Adsorption-elution Technique Research Articles

Dealing with blood group discrepancies in donor populations in a resource-poor blood center, practical approach: A single blood center study from Eastern India

Abstract BACKGROUND: ABO and RhD grouping are the most important pretransfusion tests. Discrepancies in blood grouping are one of the major causes of transfusion reactions. Although ABO and RhD blood group discrepancies are not a very frequent phenomenon, an overall prevalence in the healthy populations was not known in the south-eastern part of West Bengal, India. AIMS: To analyze the overall prevalence of ABO-Rh (D) blood group discrepancies in healthy blood donor populations, serological methods to resolve them and to supply blood in emergency circumstances to such persons. MATERIALS AND METHODS: In this study, a total of 8000 voluntary blood donors were chosen by the simple random sampling from South-Eastern part of West Bengal and blood grouping was done by the conventional tube technique method. Discrepancies were found among 24 blood donors with overall incidence of 0.3%. Resolution of group discrepancies done by extended incubation, weak D testing, subgroup detection with specific antisera, adsorption elution technique, secretor status by inhibition technique, etc., Direct immunoglobulin test (DAT) was done on all the samples by the polyspecific gel card method. RESULTS: Most of the discrepancies were due to weak or missing serum reactivity, (11 samples, incidence 0.13%), extra serum or red cell activity found in 6 samples (incidence 0.07%), subgroups causing weak red cell activity found in 4 samples (incidence 0.05%), and weak D antigen found in three samples (incidence 0.03%). One sample was DAT positive with no grouping anomaly. CONCLUSION: Here, in this study, blood group discrepancies were resolved using simple serological techniques. This study may help to resolute group discrepancies in day to day transfusion practices where modern facilities are not available.

Infectious Pepper Mild Mottle Virus and Human Adenoviruses as Viral Indices in Sewage and Water Samples

The objective of this study was to compare human adenoviruses (HAdVs) genome and infectivity, polyomaviruses (JC and BK) genome (JCPyVs) and (BKPyVs), Pepper Mild Mottle Virus (PMMoV) genome and infectivity, and infectious bacteriophages as viral indices for sewage and water samples. One hundred and forty-four samples were collected from inlets and outlets of water and wastewater treatment plants (WTPs), and WWTPs within Greater Cairo from October 2015 till March 2017. Two methods of viral concentration [Aluminium hydroxide (Al(OH)3) precipitation method and adsorption-elution technique followed by organic flocculation method] were compared to determine which of them was the best method to concentrate viruses from sewage and water. Although samples with only one litre volume were concentrated using Al(OH)3 precipitation method and the same samples with larger volumes (5–20 L) were concentrated using the adsorption-elution technique followed by the organic flocculation method, a non-significant difference was observed between the efficiency of the two methods in all types of samples except for the drinking water samples. Based on the qualitative prevalence of studied viruses in water and wastewater samples, the number of genome copies and infectious units in the same samples, resistance to treatment processes in water and wastewater treatment plants, higher frequency of both adenoviruses and PMMoV genomes as candidate viral indices in treated sewage and drinking water was observed. The problem of having a viral genome as indices of viral pollution is that it does not express the recent viral pollution because of the longer survivability of the viral genome than the infectious units in water and wastewater. Both infectious adenovirus and infectious phiX174 bacteriophage virus showed similar efficiencies as indices for viral pollution in drinking water and treated sewage samples. On the other hand, qualitative detection of infectious PMMoV failed to express efficiently the presence/absence of infectious enteric viruses in drinking water samples. Infectious adenoviruses and infectious bacteriophage phiX174 virus may be better candidates than adenoviruses genome, polyomaviruses genome, and PMMoV genome and infectivity as viral indices for water and wastewater.

To Evaluate the Sensitivity of ABO Antigen Determination from Dental Pulp at Various Temperatures over a Duration of 3 Months - A Diagnostic Study

Introduction: Every individual either living or dead has an unique identity as per theory of uniqueness. Medico legal examination recognizes the blood group as an unique indicator as it remains unchanged, forensic study uses teeth as the unique biological indicator, as they are the least destructible part of the body.. The present study was conducted to evaluate the efficacy in retrieving ABO blood group antigens from pulp tissue at various temperatures like freezing temperature, 100°C and 200°C with room temperature being the baseline for the study and at various time intervals (same day, 14th, 30th and 90th day) post extraction by adsorption –elution method. Materials and Methods: A diagnostic study of 80 cases was included which involved extraction procedures under local anesthesia following the aseptic protocol. Sterile cotton/gauze was compressed in the socket and later the blood collected from it served as a control group for the study. The extracted teeth were washed and kept, stored in numbered bottles for different time periods after being subjected to different degrees of temperatures i.e. at room temperature, freezing temperature, 100°C and 200°C for 60mins respectively. Results: Goodman Kruskal Gamma test was used for statistical analysis to obtain the correlation between blood groups at different temperatures. After analyzing the ABO antigen for sensitivity at various degree of temperature over different time intervals it was found that at freezing temperature and room temperature, antigens can be assessed , while at higher temperatures it is difficult to appreciate. Conclusion: Adsorption-Elution technique can be used as a reliable tool for determination of ABO blood group from the dental pulp of deceased person.

Detection of Rare Blood Group Ax Phenotype in Blood Donor

Background: Karl Landsteiner discovered ABO blood group system in early 20th century, but still, uncertainty remains in immune-hematology while detection of ABO subgroups or weaker variants. The presence of weak subgroups in blood donor samples gives rises to discrepancy in forward (cell) and reverse (serum) grouping. Methods and Materials: We here report a case of the 'A' weak Rh 'D' Positive, Probably Ax Phenotype in a blood donor who came for replacement donation at our blood bank. The blood group discrepancy was resolved by using serological testing, Adsorption elution technique and saliva secretor study. Results: Blood grouping by the tube technique showed no reaction was shown with anti-B, a faint reaction with anti-A, 1+ agglutination with Anti-AB, and a significant reaction (4+) with anti-D and anti-H. The patient's serum showed the presence of anti-B antibody as well as anti-A1. Result for eluate showed microscopic agglutination (1+) with group A cells and a negative reaction with group B cells and saliva secretor study showed having only H substance. Conclusion: This report highlights the importance of cell and serum grouping in solving blood group discrepancy in blood donors. This rare phenotype in a donor is first of its kind reported from India.

Evaluation of adsorbents and eluents for application in virus concentration and adsorption-desorption isotherms for coliphages

Adsorption elution technique is widely used for virus preconcentration before detection and quantification. However, the existing methods do not provide adequate recovery of viruses. DEAE cellulose, and Moringa oleifera seed protein functionalized rice husk ash (FaRHA) adsorbents were evaluated for the concentration of an enteric virus, Rotavirus A (RVA), an F-specific coliphage, MS2, and a somatic coliphage, SUSP2. Recovery with various adsorbent-eluent pairs was tested using initial coliphage concentration (Co) 104 PFU/mL. An eluent composed of 1.5 M NaCl, 2% Tween 80, and 0.05 M KH2PO4 (pH 9.2) yielded a high recovery of MS2 from DEAE cellulose (82%) and it also yielded high recovery of RVA. However, SUSP2 recovery from DEAE cellulose was ~61%, even after eluent optimization. An eluent comprised of glycine 3X broth, 1.5 M NaCl, 3% Tween 80, and 0.05 M KH2PO4 (pH 10.2) yielded high recovery of SUSP2 from FaRHA (88%). The maximum recovery of MS2 and RVA from FaRHA was lower (77% and 32%, respectively). The Freundlich model provided a good fit to the adsorption-desorption isotherms for the coliphages. For both the coliphages, the Freundlich capacity parameter, KF, was two orders of magnitude higher for DEAE cellulose compared to FaRHA. MS2 recovery from DEAE cellulose was minimally affected by antichaotrophic ions and dissolved organic matter, and higher sorption could be achieved over a wide pH range. For FaRHA, pH variation and various water matrices had a significant adverse effect on coliphage recovery. Thus, DEAE cellulose is a superior adsorbent for virus preconcentration from water samples.

Serological Detection of Rh-Del Phenotype among Rh-Negative Blood Donors at National Blood Center, Yangon, Myanmar.

Background Red cell Rhesus (Rh) antigen expression is influenced by the genetic polymorphism of RHD and RHCE genes and reveals serologically different reactions of RhD variants such as partial D, weak D, and Rh-Del. Serologically, Rh-Del type can only be detected by an adsorption-elution technique, and it might be mistyped as Rh-negative. The prevalence of Rh-Del has not been reported yet in Myanmar. Method A total of 222 Rh-negative blood donors in the National Blood Center were tested for weak D and Rh-Del by indirect antihuman globulin and adsorption-elution method, respectively. RhCE typing was performed among Rh-negative and Rh-Del. Results Of them, 75.2% (167/222) were Rh-negative, 15.8% (35/222) were Rh-Del, and 9% (20/222) were weak D. Of 202 blood donors (167 true Rh-negative and 35 Rh-Del), all of the Rh-Del positives were C-antigen-positive with 94.3% Ccee phenotype (33/35) and 5.7% CCee (2/35). Most of the Rh-negative donors (80.2%) were ccee phenotype (134/167). Conclusion About half of Rh-Del subjects were repeated donors, and attention was needed to avoid transfusion of truly Rh-negative patients to prevent alloimmunization. It is recommended to do Rh-Del typing of Rh-negative donors who are C-antigen-positive and consider moving them to the Rh-positive pool. Further study is needed to clarify the alloimmunization status for transfusion of Rh-Del blood to Rh-negative recipients. Molecular markers for RhD-negative and D variants should be established in the Myanmar population to improve selection of antisera for Rh typing and enhance safety of the transfusion services.

A new adsorption-elution technique for the concentration of aquatic extracellular antibiotic resistance genes from large volumes of water

Extracellular antibiotic resistance genes (eARGs) that help in the transmission and spread of antibiotic-resistant bacteria are emerging environmental contaminants in water, and there is therefore a growing need to assess environmental levels and associated risks of eARGs. However, as they are present in low amounts, it is difficult to detect eARGs in water directly with PCR techniques. Here, we prepared a new type of nucleic acid adsorption particle (NAAP) with high capacity and developed an optimal adsorption-elution method to concentrate eARGs from large volumes of water. With this technique, we were able to achieve an eARG recovery rate of above 95% from 10 L of water samples. Moreover, combining this new method with quantitative real-time PCR (qPCR), the sensitivity of the eARG detection was 104 times that of single qPCR, with the detection limit lowered to 100 gene copies (GCs)/L. Our analyses showed that the eARG load, virus load and certain water characteristics such as pH, chemical oxygen demand (CODMn), and turbidity affected the eARGs recovery rate. However, high eARGs recovery rates always remained within the standard limits for natural surface water quality, while eARG levels in water were lower than the detection limits of single qPCR assays. The recovery rates were not affected by water temperature and heterotrophic plate counts (HPC). The eARGs whatever located in the plasmids or the short-length linear DNAs can be recovered from the water. Furthermore, the recovery rate was high even in the presence of high concentrations of plasmids in different natural water (Haihe river, well water, raw water for drinking water, Jinhe river, Tuanbo lake and the Yunqiao reservoir). By this technology, eARGs concentrations were found ranging from (2.70 ± 0.73) × 102 to (4.58 ± 0.47) × 104 GCs/L for the extracellular ampicillin resistance gene and (5.43 ± 0.41) × 102 to (2.14 ± 0.23) × 104 GCs/L for the extracellular gentamicin resistance gene in natural water for the first time, respectively. All these findings suggest that NAAPs have great potential for the monitoring of eARGs pollution in water.

Prevalence of weak D phenotype among D negative C/E+ blood donors in Morocco

Aims: Determination of D variants is important for selection of blood products and to prevent anti-D-related hemolytic disease of the fetus and newborn. The prevalence of weak D phenotypes varies significantly among different ethnic populations. In Morocco, D variants have not been fully characterized. The purpose of this study was to determine prevalence of weak D among D negative and C/E+ blood donors and distribution of Rhesus blood group antigens using serologic methods. Methods: A total of 15,865 blood samples from Moroccan blood donors were tested for D, C, c, E, e antigens by routine serologic methods. Among blood donors serologically D negative C+ and/or E+, 63 samples were tested for weak D by indirect antiglobuline test, enzymatic treated cells test and adsorption elution technique. Results: Among 63 samples tested, 10 were positive by serologic methods (15.87%). Six samples identified as weak D by enzymatic test and four samples identified as Del by adsorption elution. Rhesus blood group antigens are distributed as DCcee: 38.85% and dccee :8.77%. Conclusion: The prevalence of D variants must be considered in transfusional and obstetrical strategy for RhD typing to ensure blood transfusion safety by optimizing the management of D-negative RBC units and Rh immune globulins.

Bombay blood group: Is prevalence decreasing with urbanization and the decreasing rate of consanguineous marriage

Context:Bombay blood group although rare is found to be more prevalent in the Western and Southern states of India, believed to be associated with consanguineous marriage.Aims:To estimate the prevalence of the Bombay blood group (Oh) in the urban population of Puducherry. To find the effect of urbanization on consanguineous marriage and to establish whether consanguinity plays a part in the prevalence of Oh group. To compare Oh group prevalence with that of other neighboring states, where population is not predominantly urban.Settings and Design:This is a descriptive study in a tertiary care hospital in Puducherry, over a period of 6 years.Materials and Methods:All blood samples showing ‘O’ group were tested with anti-H lectin. Specialized tests like Adsorption Elution Technique, inhibition assay for determination of secretor status were performed on Oh positive cases. Any history of consanguineous marriage was recorded.Statistical Analysis Used:All variables were categorical variable and percentage and proportions were calculated manually.Results:Analysis of the results of 35,497 study subjects showed that the most common group was ‘O’ group constituting 14,164 (39.90%) of subjects. Only three “Oh” that is, Bombay phenotype (0.008%) were detected. Consanguinity was observed in two cases (66.66%).Conclusions:This study shows the prevalence of Bombay blood group representing the urban population of Puducherry, to be high (0.008%) and associated with consanguineous marriage (66.66%). Thus, consanguinity is still an important risk factor present, even in an urban population in Southern India.

Norovirus GII.17 Predominates in Selected Surface Water Sources in Kenya.

In this study, the prevalence and genotypes of noroviruses (NoVs) in selected water sources from rural, urban and refugee settings in Kenya were investigated. Ten litres each of river, household and borehole water was collected in rural (Mboone River), urban (Nairobi and Mutoine River) and refugee (Dadaab refugee camp) settings. NoVs were recovered from the water samples by a glass wool adsorption-elution technique and/or PEG/NaCl precipitation. Nucleic acid was extracted using the automated MagNA Pure platform. NoVs were detected with singleplex real-time reverse transcription-polymerase chain reaction assays and characterised by nucleotide sequence analysis. NoVs were detected in 63% (25/40) of the selected water samples comprising GII (42.5%), GI (2.5%) and mixed GI/GII (17.5%) positive samples. The prevalence of NoVs in the Mutoine River (urban area) was higher than in the Mboone River (rural area) (P = 0.0013). Noroviruses GI.1, GI.3, GI.9, GII.4, GII.6, GII.12, GII.16 and GII.17 were identified, with GII.17 accounting for 76% (16/21) of the typed strains. The NoV GII.17 predominance differs to other studies in Africa and further surveillance of NoVs in clinical and environmental settings is required to clarify/elucidate this observation. As information regarding NoVs in Kenyan water sources is limited this report provides valuable new data on NoV genotypes circulating in environmental water sources and the surrounding communities in Kenya.

Quantitative characterization of the inhibitory effects of salt, humic acid, and heavy metals on the recovery of waterborne norovirus by electropositive filters

The virus adsorption-elution technique (VIRADEL) using electropositively charged filters is used frequently for recovering enteric viruses from water. The filter-absorbed virus is typically eluted, concentrated, and subsequently detected by culture or molecular methods. Human norovirus (HuNoV), one of the most important waterborne pathogens, cannot be cultivated by conventional culture methods and is typically detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. However, it is plausible that various inhibitors could be concentrated simultaneously during the VIRADEL process and affect RT-PCR assays. In this study, we evaluated the effect of typical inhibitors, including humic acid, heavy metals, and salt, on the recovery of norovirus by two different electropositive filters: 1MDS and Nanoceram. Known amounts of HuNoV and murine norovirus were inoculated in 1 L of surface water containing various concentrations of humic acid, heavy metals (cadmium and lead), or NaCl. Our results indicate that the presence of heavy metals or salt significantly reduced the recovery of virus from the electropositive filters. Thus, care should be taken when analyzing waterborne norovirus using electropositive filters in environments with high concentrations of heavy metal inhibitors or salts.

RHD*Weak D Type 38: A Family Study

AbstractAbstract 5155 Introduction:The RHD*weak D type 38 is produced by the single nucleotide change (833G>A) in RHD exon 6, carrying a Gly278Asp substitution in the transmembraneous RhD protein. This RHD allele shows a reduced expression of the D antigen on the red blood cell (RBC) surface and may be erroneously typed as D− by standard serologic methods, including the indirect antiglobulin test (IAT). The frequency of the RHD*weak D type 38 in Caucasians is reported to be 1. 5% in Ccee phenotype, while we have recently detected a relatively higher frequency of 2. 6% among Ccee phenotype in Brazil. In this study we performed a serologic and molecular analysis of three generations from a family with the rare RHD*weak D type 38. Study design and methods:The RBCs from 45 years-old woman blood donor tested D negative by routine serologic tests (Ccee phenotype) but were positive by the adsorption-elution technique. Further serologic investigation to establish the patterns of reactivity of the RBCs was preformed using anti-D Moabs IgM (clones HM10, P3X63, P3X21211F1, P3X21223B10; Diagast, Loos, France) and anti-D Moabs IgG/IAT (clones HM16, P3X35, P3X241, P3X249, P3X290; Diagast, Loos, France, and MS-26, ESD1; DiaMed, Latino América S. A.). Flow cytometric methods were employed to evaluate the D antigen densities, and molecular sequencing studies were done using a sequencing Kit (Big Dye Terminator v1. 1, applied Biosystems, Weiterstadt, Germany) and a genetic analyser (ABI 3100, Applied Biosystems, Foster City, CA, USA). Results:We found that the RBCs reacted negatively with 4/4 IgM anti-D, but showed weakly positive results with 4/7 IgG anti-D reagents. In face of such results we studied 12 members of her family and found that 6 more individuals tested D negative by standard serologic tests (Ccee phenotype) but were positive by adsorption-elution technique. The flow cytometry investigation of samples from the 7 subjects showed D antigen density in a range of 60 – 80 sites per cell and a Rhesus Index of 0. 1 – 0. 6. DNA sequencing analysis from 7 family members was performed showing that all descendants (siblings) had the same RHD*weak D type 38 genotype as the mother, also inherited by the grandson. Conclusions:The molecular analysis identified the presence of RHD*weak D type 38 in the three generations of a Brazilian family. The study revealed maternal inheritance (first generation) of the weak D type 38, with Ccee phenotype by her six children and the second generation showed paternal inheritance in a son. We also emphasize the significant prevalence of RHD*weak D type 38 in the Brazilian population, often showing a low antigen D density. Disclosures:No relevant conflicts of interest to declare.

Diverse norovirus genotypes identified in sewage-polluted river water in South Africa

This study aimed to assess norovirus (NoV) contamination and genotype diversity in surface water in Gauteng, South Africa. Between January 2008 and December 2010, three rivers, namely Klip, Suikerbosrant, and Rietspruit were monitored for NoV genogroup (G)I and GII. Viruses were recovered using the glass wool adsorption-elution technique and detected by real-time reverse transcription-polymerase chain reaction. From 2008 to 2010, NoVs were detected in 66% (70/106) of Klip river samples. The Rietspruit and Suikerbosrant rivers were contaminated with NoV in 95% (20/21) and 21% (5/24) of samples, respectively. NoV-positive samples comprised of 33% GI, 29% GII and 38% of both GI and GII strains. Based on partial capsid gene analysis (region C), 16 NoV genotypes (6 GI, 10 GII) were identified. The major genotypes detected were GI.4, GI.5 and GII.4. These rivers could be a potential source of NoV infection for communities using the water for domestic or recreational purposes.

Evaluation of electropositive filtration for recovering norovirus in water

The virus adsorption-elution (VIRADEL) technique has been widely used in the recovery of various enteric viruses in water, and an electropositive filter such as 1 MDS has been commonly applied. However, effective methods of monitoring waterborne norovirus (NoV) have not yet been well characterized and optimized. Hence, in this study, the VIRADEL technique was evaluated and optimized for effectively detecting NoV in water by two commonly used electropositive filters (1MDS and NanoCeram filter). Various elution and concentration methods were evaluated by using both murine norovirus (MNV) and human NoV. Among the tested elution buffers, the most effective was 1.5% beef extract plus 0.01% Tween 80 for both 1MDS (67.5%) and NanoCeram (85.7%) microfilters. The recovery rate of GII-4 human NoV was higher by organic flocculation (86.6%) than by polyethylene glycol (PEG) precipitations (11.6~73.6%). When both 1MDS and NanoCeram filters were tested to detect NoV in surface and groundwater, the sensitivity of NoV recovered by these filters appeared to depend on the types and conditions of environmental water. The results of this study will help to set a standard of detection method for NoV in water.

Comparison of Hollow‐Fiber Ultrafiltration to the USEPA VIRADEL Technique and USEPA Method 1623

Hollow-fiber ultrafiltration (UF) is a technique that is increasingly viewed as an effective alternative for simultaneously recovering diverse microbes (e.g., viruses, bacteria, parasites) from large volumes of drinking water. The USEPA has organism-specific methods, including Method 1623 for Cryptosporidium and Giardia and the virus adsorption-elution (VIRADEL) technique using 1MDS electropositive filters. In this study, we directly compare the performance of a previously published UF method to that of the USEPA Method 1623 (for recovering Cryptosporidium parvum and Giardia intestinalis) and the 1MDS VIRADEL method (for bacteriophages and echovirus) using 100-L dechlorinated tap water samples. The UF method produced significantly higher recoveries of C. parvum versus Method 1623 (83% mean recovery for UF versus 46% mean recovery for Method 1623), while recoveries for G. intestinalis were similar for both methods. Results of the virus method comparison showed the UF method (including secondary concentration using microconcentrators) to be very effective for the recovery of echovirus 1, bacteriophage MS2, and bacteriophage phi X174, with mean recovery efficiencies of 58, 100, and 77%, respectively. The VIRADEL technique (including secondary concentration by organic flocculation) recovered significantly less echovirus 1, and the bacteriophages could not be quantified by the method due to phage inactivation and/or assay inhibition. The results of this study demonstrate that the UF technique can be as effective, or more effective, than established USEPA methods for recovery of viruses and protozoan parasites from 100-L tap water samples.

McLeod phenotype without the McLeod syndrome

McLeod neuroacanthocytosis syndrome is a late-onset X-linked multisystem disorder affecting the peripheral and central nervous systems, red blood cells (RBCs), and internal organs. A variety of mutations have been found in the responsible gene (XK) including single nonsense and missense mutations, nucleotide mutations at or near the splice junctions of introns of XK, and different deletion mutations. To date no clear phenotype-genotype correlation is apparent. The clinical details of one case of McLeod phenotype without apparent neuromuscular abnormalities have been reported. Here the clinical details of two additional cases are presented, of which the genetic details have previously been published. Two asymptomatic or minimally symptomatic cases at ages expected to manifest the McLeod syndrome (MLS) were evaluated. The first case had been authenticated as a genuine McLeod both by serology and by genotyping (R222G missense mutation) and the second case had a mutation in XK (IVS2+5G>A) and by serology exhibited very weak Kx antigen and no detectable Kell antigens, except extremely low k antigen by adsorption-elution technique. The patients were examined for hematologic, neurologic, and other clinical abnormalities. Despite documented McLeod phenotype on RBCs, and identified mutations of XK, neurologic and other clinical findings were minimal at ages expected to manifest MLS. The different XK mutations may have different effects upon the XK gene product and thus may account for the variable phenotype.

Evaluation of 1MDS electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water

The 1MDS electropositive microfilter was designed specifically for virus capture and recovery from water, but its electrostatic properties raise the possibility that 1MDS filters can also effectively capture bacteria and parasites present in water samples. This filter is recommended by United States Environmental Protection Agency (USEPA) for recovering human enteric viruses from water matrices through the Virus Adsorption-Elution (VIRADEL) technique. If bacteria and parasites can also be concentrated and recovered using 1MDS filters, this sampling technique would have greater utility and cost-effectiveness for microbial water quality testing. In this study, both 142-mm flat and 25.4-cm cartridge 1MDS filters (Cuno) were tested to determine their effectiveness for recovery of MS2 and phi X174 bacteriophage, Salmonella enterica (serovar Typhimurium), Bacillus globigii endospores, and Cryptosporidium parvum oocysts from a tap water matrix. By amending the USEPA standard beef extract/glycine eluent with a surfactant (Tween 80) and dispersant (sodium polyphosphate) and varying the pH and temperature, multiple eluent conditions were compared in order to identify an optimum eluent for all organisms. While viruses, bacteria, and parasites are effectively retained by the 1MDS filter, elution efficiencies and associated recovery efficiencies varied for each organism.

Application of a molecular method for the detection of group A rotaviruses in raw and treated water

Group A human rotaviruses (HRVs) are the most important aetiological agents of acute viral gastroenteritis in infants and young children in both developing and industrialised countries. Rotaviruses are resistant to many chemical disinfectants and reportedly survive well in treated tapwater and sewage. In this study a group A specific reverse transcriptase-polymerase chain reaction (RT-PCR) followed by a nested-PCR was applied for the detection of HRVs in raw and treated drinking-water samples drawn at a water reclamation plant. For a period of two years (July 2000 to June 2002), borehole, raw and treated drinking-water samples were collected weekly. Viruses were recovered from the water samples using a glass wool adsorption-elution technique followed by secondary concentration using precipitation with polyethylene glycol. In the first year of the study group A HRVs were detected in 11% sewage samples, 8% partially treated waters and 5% final treated drinking waters. The results of the second year of the study showed the presence of group A HRVs in 11% sewage and untreated surface water samples, 15% partially treated water and 6.5% final treated drinking waters. No HRVs were detected in the water samples from the boreholes. The presence of group A HRVs in treated drinking-water samples suggested that this water could be a potential source of infection to consumers. The data also implied that either the water treatment did not remove HRVs or the treated water was contaminated post-treatment.

Recovery of feline calicivirus infectious particles and genome from water: comparison of two concentration techniques

The aim of this work was to determine the recovery rate of feline calicivirus (FCV-F9) infectious particles and genome from water after a concentration step using either adsorption elution on glass wool or filtration through an electropositive membrane. The results showed that the membrane filtration technique allowed a 75% recovery rate of FCV-F9 infectious particles while the yield was only 5.3% for FCV-F9 genome. Using the glass wool adsorption-elution technique, the recovery rate of FCV-F9 infectious particles was 0.5% while the yield was 102.5% with Poliovirus 1.

Isolation of a RecombinantBacillus sp. TS-23 α-Amylase by Adsorption-Elution on Raw Starch

A Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCI buffer (pH 8.5). The adsorption-elution procedure resulted in a yield of 53% α-amylase activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α-amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption-elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption-elution cycles. These results suggest that the raw starch adsorption-elution technique has a great potential in the isolation of Bacillus sp. TS-23 α-amylase from the culture broth of recombinant E. coli.