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Polioviruses (PVs) are not associated with waterborne transmission to the same extent as many other enteric viruses. However, they are typically transmitted by the faecal-oral route, which implies that the risk of infection by exposure to the viruses in water cannot be underestimated. The risk appears particularly high for rural communities, which use sewage-polluted river water for domestic purposes. Thus, the presence in the environment of highly evolved, neurovirulent vaccine-derived poliovirus (VDPV) strains in the absence of polio cases would have important implications for strategies to terminate immunisation with oral poliovirus vaccine (OPV) following global polio eradication. The aim of the current study was to determine the prevalence of VDPVs in selected sewage and river water samples collected from 2001 to 2003, and to construct phylogenetic trees of the partially sequenced 5′untranslated region (5′UTR) and the VP1 region of the genomes to deduce the genetic relatedness between the PV strains. Using the monolayer plaque assay, 703 plaques from sewage and 157 plaques from river water samples were analysed. Application of a RT-multiplex PCR revealed that 176 of these plaques were non-polio enteroviruses, and 49 were PV isolates. The Sabin-specific RTtriplex PCR revealed the presence of 29 Sabin PV type 1, 8 Sabin PV type 2 and 12 Sabin PV type 3 isolates. The 5′UTR and the VP1 region of 13 PV type 1, 7 PV type 3 and 6 PV type 2 isolates were partially sequenced. The majority of the OPV isolates (24 out of 26) displayed close sequence relationships (>99% VP1 sequence identity) to the parental Sabin PV vaccine strains and were classified as "OPV-like viruses". Two isolates (D1 08/28 and OF1 05/21) were found to be highly divergent and were classified as "suspected" VDPVs. Isolate OF1 05/21 (a "suspected" VDPV type 1) showed more than 0.9% divergence in VP1, whereas isolate D1 08/28 (a "suspected" VDPV type 2) showed 1.4% divergence in VP1 from the parental Sabin PV vaccine strains. As with most of the other OPV-like isolates, these "suspected" VDPVs were carrying mutations, which have previously been associated with reversion of the attenuated Sabin PV strains to increased neurovirulence. It was estimated that the total period of replication for the two "suspected" VDPVs was between 12 and 16 months. In conclusion, this study provided new and relevant information on the prevalence of "suspected" VDPVs in sewage and openUP river water, and opened the way to assess the possible broader significance of the findings reported here.

After exposure to the oral poliovirus vaccine (OPV), immunocompetent persons excrete poliovirus (PV) vaccine strains for a limited period. In contrast, immunodeficient individuals remain sometimes chronically infected, and in some cases, PV excretion times as long as 10 years have been reported. During prolonged replication in the human intestine, the PV vaccine strain almost invariably reverts its attenuated character and acquires neurovirulent properties (vaccine-derived PVs, or VDPVs), which resemble wild-type PV strains. The aim of this study was to determine the occurrence of OPV strains in stools of immunodeficient children from a selected area in South Africa, as a first step toward future research on the prevalence and potential health impact of VDPVs. In a period of 1 year, a total of 164 stool samples of HIV-positive children aged 4 months to 8 years were studied for the excretion of OPV strains. In addition, 23 stool samples from healthy immunocompetent children were analyzed after receiving their OPV immunization. By applying a reverse transcription-polymerase chain reaction in combination with a nested PCR, a total of 54 enteroviruses (EVs) were detected in the stool specimens of the immunodeficient children. Using restriction enzyme analysis, 13 PVs were distinguished from 41 nonpolio EVs (NPEVs). A Sabin-specific RT-triplex PCR confirmed the presence of 7 Sabin PV type 1, 4 Sabin PV type 3, and 2 Sabin PV type 2 isolates. The majority of the NPEV group was made up of 7 coxsackievirus B3 (CBV3), 6 echovirus 11 (ECV11), 5 ECV9, and 3 coxsackievirus A6 (CAV6) isolates. According to the results, two of the immunodeficient patients (P023 and P140) who had received their last OPV immunization more than 15 months before (vaccinated at 14 weeks of age) tested positive for Sabin PVs types 3 and 1, respectively. A 5-year-old immunodeficient patient (P052) who had received her last OPV immunization more than 42 months before (vaccinated at 18 months of age) tested positive for Sabin PV type 1. These results suggested that immunodeficient patients vaccinated with OPV might excrete potentially pathogenic VDPVs for a prolonged period. These VDPVs may circulate in the community, resulting in possible infections in the unvaccinated population. Therefore, the information obtained in this study would be essential for strategies aimed at the protection of both immunodeficient as well as immunocompetent individuals against complications of vaccination with OPV. Article Outline

Due to the increased demand and consumption of bottled water in South Africa, there has been a growing concern about the microbiological quality of this product. Retail outlets sell local as well as imported bottled water to consumers. The microbiological quality of 10 different (8 local and 2 imported) bottled water products were tested over a period of three months on days 1, 30 and 90. Tests for the detection of heterotrophic plate count (HPC) bacteria, total and faecal coliform bacteria, spore-forming Clostridium perfringens, somatic and F-RNA coliphages were performed on the samples. In addition samples were analysed for three selected enteric viruses, caliciviruses, enteroviruses and rotaviruses using the reverse transcriptase-polymerase chain reaction (RT-PCR). The results indicated that 8/10 of the bottled water samples analysed, met the requirements set by the South African Bureau of Standards (SABS) for HPCs in bottled water of less than 100 counts per ml. However, in two bottled water samples the average HPC bacteria counts were 2.64 x 10 2 cfu•ml-1 and 8.89 x 10 3 cfu•ml-1 respectively which exceeded the recommended SABS guideline. HPC counts showed a slight variation during the three-month period in the bottled water samples. Total and faecal coliform bacteria, enterococci, C. perfringens, bacteriophages or enteric viruses were not detected in any of the ten bottled water samples analysed. It can be concluded that the microbial quality of eight of the ten selected bottled water samples analysed was within the acceptable limits set by the SABS guidelines and therefore, was safe for human consumption.

Aims: The aim of the study was to determine the prevalence of vaccine-derived polioviruses (VDPVs) in stool specimens of immunodeficient patients such as HIV-positive children (including those with an AIDS indicator condition, according to the Centres for Disease Control and Prevention classification) by applying various molecular techniques. Methods and Results: A total of 164 stool samples from HIV-positive children and 23 stool samples from healthy immunocompetent children (the control group) were analysed during 2003 and 2004. By applying a reverse transcription polymerase chain reaction (RT-PCR) in combination with a nested PCR, a total of 54 enteroviruses were detected in the stool specimens of the immunodeficient children. The use of restriction enzymes and a Sabin specific RT-triplex PCR confirmed the presence of 13 polioviruses (PVs), such as seven Sabin PV type 1, four Sabin PV type 3 and two Sabin PV type 2 isolates. The 5¢untranslated region and the VP1 capsid-encoding protein of the 13 PVs and the three PVs from the stools of the immunocompetent children were partially sequenced and their genetic relatedness was deduced from the constructed phylogenetic trees. The majority of the PVs isolated from the stools of the immunodeficient children (10 of 13 isolates) were classified as 'oral poliovirus vaccine (OPV)-like viruses', as these isolates had close sequence relationships (>99% in VP1 nucleotide sequences) to the original Sabin PV vaccine strains. Three PVs showed £99% VP1 sequence identity to the Sabin PV vaccine strains and were classified as 'suspected' immunodeficient VDPVs (iVDPVs). All of the OPV-like isolates and the 'suspected' iVDPVs carried mutations at specific positions in their partially sequenced regions, which have been associated with reversion of the attenuated Sabin PV vaccine strains to increased neurovirulence. Conclusions: Thus, this study adds further evidence to the observation that immunodeficient individuals may excrete OPV strains with potential neurovirulent phenotypes. Significance and Impact of the Study: Prolonged excretion of PVs by immunodeficient individuals is of major concern, because continued replication of PVs in the human gut could result in the reversion of these viruses to greater neurovirulence. When exposed to OPV, immunodeficient patients may become chronically infected, spreading potentially neurovirulent VDPVs for many months or years to close contacts and children who are no longer being vaccinated after termination of OPV vaccination in the near future.

Since the initiation of the global poliomyelitis eradication program in 1988, the number of wild-type polio cases decreased from 350,000 to fewer than 500, and the number of polio endemic countries declined from more than 125 to 10. The last case of polio in South Africa caused by a wild-type poliovirus (PV) occurred in 1989. The live attenuated oral poliovirus vaccine (OPV) has been effectively used in the reduction and control of poliomyelitis. However, as OPV strains are excreted in stools after vaccination, this vaccine could become a source of dissemination of PVs in the environment and the potential cause of poliomyelitis. Therefore, the aim of the study was to determine the occurrence of OPV strains in selected sewage and river water samples. During the period between 2001 and 2003, 138 samples of river water and 213 samples of settled sewage were collected from selected areas of South Africa. A total of 860 plaques were analysed, which consisted of 703 plaques from the sewage and 157 plaques from the river water samples. Using a reverse transcriptase (RT)-multiplex PCR, 49 PVs were successfully distinguished from 176 non-polio enteroviruses (NPEVs). The 176 NPEVs consisted of 50 coxsackie B2 viruses (CBV2), followed by 39 echoviruses 11 (ECV11), 25 CBV5, 21 CBV3, 15 CBV4, 14 coxsackie A6 viruses (CAV6), 7 CBV6, 2 CAV5, 2 CBV1, and 1 ECV19, which was in agreement with the prevalence of these EVs in other parts of the world. The Sabin-specific RT-triplex PCR revealed the presence of 29 Sabin PV type 1, 8 Sabin PV type 2, and 12 Sabin PV type 3 isolates. Buffalo green monkey kidney and primary liver carcinoma cell cultures allowed the amplification of a broad spectrum of EVs, whereas human epidermoid carcinoma cells were more selective for PVs. This study addressed some of the issues regarding the prevalence of OPV strains in the environment. The identification of 49 viable OPV isolates confirmed the presence and circulation of PV vaccine strains in sewage and river water. The extent of the potential health risk constituted by these OPV isolates remains to be investigated.

Evidence has been presented that some heterotrophic bacteria often detected in drinking water supplies possess features associated with pathogenicity. This suggests that even the low numbers of heterotrophic bacteria considered acceptable by drinking water specifications may constitute a health risk, particularly to immunocompromised consumers. In this study, 339 bacteria were isolated at random from routine heterotrophic plate count (HPC) tests on selected drinking water supplies in South Africa. In a first screen for potentially pathogenic properties, 188 of the isolates (55.5%) displayed a- or b-haemolysis on blood agar. Further analysis of the haemolytic isolates for enzymes associated with virulence revealed the presence of chondroitinase (5.3%), coagulase (16.0%), DNase (60.6%), elastase (33.0%), fibrinolysin (53.7%), gelatinase (62.2%), hyaluronidase (21.3%), lecithinase (47.9%), lipase (54.8%) and proteinase (64.4%) of the isolates. No fluorescein or pyocyanin was detected in any of the isolates. Among the haemolytic isolates 68.6% were resistant to oxacillin (1 μg), 59.6% to penicillin G (2 units), 47.3% to penicillin G (10 units), 53.7% to ampicillin (10 μg) and 42.6% to ampicillin (25 μg). Cytotoxicity, invasiveness and adherence properties of the haemolytic isolates was determined on HEp-2 and Caco2 cell lines. Among the haemolytic isolates 96% were cytolytic on the HEp-2 cell line. All the haemolytic isolates adhered to HEp-2 and Caco2 cells but gram-negative isolates tended to adhere in larger numbers than gram-positive isolates. HEp-2 cells were invaded by 42% of the haemolytic isolates. Heterotrophic bacteria, which most frequently revealed the above features associated with pathogenicity included species of the following genera: Aeromonas, Acinetobacter, Aureobacterium, Bacillus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, Tsukamurella and Vibrio. The results obtained in this study support earlier indications that bacteria detected by routine heterotrophic plate counts on drinking water supplies may include bacteria associated with potentially pathogenic properties. The extent to which these bacteria in drinking water supplies may constitute a health risk remains to be investigated.