Occurrence of noroviruses in recycled water and sewage sludge: emergence of recombinant norovirus strains.

AIMS: To investigate the presence of noroviruses in recycled water and sewage sludge obtained from a wastewater treatment plant in Bangkok Metropolitan Region, Thailand. METHODS AND RESULTS: Twenty-seven recycled water and twenty-three sewage sludge samples were tested for the presence of norovirus genogroup (G)I and GII using RT-nested PCR. Molecular characterization of noroviruses was undertaken by DNA sequencing and phylogenetic analysis. The level of the RNA genome of the noroviruses was determined using quantitative real-time RT-PCR. Noroviruses were detected in 44·4% of recycled water samples and 73·9% of sewage sludge samples. Norovirus GI.2 and GII.4 were identified in recycled water samples at levels of 2·19 × 101 and 3·26 × 104 RNA copies per litre. Six different genotypes of GI (GI.1, GI.2, GI.5a, GI.5b, GI.6b and GI.7) and GII.17 were identified in sewage sludge samples at levels ranging from 1·99 × 101 -1·43 × 105 RNA copies per gram wet weight. Four recombinant norovirus strains were detected in sewage sludge samples, namely GII.P16-GII.2, GII.P16-GII.4, GII.P16-GII.13 and GII.P21-GII.13. CONCLUSIONS: These findings provide evidence that noroviruses may be spread to the community and environment via the use of recycled water for plant areas, and sewage sludge for land application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating recombinant norovirus strains in sewage sludge samples. The presence of noroviruses in recycled water and sewage sludge contributes to a health risk of environmental exposure.

Assessment of a rapid immunochromatographic test for the diagnosis of norovirus gastroenteritis.

Noroviruses are the leading cause of acute gastroenteritis in people of all ages. Since the viruses are highly infectious, rapid and early diagnosis is important to prevent and control the disease. The present study aimed to evaluate the commercial immunochromatographic test RIDA® QUICK Norovirus for the detection of norovirus in stool samples from patients with acute gastroenteritis in Thailand. As compared with reference RT-PCR results, the RIDA® QUICK Norovirus assay provided a sensitivity of 48.2 and 83.3% with a specificity of 87.5%. False positive results were observed in 12.5% of norovirus-negative stool samples. Based on commercial quantitative real-time RT-PCR, the RIDA® QUICK Norovirus assay revealed a highly significant association, p-value <0.001, and good agreement (kappa = 0.6). The assay could detect norovirus in stool samples ranging from 3.22 × 10(6) to 3.26 × 10(8) copies/ml. False negative results occurred in the stool samples containing 5.9 × 10(6) copies/ml of norovirus GI or 1.85 × 10(4) - 4.28 × 10(5) copies/ml of GII. The immunochromatographic RIDA® QUICK Norovirus assay may be useful for rapid screening of norovirus infections in patients with acute gastroenteritis in both developed and developing countries where the RT-PCR method has not been established for routine diagnosis.

Detection and characterization of hepatitis A virus in water samples in Thailand.

AIMS: Outbreaks of hepatitis A in Thailand have been reported continuely and associated with water supply. However, the genetic analysis of hepatitis A virus (HAV) in water is limited. This study described the application of virus concentration method and reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) to detect HAV RNA and analyse the genetic sequence of the virus in environmental water samples. METHODS AND RESULTS: The HAV from water samples was concentrated by using a developed virus concentration method (adsorption-elution and subsequent speedVac reconcentration) and the viral RNA was detected by RT-nested PCR followed by sequencing of the amplified DNA products. Detection limit of HAV determined by the RT-nested PCR was 1.29 radioimmunofocus assay (RIFA) units ml(-1). The DNA band appeared at 183 basepairs. No cross-reactivity was observed in the presence of other enteric viruses (poliovirus and rotavirus). A total of 180 water samples were collected, concentrated, and detected for HAV. The HAV was found in 6/40 (15%) of water samples collected from a swamp and 3/30 (10%) collected from a canal. Ten river samples and 100 tap water samples stored in containers for drinking and domestic uses were negative for HAV. In sequence analysis of the DNA products and alignment with the HAV sequence deposited in the GenBank, six water samples showed the nucleotide sequence associated with HAV. The 120 nucleotides in the N-terminal VP1 region obtained from two swamp samples showed 95 and 96.7% identity to HAV genotype IA. In nearly all water samples where HAV was present bacterial indicators (faecal coliforms and Escherichia coli) were found for faecal contamination. CONCLUSIONS: A coupled virus concentration method and RT-nested PCR was successfully applied to examine HAV in water samples collected from various sources. DNA sequencing of nested PCR products showed the genotype IA associated with HAV that is predominate in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY: This research is the first study of genetic sequence of HAV in water samples in Thailand. The presence of naturally occurring HAV might pose a potential health risk for people.

Use of a rapid immunochromatographic test for early diagnosis of dengue virus infection.

Acute serum samples collected from 92 patients were analysed for the presence of immunoglobulin M and G antibodies against dengue viruses using a rapid immunochromatographic (IC) test. Of 52 patients with dengue virus infection diagnosed using a standard hemagglutination inhibition test, 41 patients demonstrated dengue virus infection using the IC test. Half of the serum samples collected on day 3 after the onset of fever were positive for dengue antibodies, and all were positive on day 6 after symptom onset. In comparison with the hemagglutination inhibition test, the rapid IC test showed 79% sensitivity and 95% specificity. The efficiency of the test was 86%. The IC test showed good agreement with the hemagglutination inhibition test (k=0.72). The IC test is quick to perform and provides early diagnosis of dengue virus infection.

An improved method for concentrating rotavirus from water samples.

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

Detection of poliovirus, hepatitis A virus and rotavirus from sewage and water samples.

A modified adsorption-elution technique for concentration of enteric viruses from sewage and water samples was developed. The viruses in water were concentrated by negatively charged membrane filtration, eluted with 2.9% tryptose phosphate broth containing 6% glycine pH 9.0, and reconcentrated using centrifugation by a speedVac concentrator. The presence of poliovirus, hepatitis A virus (HAV) RNA, and rotavirus antigen was determined by cell culture isolation, nested polymerase chain reaction (nested PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. A total of 100 sewage and water samples were collected from various sources in congested communities in Bangkok, concentrated and examined for those enteric viruses. Of 20 surface water samples from canals which located near sewage drains, 15% were positive for HAV RNA by nested PCR. Of 48 domestic sewage samples from man-holes of underground sewers, 8% were positive for rotavirus antigen by ELISA. Even though the samples were concentrated 256-2,000 fold, poliovirus was not found by isolation in cell culture.

Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.

Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay.

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-alpha) in serum samples of children infected with dengue virus (n=99) and healthy controls (n=41). The minimum detectable concentration of TNF-alpha by the BS-ELISA was 3.3 pg/ml. The mean TNF-alpha level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44+/-42.0 pg/ml). Lower levels were found in DHF grade I (28.44+/-42.7 pg/ml), DHF grade II (24. 21+/-25.4 pg/ml) and dengue fever (DF) (14.10+/-24.0 pg/ml). TNF-alpha in the sera of DF and DHF patients could be detected on days 2-6 after the onset of fever, the high level occurring on day 5. TNF-alpha was detected in 41.4% (24.01+/-35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2+/-15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-alpha than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-alpha than DF patients (P-value=0.020) but no difference in the TNF-alpha levels from sera of DHF grades I-III patients was observed (P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-alpha in serum samples of DF and DHF patients. The TNF-alpha levels might be associated with dengue virus infection and related to disease severity of DHF.

Immunoglobulin M-capture biotin-streptavidin enzyme-linked immunosorbent assay for detection of antibodies to dengue viruses.

A biotin-streptavidin system was adapted to an IgM-capture ELISA for detection of dengue antibodies in human sera. To develop this assay, high titers of antibodies to flavivirus were purified by ion-exchange chromatography (DEAE-cellulose) and labeled with biotin. Heavy chain-specific goat anti-human IgM was first bound to the wells of a polystyrene microtiter plate, followed by binding of IgM in test specimens, and the use of tetravalent dengue antigens (dengue 1-4), biotin-labeled anti-flavivirus IgG, and streptavidin-peroxidase conjugate. The sensitivity and specificity of the IgM-capture biotin-streptavidin ELISA (IgM-BS-ELISA) in acute sera were 83.3% of patients with dengue infection and 95.3% of nondengue-infected cases, respectively. The positive predictive value was 92.4% and the negative predictive value was 89.2%. The efficiency of test was 90.4%. In convalescent sera, the sensitivity and specificity of IgM-BS-ELISA were 100% and 92.6%, respectively. The predictive values of positive and negative results were 90.3% and 100%, respectively. The efficiency of test was 95.6%. The agreement rate of IgM-BS-ELISA and standard hemagglutination inhibition test was good: kappa (kappa) values were 0.79 for acute sera and 0.91 for convalescent sera. The correlation between two methods was quite good, with correlation coefficients (r) of 0.76 for acute sera and 0.85 for convalescent sera (P < 0.001). The results indicate that the IgM-BS-ELISA is highly sensitive, specific, simple to perform, and rapid.

Comparison of dengue virus antigens in sera and peripheral blood mononuclear cells from dengue infected patients.

The presence of dengue virus antigens in acute sera and peripheral blood mononuclear cells (PBMC) from dengue infected patients were determined by a biotin-streptavidin enzyme-linked immunosorbent assay (BS-ELISA). The frequency of the antigens detected in PBMC was higher than that in sera (53.8% vs 18.9%). In comparison with sera, the detection rate in PBMC was greater than six times: 7 cases were positive only in sera whereas 44 cases were positive only in PBMC, p < 0.001. The presence of the antigens in the sera did not depend on the severity of the disease, i.e. dengue fever, dengue hemorrhagic fever (grades I and II) or dengue shock syndrome (grades III and IV). In contrast, the presence of the antigens in PBMC increased from 36.8% to 100% when the infection was more severe. The dengue virus antigens could be detected in the samples collected between day 2 and day 7 after onset of the disease with the highest rate of detection (68.8%) in PBMC collected on day 4. The data suggest the use of PBMC with access to the appropriate acute-phase specimen for detection of dengue virus antigens.

Hepatitis B sero-prevalence and risk factors among school-age children in a low socioeconomic community, Bangkok.

A cross-sectional study of 165 school-age children who had no history of HBV vaccination was carried out in a low socioeconomic community of Din-Daeng, Bangkok. Blood specimens were collected for determination of HBV seromarkers (HBsAg, Anti-HBs and Anti-HBc) by EIA commercial kits. The results showed that the prevalence of HBV seromarkers was 24.85%, the HBsAg carrier rate was 3.64%, the anti-HBs positive rate was 15.15%, and the prevalence of only anti-HBc was 6.06%. To investigate factors associated with the positivity of HBV seromarkers, children were divided into two groups--the first group consisted of 41 children with HBV seromarkers and the second consisted of 124 children without HBV seromarkers. The study variables between the two groups were compared and analysed. The results revealed that factors associated with HBV positivity were (a) child factors: child's age, child's sex, ear piercing in female, sharing blade during haircutting, contact wound from other persons, using wares with other persons, searching things in garbage, and (b) family factors: older parent, low education in parent, low family income per month, low parent's knowledge and attitude about HBV infection and vaccination, (P < 0.05). After using stepwise regression analysis, the factor of ear piercing in female was only one significant variable.

Reverse passive hemagglutination tests for rapid diagnosis of snake envenomation.

Reverse passive hemagglutination (RPHA) tests for the detection of six major poisonous snake venoms of Thailand were studied. Three different species of red blood cells i.e., sheep (SRBC), human (HRBC) and chicken (CRBC) were sensitized with protein A-affinity purified rabbit antivenom IgG using chromic chloride as a coupling reagent. The properties of these sensitized erythrocytes with regard to sensitivity, specificity, stability to venom enzymes and storage etc., were studied and compared. The sensitivities of the RPHA tests in venom detection were 2 to 635 ng/ml. Cross-reactions were observed with heterologous venoms at concentrations at least 62 times higher than those observed with homologous venoms. After treatment with glutaraldehyde, the coupled red blood cells showed reduced sensitivity but were stable at 4 degrees C from 1 to 12 months depending upon the antibody and the species of erythrocytes. The entire test required 60 to 120 min. The RPHA using fresh SRBC correctly identified various venoms in 48 of 59 (81.3%) serum samples and 16 of 26 (61.5%) wound swabs. Venom mis-identifications were made in 2 sera (3.4%). In a comparison of 24 paired serum and wound swab samples, more positive identifications were made with serum than with swab samples but the difference was not statistically significant (p > 0.05).

Clinical study on antithrombotic effects of ticlopidine in ischemic stroke.

The investigators conducted a clinical study on antithrombotic effectiveness in ischemic stroke at Siriraj Hospital Medical School, Mahidol University from May 1987 to May 1989. Twenty-nine patients, 16 males and 13 females were enrolled in the study. The ages of the patients ranged from 30-87 years with a mean age of 63 +/- 11 years. Ticlopidine (250 mg) could significantly inhibit platelet aggregation induced by ADP and collagen within 24 hours of drug administration. After 1 week to 6 months, only aggregation by ADP was still inhibited significantly without significant effects on fibrinolytic activity and prostacyclin. Hematocrit was significantly decreased at the 1st and 2nd month of treatment. Serious side effects were skin rash and severe headache while the other common ones were dizziness, and diarrhea but these effects disappeared without discontinuing the drug. Most patients who suffered from nausea, diarrhea and headache, had temporary elevated SGPT. It may be concluded that only half of the recommended dose of ticlopidine has inhibitory effects on both phases of ADP-induced aggregation without interfering with fibrinolytic activity and can maintain prostacyclin. However, it also possesses either serious or common side-effects. This drug, therefore, should be used with the awareness of the clinician.