Evaluation of methods for concentrating hepatitis A virus from drinking water

By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture-adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission.     

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

Round robin investigation of methods for the recovery of poliovirus from drinking water

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inactivation of Norwalk virus in drinking water by chlorine.

Norwalk virus in water was found to be more resistant to chlorine inactivation than poliovirus type 1 (LSc2Ab), human rotavirus (Wa), simian rotavirus (SA11), or f2 bacteriophage. A 3.75 mg/liter dose of chlorine was found to be effective against other viruses but failed to inactivate Norwalk virus. The Norwalk virus inoculum remained infectious for five of eight volunteers, despite the initial presence of free residual chlorine. Infectivity in volunteers was demonstrated by seroconversion to Norwalk virus. Fourteen of 16 subjects receiving untreated inoculum seroconverted to Norwalk virus. Illness was produced in four of the eight volunteers and in 11 of 16 control subjects. A similar Norwalk virus inoculum treated with a 10 mg/liter dose of chlorine produced illness in only one and failed to induce seroconversion in any of eight volunteers. Free chlorine (5 to 6 mg/liter) was measured in the reaction vessel after a 30-minute contact period. Norwalk virus appears to be very resistant to chlorine which may explain its importance in outbreaks of waterborne disease.     

Purification of hepatitis A virus from chimpanzee stools.

Hepatitis A virus antigen was purified from early acute-phase chimpanzee stools by a rapid three-step procedure using 7% polyethylene glycol precipitation, CsCl banding, and Sepharose 2B column chromatography. Electron microscopic examination of the hepatitis A virus entigen preparation revealed highly purified hepatitis A virus particles.     

Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

A piezoelectric immunobiosensor for atrazine in drinking water.

A piezoelectric crystal immunobiosensor has been developed for the assay of atrazine herbicides in drinking water. Determinations from 0.03-100 micrograms l-1 (parts per billion) of atrazine can be made with a relative SD of about +/- 8%. Atrazine antibodies (polyclonal from sheep) are layered onto the gold electrode of 10 MHz piezoelectric crystals, which are precoated with protein A. The sensor is reversible, being reusable for about eight or nine assays.     

Isolation of Legionella species from drinking water.

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Evaluation of factors affecting the membrane filter technique for testing drinking water.

The following studies were done in response to questions regarding the adoption and use of the membrane filter (MF) technique for testing drinking water for the total coliform indicator group. A comparison with the most-probable-number technique showed that MF procedures with m-Endo agar LES were somewhat superior to the most-probable-number methods in terms of numbers of coliform organims recovered. Medium preparation and storage studies indicated that rehydration of m-Endo agar LES should be done with boiling water for less than 15 min, that m-Endo agar LES should not be exposed to light for more than 4 to 6 h, and that m-Endo agar LES plates may be used for up to 4 weeks and broth verification media for up to 3 weeks under given storage conditions. MF culture colonies were commonly found which did not produce sheen as expected for coliforms and yet were verified as coliforms. The occurrence and morphology of these atypical colonies were studied. Parallel inoculation of both lauryl tryptose (LT) and brilliant green bile (BGB) broth was found to be a better colony verification approach than recommended LT preenrichment before transfer to BGB. Comparison of parallel verification results indicated very little justification for the use of LT medium in MF verification procedures. In the case of overgrown or confluent cultures, the best coliform recoveries resulted from swabbing the MF plate and directly inoculating BGB medium with the swab. The occurrence of overgrowth was defined and evidence was collected suggesting that overgrowth is a function of sample holding time. Evaluation of routine test data and bacterial population reductions as a function of time indicated that nonquantitative recovery of coliforms may not be significantly affected for at least a 72-h sample holding time.     

四种丙型肝炎病毒抗体试剂检测方法的性能评估

对血液筛查的四种丙型肝炎病毒(Hepatitis C virus,HCV)抗体试剂检测方法的性能进行评估。方法采用协作标定方式,应用四种检测方法(间接ELISA法、双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法)16种HCV抗体试剂,对经确证筛选出的70份(HCV抗体阳性35份、阴性35份)血浆样本进行检测,通过分析阳性、阴性符合率,评估四种方法 HCV抗体试剂的性能。结果间接ELISA法试剂检测阳性符合率为88.6%(31/35)~94.3%(33/35),双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法试剂检测阳性符合率为91.4%(32/35)~94.3%(33/35),四种检测方法 HCV抗体试剂阳性符合率之间的差异无统计学意义(P0.05);间接ELISA法和间接CLIA法试剂弱阳性样本检出率为11.4%(4/35)~31.4%(11/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂弱阳性样本检出率为5.7%(2/35)~11.4%(4/35),四种检测方法 HCV抗体试剂对弱阳性样本的检出率之间的差异有统计学意义(P0.05);间接ELISA法试剂检测阴性符合率分别为94.3%(33/35)~100%(35/35),间接CLIA法试剂阴性符合率分别为94.3%(33/35)~97.1%(34/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂阴性符合率均为97.1%(34/35),四种检测方法 HCV抗体试剂阴性符合率之间的差异无统计学意义(P0.05)。结论四种检测方法 HCV抗体试剂的性能基本一致,不同方法各有优缺点,建议应用多种检测方法对弱阳性样本进行确认检测。     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Statistical approach for comparison between methods of bacterial enumeration in drinking water

A general statistical procedure based on the likelihood ratio test is presented for the purpose of comparing estimates of mean bacterial density derived from different sets of data. This approach is much more appropriate than the conventional ways of analyzing bacteriological results (e.g., analysis of variance) which usually require previous transformation of the data. An illustrative application of the method compares three distinct titration techniques for enumerating heterotrophic bacteria in drinking water at 20°C incubation temperature. It was shown that both the standard plate count (SPC) and the membrane filter (MF) procedures supplied substantially the same information, whereas the microplate technique using the most probable number (MPN) for total bacterial enumeration could yield considerably different estimates: MPN values were significantly lower in three cases and significantly higher in one case out of a total of five experiments. The results consistently indicate a strong interaction between the technique used and the sample analyzed. Three different media (nutrient agar, R-2A low nutrient agar and m-SPC agar) were then evaluated for enumerating heterotrophic bacteria, using the MF technique at 48, 72 and 96 h of incubation time at 20°C. Although the media recovered approximately the same numbers of bacteria after 96 h of incubation, statistically significant discrepancies occurred after intermediate periods of incubation, perhaps because the relative rates of bacterial growth differed among media.