Validation of the bag-mediated filtration system for environmental surveillance of poliovirus in Nairobi, Kenya.

AIMS: This study compared the bag-mediated filtration system (BMFS) and standard WHO two-phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin-like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. METHODS AND RESULTS: Environmental samples were collected in Nairobi, Kenya (Sept 2015-Feb 2017), concentrated via BMFS and two-phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two-phase samples (P < 0·05). In BMFS samples, SL PV detection did not significantly differ with volume filtered, filtration time or filter shipment time (P > 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two-phase samples (P < 1 × 10-5 ). CONCLUSIONS: Poliovirus was detected at higher rates with the BMFS, a method that includes a secondary concentration step, than using the standard WHO two-phase method. SL2 disappearance from the environment was commensurate with OPV2 withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: The BMFS offers comparable or improved PV detection under the conditions in this study, relative to the two-phase method.

Molecular characterisation of hepatitis A virus strains from water sources in South Africa.

Hepatitis A virus (HAV) strains found in selected South African (SA) surface waters were characterised to establish what HAV types are circulating in the environment, thus reflecting circulation in the surrounding communities. Surface water samples used for irrigation or domestic purposes, and water samples from the outflow of wastewater plants were collected from six provinces. Viruses were recovered from the samples using a glass wool adsorption-elution method and then further concentrated using polyethylene glycol/sodium chloride precipitation. After automated nucleic acid extraction, samples were analysed for HAV by real-time reverse-transcriptase polymerase chain reaction. HAV strains were genotyped by nucleotide sequence analysis of the capsid gene VP1 and the VP1/P2B junction. HAVs were detected in 76% (16/21) of the surface water samples and in 37% (19/51) of the samples from the wastewater plants. Strains were characterised from 32 of the 35 samples and classified within genotype IB. The presence of genotype IB in the water sources confirms human faecal contamination. Hence, these faecally-contaminated water sources may be a potential transmission route of HAV infection and a potential source of contamination of irrigated fresh produce in SA.

Retrospective determination of HIV-1 status by a PCR method on paraffin wax embedded sections.

AIM: To develop a simple but reliable polymerase chain reaction (PCR) method to determine the HIV-1 status of patients on formalin fixed, paraffin wax embedded lymph node tissue. METHODS: Fifty lymph node specimens, 20 from HIV-1 seropositive and 30 from HIV-1 seronegative patients, were analysed. Lymph nodes with a variety of disease conditions were included in the study. Tissue sections were treated with a DNA extraction buffer containing proteinase K and the crude cell lysate was used in PCR analysis. Nested primers were used to amplify HIV-1 DNA sequences coding for gag, pol and env proteins. PCR products were demonstrated by polyacrylamide gel electrophoresis. Results were then compared with HIV-1 serology of the patients from whom the tissue was obtained. RESULTS: The PCR method yielded a specificity of 100%, a sensitivity of 95%, a positive predictive value of 100%, and a negative predictive value of 97% when compared with HIV-1 serology. The kappa statistic (0.958) showed an excellent agreement between the PCR method and serology. Furthermore, HIV-1 DNA was demonstrated in lymph node tissue from a serologically unconfirmed acquired immunodeficiency syndrome case necropsied in 1982. CONCLUSION: This PCR method is a simple and reliable means of retrospectively determining the HIV-1 status of patients using formalin fixed, paraffin wax embedded lymph node tissue.

Molecular characterisation of small round structured viruses associated with gastroenteritis in South Africa.

The application of the reverse transcriptase polymerase chain reaction (RT-PCR) has enabled several morphologically and physically similar small round structured viruses (SRSVs), including the prototype Norwalk virus (NV), to be classified within the Caliciviridae. This technique, using primers directed to the RNA-dependent RNA polymerase region within the ORF1 of NV, was used to characterise SRSVs associated with epidemic gastroenteritis in adults and sporadic paediatric gastroenteritis in South Africa. Genomic variation was investigated by sequence analysis of the amplified 209bp cDNA region from six isolates and comparison with other characterised SRSVs including NV. Antigenic variation was investigated by the use of the recombinant enzyme immunoassay described recently for the detection of Snow Mountain agent-like antigen in stool specimens. Two distinct antigenic groups were evident with NV-like viruses associated with adult gastroenteritis, and Mexico viruslike viruses associated with paediatric gastroenteritis. Viral isolates from two of the outbreaks of adult gastroenteritis showed a high degree of nucleotide sequence identity with NV, i.e., 84% and 98%, respectively, whereas the paediatric isolates showed 92-95% sequence similarity with the Snow Mountain-like virus, MxV. These data show concordance between antigenic and genomic analyses.

Phylogenetic analysis of the Caliciviruses.

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo-like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA-dependent RNA polymerase (approximately 470nt) and capsid hypervariable regions (approximately 1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo-like strains ranged from 61% to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round-structured viruses (SRSV), Sapporo-like, and hepatitis E virus (HEV)-like HuCVs and rabbit-, and vesicular exanthema of swine virus (VESV)-like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo-like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies.

Incidence of human calicivirus and rotavirus infection in patients with gastroenteritis in South Africa.

Human caliciviruses (HuCVs) are reportedly responsible for 2.5-4% of nonbacterial sporadic gastroenteritis. The incidence of HuCV infection in South Africa is unknown. Stool specimens from 1,296 South African patients with sporadic gastroenteritis were screened for the presence of HuCVs using electron microscopy, recombinant enzyme immunoassays for Norwalk (NV) and Mexican (MX) viruses, and the reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced to ascertain which HuCV genogroups were present. HuCVs were detected in 43/1,296 (3.3%) specimens examined, with RT-PCR proving to be the most sensitive detection method. Genetic analysis of the isolates indicated that 81% were Snow Mountain Agent, or MX-like; 8% were NV-like; and 11% were HuCV/Sapporo-like. This study indicates that a combination of assays is needed for the accurate detection of HuCVs. Comparative data on hospitalised patients showed that the incidence of rotavirus infection was approximately ten times greater than that of HuCV infection.

Experience with a provocative test of calcitonin release as a prospective screening for preclinical medullary thyroid carcinoma in MEN type 2A family members.

We report our experience with a provocative test of calcitonin (CT) release using a combined stimulus of intravenous 10% CaCl2 solution and pentagastrin on 34 normal adults (15 females: age 41 +/- 12.3 years and range 22-65 years; and 19 males: age 43 +/- 9.1 years and range 23-60 years) and in 44 family members of three proven multiple endocrine neoplasia type 2A syndrome (MEN 2A) patients. A commercial radioimmunoassay was used to determine the serum CT levels. Peak CT levels were reached within 2 to 5 minutes after administration of the stimulus in all subjects tested. In the group of normal subjects there was no significant difference in the mean basal CT levels between males (54.8 +/- 21.7 pg/ml) and females (56.5 +/- 34.8 pg/ml), whilst the mean peak response values for males was 146.3 +/- 120.6 pg/ml, which was significantly different from the mean value of females, namely 71.6 +/- 39.0 pg/ml. We did not find significant correlations between the basal CT level, peak CT response, and age. Of the 44 family members tested, 9 showed an exaggerated CT response to the combined stimulus and subsequently had a total thyroidectomy. Histological examination confirmed C-cell hyperplasia (CCH) in one and medullary thyroid carcinoma (MTC) in the other 8. Three of the 9 had high basal plasma CT levels. The 9 patients were retested postoperatively and all showed a flat response to the combined stimulus. Those family members with histological proof of MTC or CCH were screened for genetic linkage to the disease gene for MEN 2A using probe MCK2, and showed correlation in each instance.(ABSTRACT TRUNCATED AT 250 WORDS)