Detection of Group A Streptococci in Throat Swabs by Direct Fluorometry

A direct fluorometric test for the rapid detection of group A streptococci from throat swabs was compared with the microscopic fluorescent-antibody test. Formalinized throat swab cultures (490) were examined by the two methods, and the results agreed on 84% of the specimens. In another comparison, 15-hr broth cultures of 103 freshly taken throat swabs were tested by both methods. Of the specimens tested, 101 (98%) were either positive or negative by both methods. In all cases, the latter results correlated with the demonstration of presence or absence of group A streptococci in the specimens by cultural isolation and precipitin grouping tests. It may be feasible to use the direct fluorometric test in a diagnostic laboratory as described or possibly to adapt it for automatic processing of throat swab cultures.     

A comparison of two microbial detection methods used in aseptic processing of musculoskeletal allograft tissues

Tissues from 78 musculoskeletal donors were concurrently tested for microorganisms using both a swab and liquid culture method. An aggregate total of 20 organisms were detected by both methods. The swab detected 4/20 organisms while the liquid culture detected 18/20 organisms. The swab method yielded sensitivity and negative predictive values of 20 and 92.3%, respectively. Comparatively, the liquid culture displayed a sensitivity of 90% and a negative predictive value of 99%. These results clearly demonstrate that the liquid culture method is superior to swab cultures in microbial detection. Additional studies are necessary to determine the optimal culture conditions for different types of tissues when utilizing the liquid culture method.     

Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.     

Accelerated Immunofluorescence Procedure for the Detection of Salmonella in Foods and Animal By-Products

An accelerated, direct immunofluorescent-antibody procedure was developed for the detection of Salmonella in food products. This method includes pre-enrichment and selective enrichment but eliminates many of the washing and smear treatments present in existing methods. Commercially available fluorescein-conjugated somatic antiserum was used in comparing this method with conventional culture, biochemical, and serological procedures. The 894 samples tested represented 39 different products. The fluorescent-antibody procedure detected Salmonella in 216 test samples as compared to 205 positives recovered by using the standard culture procedures. In no instance did the fluorescent-antibody procedure fail to detect a Salmonella positive which had been detected by the standard procedure. With a three-tube, most-probable-number procedure, the fluorescent-antibody method was able to detect Salmonella at a level of 0.036 organism per g. In addition to being a more rapid method for the detection of Salmonella, it has proven to be comparable to conventional culture procedures.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

One-Day Fluorescent-Antibody Procedure for Detecting Salmonellae in Frozen and Dried Foods

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater.

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

Three-Day Assay for Human Cytomegalovirus Applicable to Serum Neutralization Tests

A fluorescing cell assay (FCA) technique utilizing the indirect fluorescent-antibody method to measure human cytomegalovirus (CMV)-infected cells has been applied to the rapid determination of CMV-neutralizing antibody. Human sera with complement fixation titers to CMV of 1/32 or greater and fluorescein-conjugated rabbit anti-human globulin are the primary and secondary reagents in the fluorescent-antibody test. FCA measured in 3 days the same number of infectious units measured by plaque assay in 2 weeks. FCA and plaque assay yielded identical neutralizing antibody titers to CMV in 20 human sera.     

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent-Antibody Technique in Detection of Salmonellae in Animal Feed and Feed Ingredients

Comparative studies of fluorescent antibody procedure and a cultural method for the detection of Salmonella were made on 1,013 feed and feed-ingredient samples. The agreement between the two methods was 92.1%. There were more false positives (5.7%) than false negatives (2.2%). Of the 22 false negatives, 15 (68%) were obtained on meat meal. Of the total number of samples, 37% were meat meal. An additional study of 73 samples of meat meal indicated that correlation between methods was better than correlation between samples.     

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P < 0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P < 0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P < 0.0001). The DGS produced significantly more (P = 0.003) intact cells than CB and LVF. Distorted cells were significantly (P = 0.001) more frequent in smears by LVF. The CB harvested significantly (P = 0.009) more fragmented cells. CB and LVF produced significantly (P < 0.0001; P = 0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P = 0.0062; P = 0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P = 0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k ≤ 0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 × 10⁰ cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

A comparison of bacterial flora isolated by transtracheal aspiration and pharyngeal swabs in Macaca fascicularis.

The pulmonary flora of 30 monkeys (Macaca fascicularis) was sampled by the transtracheal aspiration technique and the pharyngeal swab method, and the results were compared. The transtracheal aspiration technique yielded lower numbers of bacteria in both aerobic and anaerobic cultures. The bacteria isolated by transtracheal aspiration were predominately pure culture, thereby lowering the possibility of contamination from commensal flora. Bordetella bronchiseptica was isolated from 23.3% of the monkeys by transtracheal aspiration, but this organism was not isolated when samples were collected with pharyngeal swabs.     

Efficacy of cotton-tipped applicators for obtaining cells from the uterine cervix for Papanicolaou smears

A study was made of the efficacy of two techniques for the preparation of Papanicolaou smears. In one technique ("swab-spatula technique"), a scraping of the endocervix with a saline-moistened cotton-tipped applicator was combined with a scraping of the ectocervix with a plastic spatula. In the second technique ("swab technique"), the endocervix and ectocervix were scraped with only a saline-moistened cotton-tipped applicator. The swab technique produced more inadequate smears with a scanty cellular yield (24/408 = 6%) than did the swab-spatula technique (9/361 = 3%). The swab technique also produced higher false-negative rates (60% for CIN I, 42% for CIN II, 16% for CIN III, 20% for invasive cancer, and 32% overall) as compared with those of the swab-spatula technique (27% for CIN I, 29% for CIN II, 14% for CIN III, 0% for invasive cancer and 18% overall). Because of the higher rates of inadequate samples and false negativity in smears prepared by cotton-tipped applicators alone, it is recommended that the spatula not be omitted in taking cervical samples for the preparation of Papanicolaou smears for the detection of dysplasia or cancer. This study also reflects a poor performance of a cervical cancer detection system in the setting of a major medical school and suggests the need for instruction and periodic evaluation of the performance of the staff taking the smears. This study also substantiates doubt on the value of a second recent smear as a follow-up procedure for cervical dysplasia.     

Response and Specificity of Antibodies for Candida albicans

Rabbit antibodies for Candida albicans, reacting in agglutination and fluorescent-antibody reactions, were present in both IgM and IgG protein fractions. The two types of immune globulins were separated from ammonium sulfate-precipitated gamma-globulin either by filtration through a column of Sephadex G-200 or by diethylaminoethyl column chromatography performed by stepwise elution with various concentrations of sodium chloride. In the fluorescent-antibody test, initial separation of the IgG fraction, prior to its conjugation with dye, proved to be essential for the high specificity of this reaction. Investigation of the specificities of the two types of antibodies revealed that the IgG was highly specific, whereas the IgM was not very specific. Each antigen fraction, extracted by various methods, demonstrated its own characteristic antibody response. Only the IgG fraction yielded serotype-specific antibody useful for detection of a serotype of C. albicans in agglutination and fluorescent-antibody tests. The results indicate the importance of IgG for specific serological reactions with the Candida species.     

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv -locus in all Yersinia spp. and the presence of the ail -gene in pathogenic Y. enterocolitica only. Hybridization results with ail -probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI⁺ strains and only two TCI^- strains were positive by hybridization with ail. Hybridization results with inv - or ail -probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv - or ail -probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv - and/or ail -probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv - and/or ail - probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.