation by vitrification and thawing. METHODS: Ovarian cortical tissues (1-mm cubes) were obtained surgically from adult female olive baboons (n = 9) maintained in captivity and vitrified using dimethyl sulphoxide and ethylene glycol protocol. The proportion of morphologically intact follicles (primordial, primary and secondary) in paired fresh and cryopreserved (vitrified-thawed) ovarian tissues was compared. RESULTS: Overall, 67.1% of follicles were morphologically normal after vitrification. When compared to fresh ovarian tissue, vitrified-thawed ovarian tissue contained a comparable number of intact primordial follicles (48.9 vs. 52.9%), and a lower number of both primary (14.8 vs. 29.5%; p < 0.05) and secondary (2.0 vs. 0.7%; p < 0.05) follicles. CONCLUSION: After vitrification and thawing, baboon ovarian tissue retains about 67% of morphologically normal follicles, which is comparable to results for human ovarian tissue, and suggests that the olive baboon is a promising animal model for preclinical assessment of ovarian vitrification, thawing and autotransplantation studies.

Multilocus sequence typing of Enterocytozoon bieneusi: Lack of geographic segregation and existence of genetically isolated sub-populations.

The population structure of Enterocytozoon bieneusi was examined by multilocus sequence typing (MLST) of 64 specimens from AIDS patients in Peru, Nigeria, and India and five specimens from captive baboons in Kenya using a combination of the ribosomal internal transcribed spacer (ITS) and four microsatellite and minisatellite markers. Parasites in different geographic locations (Peru, India, and Nigeria) all had strong and significant linkage disequilibrium (LD) and only limited recombination, indicative of a clonal population structure in E. bieneusi from each location. When isolates of various geographical areas were treated as a single population, phylogenetic analysis and substructural analysis using STRUCTURE found no evidence for the existence of geographically segregated sub-populations. Nevertheless, both analyses revealed the presence of two major genetically isolated groups of E. bieneusi: one (sub-population 1) contained all isolates of the anthroponotic ITS genotype A, whereas the other (sub-population 2) harbored isolates of multiple ITS genotypes with zoonotic potential. This was also supported by FST analysis. The measurement of LD and recombination rates indicated that sub-population 2 had a clonal population structure, whereas sub-population 1 had an epidemic population structure. The data confirmed the existence of genetic sub-populations in E. bieneusi that may be transmitted differently in humans.

Short-term effects of high-dose khat on sperm parameters and reproductive hormonal levels in olive baboons (Papio anubis).

The biological effects of khat (Catha edulis) on reproduction and fertility are inadequately investigated and controversial, hence we determined the effects of oral administration of high-dose khat on sperm parameters and male hormonal levels in olive baboons. In this study, 6 male baboons received a high dose of khat (500 g/week) during 1 month. Electroejaculation for sperm studies (concentration, motility and chromatin integrity) and plasma collection for hormonal analysis (testosterone, prolactin and cortisol) were done weekly during 1 month before and 1 month during khat administration as well as 2 weeks after the last dose of khat administration. Administration of khat extract induced a significant reduction in sperm motility (p = 0.008), sperm count (p = 0.041), sperm chromatin integrity (p = 0.0003), testosterone levels (p = 0.035) and prolactin levels (p = 0.0115), but not in cortisol levels and sperm volume (p > 0.05). The results suggest that high-dose khat decreases sperm quality and testosterone and hence may contribute to male infertility.

Baboon spermatology: basic assessment and reproducibility in olive baboons (Papio anubis).

BACKGROUND: Development of a reproducible baboon in vitro fertilization (IVF) system require optimized and reproducible sperm parameters. The objective of this study was to document basic spermatology values and investigate the reproducibility of these variables in the same baboons 1 or 3 months later in a larger number of baboons. METHODS: In this prospective study, sperm quality (semen volume, pH, concentration, motility, morphology and size) was evaluated in 27 sperm samples obtained from 9 baboons electroejaculated three times with a time interval of 1 month (between first and second sample collection) and 3 months (between second and third round sample collection). RESULTS: Baseline sperm values for semen volume (0.5 ± 0.3 ml), pH (7.5 ± 0.3), concentration (54.2 ± 19.3 million/ml), motility (67.3 ± 18.5%) and morphology (89.2 ± 4.8%) were similar to sperm samples obtained after 1 or 3 months (P > 0.05). Head, midpiece and tail abnormalities were rarely observed (0-9%). Sperm dimensions were characterized by a tail length of 69.6 ± 13.9 µm, a head width of 2.41 ± 0.43 µm and a head length of 3.49 ± 0.6 µm. CONCLUSION: Sperm quality was not affected by repeated electroejaculation with time intervals of 1 or 3 months, suggesting that the same baboon can participate multiple times in reproductive research.

Cyclospora papionis, Cryptosporidium hominis, and human-pathogenic Enterocytozoon bieneusi in captive baboons in Kenya.

Cyclospora papionis, Cryptosporidium hominis, and Enterocytozoon bieneusi were detected in 42 (17.9%), 6 (2.6%), and 29 (12.3%) of 235 newly captured baboons in Kenya, respectively. Most C. hominis subtypes and E. bieneusi genotypes found have been detected in humans in the area, suggesting that cross-species transmission of cryptosporidiosis and microsporidiosis is possible.

Evidence of a recombinant wild-type human astrovirus strain from a Kenyan child with gastroenteritis.

A human astrovirus (HAstV) strain from Kenya was characterized by nucleotide sequence analysis. Sequences from open reading frame 1a (ORF1a) clustered with genotype 6/7, those from ORF1b clustered with genotype 3, and those from ORF2 clustered with genotype 2. A recombination point in the ORF1b-ORF2 junction was identified, with a second possible recombination point within the ORF1a region.

Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network.

Severe rotavirus diarrhea in children <5 years of age is a major public health problem; however, limited regional and country specific data on rotavirus disease burden are available from sub-Saharan Africa. In June 2006, the World Health Organization Regional Office for Africa initiated rotavirus surveillance in selected African countries. With use of standardized methodology developed by the World Health Organization, children <5 years of age who were hospitalized with severe diarrhea were enrolled, and stool specimens were collected for detection of rotavirus strains with use of a commercial enzyme immunoassay. Rotavirus strains were further characterized for G and P types with use of a reverse-transcriptase polymerase chain reaction. From June 2006 through December 2008, rotavirus surveillance was established at 14 sites in 11 African countries. Of 5461 stool samples collected from children enrolled in 8 countries with 1 or 2 complete years of data, 2200 (40%) were positive for rotavirus. Ninety percent of all rotavirus hospitalizations occurred among children aged 3-12 months. Predominant types included G1P[8] (21%), G2P[4] (7%), and P [8] (29%); however, unusual types were also detected, including G8P[6] (5%), G8P[8] (1%), G12P[6] (1%), and G12P[6] (1%). A high percentage of mixed rotavirus infections was also detected. These preliminary results indicate that rotavirus is a major cause of severe diarrheal disease in African children.

Separate and combined effects of caffeine and dbcAMP on olive baboon (Papio anubis) sperm.

BACKGROUND: Improvement of baboon sperm capacitation is necessary for achieving high in vitro fertilization (IVF) rates in baboons. In this study, we evaluated separate and combined effects of caffeine and dbcAMP on baboon sperm capacitation. METHODS: Sixteen male baboons (n = 16) were electroejaculated. Each sperm sample was divided into two aliquots: one for chemical activation and the other untreated control. Group 1: dbcAMP (n = 6); Group 2: caffeine (n = 6) and Group 3: combination of caffeine and dbcAMP (n = 4). In each aliquot, sperm motility after 30 minutes of incubation was evaluated as well as zona pellucida (ZP) binding ability after overnight incubation with 4-5 ZP from unfertilized human oocytes. RESULTS: Sperm motility and ZP binding ability in all chemically activated groups increased significantly as compared to their respective controls (P < 0.05). CONCLUSION: Combined and separate effects of caffeine and dbcAMP increases baboon sperm motility and ZP binding ability and may improve baboon IVF.

High prevalence of species D human adenoviruses in fecal specimens from Urban Kenyan children with diarrhea.

Human adenoviruses (HAdVs) cause a wide range of clinical syndromes and are classified in seven species, A-G, comprising 52 serotypes. HAdV-A31, -F40, and -F41 have been associated with diarrhea in infants and young children. In developing countries gastroenteritis is a major cause of morbidity and mortality in children and, in comparison to rotaviruses, there are no data on the HAdVs associated with diarrhea in pediatric patients in Kenya. This study investigates the prevalence and genotypes of HAdVs in 278 stool specimens (211 diarrheal; 67 non-diarrheal) from children < or =14 years of age in urban and rural areas in Kenya. Stool specimens were screened for HAdVs using a nested polymerase chain reaction and the HAdVs genotyped by sequence analysis of a conserved hexon gene fragment. HAdVs were detected in 104/278 (37.4%) of the stool specimens: 35/43 (81.4%) of diarrheal and 10/61 (16.4%) of non-diarrheal stool specimens from children in an urban hospice; 25/94 (26.6%) of diarrheal specimens from urban children and 34/80 (42.5%) of diarrheal specimens from children in a rural area. Species D HAdVs were identified as the most prevalent HAdV species in diarrheal stool specimens from urban children comprising 18/37 (48.6%) of the strains identified. In contrast HAdV species F predominated in pediatric diarrheal specimens from the rural area, being identified in 7/16 (43.8%) of the characterized strains. This study provides valuable new data on the prevalence and distribution of HAdV genotypes in diarrheal stool specimens in Kenya and Africa, and highlights the necessity for further investigations.

Rotavirus infections among HIV-infected children in Nairobi, Kenya.

Human rotaviruses have emerged as a leading cause of acute diarrhea in children <5 years of age worldwide. Although there are previous reports relating to various aspects of rotaviruses, there is limited data on the involvement of rotavirus infection in HIV-infected children. We therefore evaluated the importance of rotavirus infections in HIV-related diarrhea in Kenyan children. Fecal samples were collected from a total of 207 children during the period February 1999 to June 2000 and screened for HRV antigen by enzyme-linked immunosorbent assay (ELISA). Positive samples were analyzed by VP6 subgroup specificity assay, by polyacrylamide gel electrophoresis (PAGE) and reverse transcriptase/polymerase chain reaction (RT-PCR). Fourteen percent (29/207) of the samples were positive. HIV-seropositive children with diarrhea were more likely than their counterparts without diarrhea to have rotaviruses [23.3% (10/43) versus 2.9% (2/70); p = 0.0001]. Rotavirus strain G3P[6] was predominant. These results indicate that rotavirus is an important viral etiological agent causing diarrhea in HIV-seropositive children.

The epidemiology of human rotavirus associated with diarrhoea in Kenyan children: a review.

Rotavirus gastroenteritis still remains a major cause of morbidity and mortality among young children in developing countries, with approximately 150,000-200,000 deaths occurring annually in sub-Saharan Africa. We reviewed papers published over the last 30 years on the epidemiology of rotavirus diarrhoea among the hospitalized and out-patient children in Kenya. The analysis shows rotavirus prevalence of 6-56% with diarrhoea occurring throughout the year and generally exhibiting distinct peaks during the dry months. Among the common genotype, G1 was the most predominant up to the year 2002 but more recently there has been an emergence of genotype G9 as the most predominant genotype and to a less extent G8. It is important to continue rotavirus surveillance in Kenya to determine accurately the burden of rotavirus disease and the emerging new genotypes. This will assist policy makers in decision making on rotavirus vaccine introduction and determining the impact of the vaccine.

Astrovirus infection in young Kenyan children with diarrhoea.

Human astroviruses (HAstV) have been commonly identified worldwide as important aetiological agents of acute gastroenteritis in all age groups including the young, elderly and immunocompromised. However, limited data exist on the prevalence of this important pathogen in Kenya. The aim of this study was therefore to determine the prevalence of astrovirus (AstV) infection in Kenyan children younger than 10 years of age with diarrhoea. During the period February 1999 to September 2005, stool samples were collected from 476 children attending clinics in Nairobi (and its environs) and the Maua Methodist Hospital, Meru North, Kenya. The faecal specimens were tested by a commercial enzyme immunoassay kit for HAstV. AstV prevalence rates were found to be 6.3%. There was significantly high prevalence of AstV infection in children 5 years [0.2% (1/476)] ( p < 0.01). Also, we showed a significantly high prevalence of AstV infection in children of