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PubMed2003

Evaluation of a rapid bacteriophage-based method for the detection of Mycobacterium tuberculosis in clinical samples.

Rapid, sensitive and low-cost methods are needed urgently for the detection of Mycobacterium tuberculosis in clinical samples, especially in developing countries. To this end, the clinical performance of FASTPlaqueTB(TM) (a bacteriophage-based method) has been studied in parallel with microscopy, standard microbiological culture and in-house IS6110-based PCR methods. A total of 64 samples, including 42 sputum samples and 22 urine samples, were tested in this study. The sensitivity, specificity and overall accuracy values for the FASTPlaqueTB assay relative to that of culture were respectively 76.5, 95 and 90 %. The corresponding values for the in-house IS6110-based PCR assay were 88, 91 and 90 % and, for Ziehl-Neelsen staining, were 59, 95 and 85 %. FASTPlaqueTB gave better clinical performance with urine samples than with sputum samples (sensitivity, specificity and overall accuracy were 100 % with urine samples and 64, 93 and 84 % with sputum samples). The 100 % sensitivity of FASTPlaqueTB was higher than that of the corresponding values for PCR (67 %) with urine samples. In conclusion, FASTPlaqueTB proved to be sensitive, cheap relative to the PCR and rapid. It is able to detect M. tuberculosis in clinical samples within 1 day, reducing the time to diagnosis in comparison with culture.

Bacteriuria↗
PubMed2004

Validation of a PCR for diagnosis of typhoid fever and salmonellosis by amplification of the hilA gene in clinical samples from Colombian patients.

Validation of a PCR test to detect hilA gene sequences of Salmonella spp. was performed in blood and faeces samples from typhoid fever and salmonellosis patients. Sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR in blood samples were performed by testing: 37 patients with clinical diagnosis of typhoid fever, 34 of them confirmed by isolation of S. Typhi from blood cultures; 35 patients infected with other pathogens corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4 and Enterobacter cloacae, 4) and blood samples from 150 healthy volunteers. To evaluate S, SP, PPV and NPV of the PCR in faeces samples we studied: 34 patients with enteritis due Salmonella spp. (S. Typhimurium, 21; S. Enteritidis, 9; S. Choleraesuis, 3 and S. Agona, 1); faeces samples from 35 patients with enteric infection due to Shigella sonnei (8), Shigella flexneri (10), enteropathogenic E. coli (12), Aeromonas hydrophila (5) and faeces samples from 150 healthy volunteers. The S, SP, PPV and NPV of the PCR in blood samples were all 100 %. PCR detected three patients with clinical diagnosis of typhoid fever and negative blood cultures. In faeces samples, S was 97 %, SP 100 %, PPV 100 % and NPV 99 %. The lowest number of c.f.u. ml(-1) detected by PCR in blood samples was 1 x 10(1) and in faeces samples 4 x 10(2).

Bacterial Proteins↗
PubMed2004

Current performance of patient sample collection in the UK.

Collection of the patient sample for pretransfusion testing begins a complex chain of events in the transfusion process. Hospitals in England and North Wales were surveyed to compare local policies against recommendations of the British Committee for Standards in Haematology (BCSH). Hospitals also measured the frequency of rejected and miscollected samples [designated as wrong blood in tube (WBIT)]. 185 of 360 (51.4%) hospitals returned questionnaires and 182 of 185 (98%) hospitals reported that a policy for sample collection existed. Apart from frequent omission of the gender of the patient, there was 96% compliance with all mandatory identifiers of the BCSH guidelines. Practice allowing additions or changes to labelling on sample tubes and request forms varied. 3.2% (14 114/445 726) of samples submitted were rejected for various reasons, the most frequent being incomplete or missing information (49.5% of the total rejected samples). The corrected mean frequency for WBIT in the 27 hospitals with one or more observed WBIT was 1 in 1501 samples (95% CI: < or =1129.09 to < or =1872.91), and the median corrected frequency for WBIT was 1 in 1303 samples. This study has identified great variation in the policy and practice for sample collection for pretransfusion testing. Regular tracking of the rates of sample rejection and WBIT could be used to identify poor performance in individual hospitals requiring investigation and action.

Blood Specimen Collection↗
PubMed2005

Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples.

AIMS: To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS: The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS: The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).

Base Sequence↗
PubMed1995

Achieving equal probability of selection under various random sampling strategies.

The underlying objective of epidemiological investigations is to extrapolate results from a sample to the relevant population. The simplest way of achieving this is to adopt a sampling strategy in which each individual in the population has the same chance of being selected--that is, to employ an 'equal probability of selection method' (epsem). The easiest ways of achieving this are to use simple random sampling or stratified random sampling with a constant sampling fraction. These strategies are often impracticable, however, particularly in large investigations covering a wide geographical area where resource implications dictate a more complex approach such as multi-stage or cluster sampling. Following detailed definitions and appropriate illustrations of these terms, the main purpose of this paper is to provide a working guide of how to achieve epsem using these various random sampling techniques. In brief, for multi-stage sampling with the rare feature of equal-sized first stage units, epsem is achieved by applying the above simple or stratified approaches to the first stage units. Even in the more realistic scenario of unequal first stage units, the same options apply provided that a fixed proportion of second stage units are to be selected (cluster sampling is in fact just one example of this, with 100% sampling of second stage units). If on the other hand a fixed number of second stage units are to be selected then for epsem the first stage units should be selected with each one having a probability proportionate to its size.

Data Collection↗
PubMed1992

The cytological detection of persistent cervical intraepithelial neoplasia after local ablative treatment: a comparison of sampling devices.

OBJECTIVE: To determine whether the cytological detection of persistent cervical intraepithelial neoplasia (CIN) after local ablative treatment is improved by the use of sampling devices other than the Ayre's spatula. DESIGN: A randomized controlled study. SETTING: Lothian Area Colposcopy Clinic. SUBJECTS: 856 patients who had received local therapy (CO2 laser or cold coagulation) for CIN II or III between 9 and 30 months earlier. INTERVENTION: Each patient had three consecutive cervical smears taken, one with the Ayre's spatula, one with either the Aylesbury, the Rocket or the Multispatula device, and finally one with the Cytobrush. The allocation of which spatula and the order of the first two was randomized. Each patient had a colposcopic examination immediately after the smears were taken. MAIN OUTCOME MEASURES: A comparison of the detection of histologically proven persistent CIN by the Ayre's spatula with the detection of persistent disease by alternative sampling devices. RESULTS: Of the 856 patients 130 had histologically proven persistent CIN. Another 98 had suspicious findings on colposcopy but punch biopsies reported as histologically normal. Of the remaining patients with normal colposcopy 130 were randomly selected to form a control group. The cervical smears from these 358 women were reported. Significantly fewer Ayre's samples contained endocervical cells than Aylesbury samples (47% vs 59%, difference 12%; 95% CI 3%-21%; P less than 0.001), Rocket samples (47% vs 67%; difference 20%, 95% CI; 12%-32%; P less than 0.001) or Multispatula samples (47% vs 76%; difference 29%, 95% CI 19-38%; P less than 0.001). When punch biopsies contained CIN, dyskaryotic cells were seen in 10% of Ayre's samples, 4.3% of Aylesbury samples, 8.3% of Rocket samples, and in no smear taken with the Multispatula. Obtaining a third smear with the Cytobrush did not substantially improve the detection rate of dyskaryosis. Neither the order of use of the spatulas, the form of initial treatment nor the size of the transformation zone had any apparent effect on the cytological detection of persistent CIN. CONCLUSIONS: We recommend that surveillance of patients who have received local ablative therapy for CIN should be by both cytology and colposcopy, and that cytological samples should be obtained using the Ayre's spatula.

Carcinoma in Situ↗
PubMed1987

Drug use among a sample of males admitted to an alcohol detoxication center.

Urine samples were obtained from 93% of a sample of 111 consecutive male admissions to a nonmedical detoxication center in Toronto. Analysis of these samples revealed that 51 (50%) had traces of drugs other than alcohol and that 12 (12%) were alcohol free. Benzodiazepines were the most frequently detected drugs and these were found in 32% of all samples. Barbiturates were detected in seven (7%) samples and cannabinoids in 10 (10%) samples. A wide range of urine alcohol concentrations was found and some samples had zero or low concentrations. Although alcohol urine concentrations were generally lower in samples containing other drugs, the distribution of urine alcohol concentrations was similar for samples containing only alcohol and those containing alcohol and some other drug. Of the 51 samples found to contain drugs other than alcohol, subjects' self-reports were concordant in 27 cases (53%). Most of the discrepancies between self-reports and urine analysis were due to the under-reporting of the use of benzodiazepines. Subjects with drugs in their urine tended to be younger than others, but they were not distinguished with respect to their behaviors while in the detoxication center or length of stay. Those with the highest urine alcohol concentrations had shorter stays in the detoxication center. Implications for further studies are discussed.

Adult↗
PubMed1991

Influence of time of sampling onset on parameters used for activation time determination in computerized intraoperative mapping.

The purpose of this work is to determine the sensitivity of the estimated time of peaks and maximum slopes, commonly used in activation time computations, to the instant at which sampling is initiated. Based on complex and quickly changing waveforms, 471 monopolar (MP) and bipolar (BP) epicardial responses in man were selected. These were decimated from 10 kHz to simulate sampling at frequencies ranging from 200 Hz to 2,000 Hz. The peak and maximum absolute slope for BP and the minimum slope for MP were computed repeatedly starting at successive 100 microseconds intervals extending throughout the sampling period and compared with these parameters computed from the waveform sampled at 10 kHz. Slopes were estimated using each of four different algorithms. The average greatest shift (AGS) due to variations in sampling onset ranged from 11.2 +/- 3.5 (200 Hz) to 0.3 +/- 0.2 msec (2,000 Hz). For bipolar algorithms, the peak performed better than the slope algorithms (AGS: 5.9 +/- 3.3 to 0.3 +/- 1.0 msec). For MP algorithms, 2 point linear, and 3 and 5 point Lagrange slope estimates performed similarly (AGS: 5.6 +/- 3.3 to 0.3 +/- 0.2 msec); a 5 point least square fit algorithm performed poorly. Sampling MP and BP electrograms below 500 and 400, respectively, often caused maximum shifts greater than 4 msec. Thus, the resolution of the peak and estimated slope is not limited to the sampling period, variations in initiation of sampling can cause significant outliers especially at low sampling rates, and MP electrograms should be sampled faster than BP electrograms for comparable accuracy.

Electrocardiography↗
PubMed2007

Analysis of forensic soil samples via high-performance liquid chromatography and ion chromatography.

Traditional forensic soil comparisons are performed via physical and/or chemical examinations of color, texture, and mineral content, leaving any organic- or water-soluble fractions unexamined. This study uses high-performance liquid chromatography (HPLC) and ion chromatography (IC) to assess the qualitative and quantitative variation in these fractions of soil. Soil samples (n=120) were collected over the course of 3 weeks from urban, suburban, and rural locations in and around Lansing, MI. Additional samples from six of these locations (two urban, two suburban, and two rural) were collected once a week for 10 weeks for temporal analysis. Nine additional samples, equally spaced over a 1 m(2) grid, from these same six locations were collected for spatial analyses. Qualitative and quantitative analysis of the resultant chromatograms separated the 120 samples into 10 groups by HPLC and 23 groups by IC. This study shows that using HPLC and IC to analyze the organic- and water-soluble fractions of soil can successfully discriminate samples. Quantitative analysis of the results eliminates some false inclusions by providing further differentiation of samples. The results of this study indicate that adding HPLC and IC analyses to traditional forensic soil analysis schemes can improve overall sample differentiation. The methods used in this study were also able to detect both qualitative and quantitative variations in soil over a relatively small geographic area. This demonstration of soil heterogeneity underscores the importance of the collection of a representative known sample population when assessing a forensic soil comparison. Significant temporal variation was also demonstrated over the course of 10 weeks of sampling; however, samples were found to be consistent over shorter periods of time. Baseline levels of inorganic anions were determined via IC; these levels may be useful in assessing the significance of anions detected in soil from cases involving low explosives.

Anions↗
PubMed1983

Antibiotic susceptibility testing of subgingival plaque samples.

The in vitro inhibitory effect of several antimicrobial agents was determined against dispensed dental plaque samples taken from periodontally diseased sites as an aid in the selection of antibiotics for adjunctive use in periodontal therapy. 2 groups of patients were sampled. 1 group of 10 patients with severely advanced disease had received periodontal treatment which included the frequent adjunctive use of an antibiotic. The second group consisted of 15 individuals with less severe periodontal disease; only 4 individuals had been previously treated with antibiotics for their periodontal disease. Bacterial samples of subgingival plaque were taken from each patient and tested against a battery of antibiotics to determine which agent was the most effective in suppressing bacterial growth. Each antibiotic was incorporated into Trypticase-soy blood agar at a concentration equivalent to that achieved in either gingival fluid or blood following recommended oral dosages. The inhibitory effect was determined by comparing the number of bacterial recovered on the antibiotic-containing medium to the total number of bacteria recovered on the basal medium. Penicillins, with the exception of cloxacillin, were the most effective in inhibiting bacterial growth. Benzylpenicillin consistently inhibited the growth of 90% of the isolates recovered on media free of antibiotics while ampicillin and amoxicillin frequently inhibited 99% or more of the bacteria recovered. Tetracycline was generally inhibitory for at least 90% of the isolates if the patients had not been previously treated with this agent. However, resistance to this drug was common in samples taken from patients previously treated with tetracycline. Doxycycline, a tetracycline derivative, did not inhibit significantly more isolates than tetracycline. Clindamycin was inhibitory for 90% or more of the organisms in most of the samples; and, was usually effective in inhibiting isolates in samples which exhibited large numbers of isolates resistant to tetracycline. Erythromycin was relatively ineffective against the isolates recovered from samples from the severely diseased group but was inhibitory to isolates in some samples taken from the more moderately diseased group. Metronidazole, at the concentration tested, was largely ineffective against the isolates in bacterial samples from both groups. No single antimicrobial agent was found to be inhibitory for greater than 90% of the bacteria recovered from all of the subgingival plaque samples with the possible exception of some penicillins.

Adolescent↗
PubMed1985

Sampling of subgingival microbiota for dark-field microscopy.

The aim of the present investigation was to study the validity of 2 different methods of sampling the subgingival microbiota. The composition of the flora was estimated using the dark-field microscope. Subgingival samples were obtained using either a curette or a Hamilton microsyringe for a subgingival washing. The method of determination of the composition of the subgingival microorganisms was found to be very reproducible when the same investigator performed the counting. Samples obtained by the washing technique had a slight but significantly higher proportion of coccoid cells when compared to samples obtained by curette. The calculation of the reproducibility of the washing technique as assessed in 6 immediately repeated samples from the same sites yielded a coefficient of variation of 40% for spirochetes and motile rods. When 9 repeated samples from each of 28 sites (14 with a curette, 14 with washing) were studied over a period of 32 days, no systemic alterations were observed in the % of spirochetes and motile rods. The variation within each individual site was high. For curette samples and washing samples, the coefficients of variation for spirochetes and motile rods were 85% and 63%, respectively. The present investigation clearly demonstrated that care should be taken in the interpretation of single samples of subgingival microbiota. A more relevant picture of the actual situation is achieved when trends of repeated samples over time are studied.

Bacteria↗
PubMed2006

Is the fluoride/creatinine ratio of a spot urine sample indicative of 24-h urinary fluoride?

OBJECTIVE: The main aim of this study was to determine how representative the fluoride/creatinine (F/Cr) ratio of a spot urinary sample is of the fluoride content of a 24-h urine sample in young children aged 12-36 months. Subsidiary objectives were to: (a) evaluate the use of 24-h urine samples in monitoring fluoride exposure and (b) estimate the retention of fluoride in very young children. METHODS: Seven healthy children residing in a fluoridated area completed the study. Dietary information was obtained using a 3-day estimated food diary followed by an interview on the fourth day. Samples of foods and drinks were analysed for fluoride content. Ingestion of fluoride from toothpaste was also measured. A 24-h urine sample and a morning spot urine sample were collected from each child. RESULTS: The mean age of the children was 32 months (range: 16-36 months). The mean total daily intake of fluoride was 0.71 (+/-0.41) mg or 0.05 (+/-0.02) mg/kg bodyweight, of which 37% came from the diet, the remainder being from toothpaste ingestion. None of the children used any other sources of fluoride such as fluoride tablets or fluoridated salt. The mean F/Cr ratio was 1.49 (+/-0.63) mg F/g creatinine. A positive correlation (Pearson's correlation=0.76, P=0.05) between fluoride excretion estimated using the F/Cr ratio in a morning spot urine sample and fluoride excretion in a 24-h urine sample was found. There was also a positive correlation (Pearson's correlation=0.83, P=0.02) between total daily fluoride intake and 24-h urinary fluoride excretion. Less than half (43%) of the total daily fluoride intake was retained in the body. CONCLUSIONS: The F/Cr ratio of a morning spot urine sample may be used to estimate mean 24-h urinary excretion of fluoride and therefore has potential application for larger-scale epidemiological studies where 24-h samples are impractical. Estimates of 24-h urinary fluoride excretion can be used to gauge fluoride exposure.

Beverages↗
PubMed2000

Feature selection and classifier performance in computer-aided diagnosis: the effect of finite sample size.

In computer-aided diagnosis (CAD), a frequently used approach for distinguishing normal and abnormal cases is first to extract potentially useful features for the classification task. Effective features are then selected from this entire pool of available features. Finally, a classifier is designed using the selected features. In this study, we investigated the effect of finite sample size on classification accuracy when classifier design involves stepwise feature selection in linear discriminant analysis, which is the most commonly used feature selection algorithm for linear classifiers. The feature selection and the classifier coefficient estimation steps were considered to be cascading stages in the classifier design process. We compared the performance of the classifier when feature selection was performed on the design samples alone and on the entire set of available samples, which consisted of design and test samples. The area Az under the receiver operating characteristic curve was used as our performance measure. After linear classifier coefficient estimation using the design samples, we studied the hold-out and resubstitution performance estimates. The two classes were assumed to have multidimensional Gaussian distributions, with a large number of features available for feature selection. We investigated the dependence of feature selection performance on the covariance matrices and means for the two classes, and examined the effects of sample size, number of available features, and parameters of stepwise feature selection on classifier bias. Our results indicated that the resubstitution estimate was always optimistically biased, except in cases where the parameters of stepwise feature selection were chosen such that too few features were selected by the stepwise procedure. When feature selection was performed using only the design samples, the hold-out estimate was always pessimistically biased. When feature selection was performed using the entire finite sample space, the hold-out estimates could be pessimistically or optimistically biased, depending on the number of features available for selection, the number of available samples, and their statistical distribution. For our simulation conditions, these estimates were always pessimistically (conservatively) biased if the ratio of the total number of available samples per class to the number of available features was greater than five.

Algorithms↗
PubMed2004

Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment.

A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.

Animal Feed↗
PubMed2007

Use of capillary blood samples as a new approach for diagnosis of Dengue virus infection.

We evaluated the use of capillary blood samples stored on filter papers for diagnosis of dengue virus infection. Venous and capillary blood samples were collected from 130 patients suspected of having dengue fever. We compared the performances of standard reference methods using capillary blood samples absorbed onto filter papers versus venous blood samples. The resulting sensitivity, specificity, and positive predictive value of tests performed on filter paper compared to those performed on venous blood samples were 81.6% (62/76; 95% confidence interval [CI], 74.9% to 88.3%), 90.7% (49/54; 95% CI, 85.7% to 95.7%), and 92.5% (62/67; 95% CI, 86.2% to 98.8%), respectively. During the acute phase of dengue virus infection (day 1 to day 4), the tests performed on capillary blood samples had a sensitivity of 88.5% (95% CI, 82.0% to 95.0%) and a specificity of 93.8% (95% CI, 88.9% to 98.7%). During the convalescent phase of infection, this method allowed the viral serotype to be determined for 4 of 15 (27%) dengue virus-infected patients for whom virological diagnosis using venous samples was negative. Capillary blood samples could therefore be a good alternative for the diagnosis of dengue virus infection in tropical areas. Indeed, these samples are convenient for storage and transport without the need for a cold chain and simplify the collection of samples from children. Moreover, our results suggest that viral particles persist longer in capillary blood than in peripheral blood. Analysis of the viability of viral particles under these conditions may give new insights into the physiopathology of dengue virus infection and the transmission of dengue virus during outbreaks.

Aedes↗
PubMed1989

Evaluation of a rapid method of extracting DNA from stool samples for use in hybridization assays.

The ability of the Extractor system (Molecular Biosystems, Inc., San Diego, Calif.) to isolate nucleic acid (NA) from stool samples for use in hybridization assays was investigated. Crude NA was recovered from 45 of 50 stool samples by using this system. The amount of NA recovered varied considerably depending on the microbial flora present in the sample (mean +/- standard deviation, 50.2 +/- 46.7 micrograms; range, 2 to 228 micrograms) but did not correlate with the consistency of the sample. Samples containing primarily gram-positive organisms or yeast cells gave lower yields of NA (less than 10 micrograms) than those containing gram-negative bacilli. The five samples which did not yield NA were sterile when cultured aerobically on blood agar plates. Samples of the 45 stools yielding NA were inoculated into broth and grown overnight, and a 10-microliters sample of broth was spotted onto nitrocellulose filters. The NA samples recovered from the Extractor column were applied to nylon membranes by using the Centri-dot system. The NA on the broth blots and the NA on the Centri-dot filters were hybridized with a 310-base-pair probe specific for the 2"-O-aminoglycoside adenylyltransferase [ANT(2")] resistance gene. The Extractor-Centri-dot system demonstrated 61.9% sensitivity and 95.8% specificity in detecting the ANT(2") gene in stool samples containing colonies demonstrating the ANT(2") phenotype. The positive and negative predictive values of the NA blot were 92.8 and 74.2%, respectively.

DNA Probes↗
PubMed1992

Reactivity patterns and infection status of serum samples with indeterminate Western immunoblot tests for antibody to human immunodeficiency virus type 1.

Serum samples with indeterminate Western blot (WB) tests from 61 individuals whose sera were positive by enzyme-linked immunosorbent assay (ELISA) were studied in order to characterize their putative reactions with the human immunodeficiency virus (HIV) proteins and to resolve the HIV infection status of these individuals. The reaction observed by WB could not be confirmed either by radioimmunoprecipitation assay and subsequent electrophoresis (RIPA) or by use of LiaTek (Organon Teknika, Turnbout, The Netherlands) in 28% of the samples. Of the 86 samples that were indeterminate by WB, 66 reacted with p24 by WB; this reaction was confirmed by RIPA in only 21 (32%) and by LiaTek in 49 (74%) of the 66 samples. On the other hand, none of the indeterminate samples that reacted with HIV envelope proteins by WB did so by LiaTek, while 50% precipitated at least some of these proteins in the RIPA. The sensitivities of the three methods for detecting the antibody reaction with the different HIV proteins, which were studied with serial dilutions of positive serum samples, were similar. Thus, a lower sensitivity of RIPA or LiaTek does not seem to be the cause for the lack of reaction of the WB-indeterminate samples by these two methods. Sequential samples from individuals whose serum samples reacted by the three methods gave reproducible results, but all showed low antibody titers. Peripheral blood mononuclear cells obtained from three of the four individuals with sequential samples that reacted with HIV env proteins by WB and RIPA were negative for HIV provirus DNA after amplification by the polymerase chain reaction.

Blotting, Western↗
PubMed1997

Detection of group B rotaviruses in fecal samples from diarrheic calves and adult cows and characterization of their VP7 genes.

Groups A, B, and C rotaviruses have been identified in cattle. Group B rotaviruses are associated with sporadic cases of diarrhea in calves and adult cows. From diagnostic submissions to our laboratory, 90 fecal samples from cases of calf diarrhea, 81 fecal samples from cases of adult cow diarrhea (winter dysentery), and 20 fecal samples from case control normal adult cows were tested for group B rotaviruses by polyacrylamide gel electrophoresis (PAGE), and reverse transcription (RT)-PCR (targeting 279 bp of the VP7 gene). In addition, 53 fecal samples from diarrheic adult cows were tested for group B rotaviruses by immune electron microscopy (IEM). By RT-PCR, five samples from calves were group B rotavirus positive (5.6%). Fifteen samples from adult cows with diarrhea were group B rotavirus positive (18.5%), and none of the control fecal samples from normal cows were positive for group B rotaviruses. By PAGE, one calf sample (RT-PCR positive) was group B rotavirus positive (short electropherotype), but none of the adult cow samples were positive for group B rotaviruses. By IEM, 5 (9.4%) of the 53 fecal samples from diarrheic adult cows were group B positive (all were also RT-PCR positive). The VP7 genes of three strains (WD653 from an adult cow and the ATI and Mebus calf strains) were sequenced. The VP7 genes from the three bovine strains showed high (over 90%) nucleotide and deduced amino acid homologies, but lower homologies (48 to 61%) were seen between these genes and the genes from rodent (IDIR) and human (ADRV) group B rotaviruses. Although there were some differences of degree, all inoculated gnotobiotic calves (n = 6) showed abnormal feces between 1 and 3 days after inoculation with each of three strains of group B bovine rotaviruses, and group B rotaviruse, were detected in the feces for up to 2 weeks by RT-PCR but for shorter periods by PAGE or IEM.

Animals↗