Feasibility of the Bag-Mediated Filtration System for Environmental Surveillance of Poliovirus in Kenya.

The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in PV eradication efforts. From April to September 2015, environmental samples were collected from four sites in Nairobi, Kenya, and processed using two collection/concentration methodologies: BMFS (> 3 L filtered) and grab sample (1 L collected; 0.5 L concentrated) with two-phase separation. BMFS and two-phase samples were analyzed for PV by the standard World Health Organization poliovirus isolation algorithm followed by intratypic differentiation. BMFS samples were also analyzed by a cell culture independent real-time reverse transcription polymerase chain reaction (rRT-PCR) and an alternative cell culture method (integrated cell culture-rRT-PCR with PLC/PRF/5, L20B, and BGM cell lines). Sabin polioviruses were detected in a majority of samples using BMFS (37/42) and two-phase separation (32/42). There was statistically more frequent detection of Sabin-like PV type 3 in samples concentrated with BMFS (22/42) than by two-phase separation (14/42, p = 0.035), possibly due to greater effective volume assayed (870 mL vs. 150 mL). Despite this effective volume assayed, there was no statistical difference in Sabin-like PV type 1 and Sabin-like PV type 2 detection between these methods (9/42 vs. 8/42, p = 0.80 and 27/42 vs. 32/42, p = 0.18, respectively). This study demonstrated that BMFS can be used for PV environmental surveillance and established a feasible study design for future research.

Detection of potentially pathogenic enteric viruses in environmental samples from Kenya using the bag-mediated filtration system.

Enteric virus environmental surveillance via a highly sensitive method is critical, as many enteric viruses have low infectious doses and can persist in the environment for extended periods. This study determined the potential of the novel bag-mediated filtration system (BMFS) to recover human enteric viruses and pepper mild mottle virus (PMMoV) from wastewater and wastewater-impacted surface waters, examined PMMoV use as a fecal contamination indicator in Kenya, and identified potential BMFS process controls. From April 2015 to April 2016, BMFS samples were collected from seven sites in Kenya (n = 59). Enteroviruses and PMMoV were detected in 100% of samples, and human adenovirus, human astrovirus, hepatitis A virus, norovirus GI, norovirus GII, sapovirus, and human rotavirus were detected in the majority of samples. The consistent detection of enteroviruses and PMMoV suggests that these viruses could be used as indicators in similarly fecally contaminated sites and BMFS process controls. As contamination of surface water sources remains a global issue, enteric virus environmental surveillance is necessary. This study demonstrates an effective way to sample large volumes of wastewater and wastewater-impacted surface waters for the detection of multiple enteric viruses simultaneously.

Genetic characterization of a novel hepatitis a virus strain in irrigation water in South Africa.

Hepatitis A virus (HAV) was detected, by real-time reverse transcription-polymerase chain reaction, in irrigation water from a dam on a commercial fresh produce farm in South Africa (SA). The virus was characterized by nucleotide sequence and phylogenetic analysis of a consensus sequence spanning the VP1 and VP1/P2B genomic regions. Amino acid sequence and phylogenetic analysis indicated that the HAV strain was closely related to HAV genotype V and possibly of simian origin. This suggests that a novel HAV may be circulating in SA and its presence in irrigation water highlights the potential for zoonotic or anthroponotic cross-species transmission via environmental food and water sources.

Molecular characterisation of enteroviruses and clinical findings from a cluster of paediatric viral meningitis cases in Tshwane, South Africa 2010-2011.

BACKGROUND: Human enteroviruses (HEVs) are the most common viral pathogen associated with paediatric aseptic meningitis. From October 2010 to February 2011 a cluster of HEV-associated meningitis cases was identified in paediatric patients who had presented at two large tertiary hospitals in Pretoria in the Tshwane Metropolitan Area, Gauteng, South Africa (SA). OBJECTIVES: The aim of this study was to review the clinical features and to characterise the HEV strains associated with this cluster of meningitis cases. STUDY DESIGN: In this retrospective study HEVs, detected by real time reverse transcription-polymerase chain reaction in acute phase cerebrospinal fluid specimens from 30 patients with aseptic meningitis, were characterised and the clinical presentations of these patients were described. RESULTS: Fever (83%), headache (70%) and vomiting (67%) were the most prominent symptoms with signs of meningeal irritation recorded in 67% of the patients. There was a neutrophil predominance in the cerebrospinal fluid of 57% of the patients with pleocytosis. Based on partial nucleotide sequence analysis of the HEV viral protein 1 gene, echovirus (E) serotype 4 (E-4) was identified in 80% (24/30) of specimens with E-9 (3/30) and coxsackie virus B5 (1/30) detected less frequently. CONCLUSION: In this cluster of aseptic meningitis cases E-4 was the predominant strain with E-9, and to a lesser extent other HEVs, identified less frequently.

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.

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.

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.

An investigation of the role of oral epithelial cells and Langerhans cells as possible HIV viral reservoirs.

BACKGROUND: The role of the oral mucosa as a target of human immunodeficiency virus (HIV-1) infection and persistence is unclear. HIV-1 has been reported in oral epithelial cells, but this has not been confirmed. Cellular reservoirs may impede antiretroviral therapies and should be identified. This study was performed to determine the presence of HIV-1 in oral epithelial and Langerhans cells (LCs) of HIV-1-positive antiretroviral naïve patients. Non-invasive brush biopsy technique for future in vivo HIV research was also evaluated. METHODS: Oral mucosal cells were harvested from the buccal mucosae, dorsal tongue and the gingiva of the mandibular teeth of 35 HIV-1-positive patients using a Cytobrush Plus cell collector. Epithelial cells were purified from the samples by flow cytometric cell sorting using cytokeratin stains after which the epithelial cell samples were further purified and divided into superficial and deep epithelial cells by laser microdissection on Pap stained cytospin smears. LCs were picked up individually by laser microdissection from CD1a stained cytospin smears. Purified epithelial and LC samples were tested for the presence of HIV-1 DNA by polymerase chain reaction analysis. RESULTS: Ten of the patients had HIV-1 DNA in one or more of the sampled anatomical locations. No HIV-1 DNA could be demonstrated in any of the purified superficial or deep epithelial or LC samples. CONCLUSIONS: HIV-DNA can be found using non-invasive oral brush biopsies and should be investigated further as an experimental model for in vivo oral HIV research. Better ways to purify the different cell types should be investigated.