bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of human immunodeficiency virus type 1 (HIV-1) antibody by western blotting and HIV-1 DNA by PCR in patients with AIDS
Authors: Jackson J B , Parsons J S , Nichols L S , Knoble N , Kennedy S , Piwowar E M
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) Western blotting (immunoblotting) band patterns and the sensitivity of an HIV-1 DNA PCR assay were determined by testing the blood of patients with AIDS. Plasma and cell pellets processed from the peripheral blood of 199 patients with absolute CD4 cell counts of less than 200 cells per mm3 were tested by a licensed enzyme immunoassay (EIA; Abbott HIV-1) and Western blot assay (Cambridge-Biotech) for HIV-1 antibody. The Roache HIV-1 AMPLICOR DNA PCR assay was used to test cell pellets from 125 of the 199 patients for HIV-1 gag DNA sequences. All plasma samples from these 199 sequential patients were reactive for HIV-1 antibody by EIA and were positive by Western blot assay using the criteria recommended by the Centers for Disease Control and Prevention. The majority of samples (192 of 199; 96.5%) displayed at least six of nine bands characteristic of the virus by Western blotting, with the lowest number of bands characteristic of the virus displayed by any sample being three. However, 39 and 48% of all patients exhibited no bands to p17 and p55 antigens, respectively, whereas 0 to 7.5% of all patients exhibited no bands to the other antigens. HIV-1 gag DNA sequences were detected in 117 (93.6%) of 125 cell pellets processed from the peripheral blood of these same patients. All eight patients initially negative by PCR tested positive when a second pellet which had been produced from the same blood sample was tested. Despite a decrease in antibody reactivity to HIV Gag and Pol proteins, patients with advanced HIV-1 infection remained positive for HIV-1 antibody by EIA and Western blot testing. Confirmation by the HIV-1 Western blot assay still appears to be the more sensitive assay for the diagnosis of HIV-1 infection in those individuals with advanced HIV-1 infection in the United States.
Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis
Authors: Vera-Cabrera L , Howard S T , Laszlo A , Johnson W M
Published: January 2020

mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans.
Food-borne disease outbreaks due to bacteria in Taiwan, 1986 to 1995
Authors: Pan T M , Wang T K , Lee C L , Chien S W , Horng C B
Published: January 2020

Between 1986 and 1995, 852 outbreaks of food-borne disease involving 26,173 cases and 20 deaths were reported in Taiwan. About 80% of the outbreaks occurred in the warmer months, i.e., between April and October. Of the 852 reported outbreaks, 555 (65%) were caused by bacterial pathogens. The three most common bacteria involved were Vibrio parahaemolyticus (35%, 197 of 555 outbreaks), Staphylococcus aureus (30%, 169 of 555 outbreaks), and Bacillus cereus (18%, 104 of 555 outbreaks).
Assessment of swab transport systems for aerobic and anaerobic organism recovery
Authors: Perry J L
Published: January 2020

Saline suspensions of 11 aerobes and anaerobes were used to inoculate swabs from Port-A-Cul (Becton Dickinson), Culturette EZ (Becton Dickinson), and Copan Amies gel (Copan Diagnostics). Swabs were removed from transport devices at 0, 24, and 48 h postinoculation and then extracted by vortexing in 1.0 ml of saline, and organism survival was determined by quantitative plate counts. For the organisms tested, Culturette EZ allowed < 1% of the original inocula to be recovered after 24 h. Port-A-Cul was only slightly better. Recovery was best with the Copan gel-containing system. Agar gel swab systems may be useful for multipurpose transport devices.
Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis
Authors: Garza-González E , Guerrero-Olazarán M , Tijerina-Menchaca R , Viader-Salvadó J M
Published: January 2020

In the present work a rapid method to determine the susceptibility of Mycobacterium tuberculosis to isoniazid and streptomycin by determining levels of mycolic acids by high-performance liquid chromatography (HPLC) was developed. Mycobacterial growth kinetics in the presence and absence of antituberculosis drugs was characterized by evaluating the total area corresponding to mycolic acid peaks (TAMA). Results show a linear relationship between the logarithm of CFU per milliliter and TAMA and show that it is possible to detect growth inhibition of M. tuberculosis in the presence of isoniazid or streptomycin by using HPLC in 3 and 4 days, respectively.
Fatty acid characterization of rapidly growing pathogenic aerobic actinomycetes as a means of identification
Authors: McNabb A , Shuttleworth R , Behme R , Colby W D
Published: January 2020

The fatty acid compositions of 39 type strains and 529 clinical or reference strains of pathogenic aerobic actinomycetes were analyzed after standardized culture by using the Microbial Identification System (MIS). Library entries for each type strain were created by using the MIS Library Generation Software, and the fatty acid profiles of clinical and reference strains were compared to these library entries. The bacteria separated into two large groups based upon major amounts of branched-chain or of saturated or monounsaturated straight-chain fatty acids. Identification of isolates was possible by using only the type strains for comparison, but fatty acid heterogeneity occurred within most species.
Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test
Authors: Mouton J W , Verkooyen R , van der Meijden W I , van Rijsoort-Vos T H , Goessens W H , Kluytmans J A , Deelen S D , Luijendijk A , Verbrugh H A
Published: January 2020

The amplified Chlamydia trachomatis test (AMP-CT; Gen-Probe), a new diagnostic test for the detection of Chlamydia trachomatis, was evaluated with urine specimens from 1,000 patients visiting the outpatient department for sexually transmitted diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands, by comparing the results to those of cell culture. From February 1996 to July 1996, urine samples for the AMP-CT test and urethral swabs for cell culture were collected from 544 men, while cervical swabs from 456 women were also taken for cell culture. Positive test results were obtained for 130 (13%) of the patients. AMP-CT test and cell culture results were discordant for 70 (7%) specimens. Analysis of the samples with discordant results was performed by an in-house PCR. After resolution of the discordant results, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, respectively, for samples from females and 100, 99.2, 93.1, and 100%, respectively, for samples from males, while for cell culture these values were 72.5, 99.2, 92.5, and 98%, respectively, for samples from females and 57.4, 99.0, 86.1, and 95.4%, respectively, for samples from males. We conclude that the AMP-CT test is a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males.
Survival of Acinetobacter baumannii on dry surfaces
Authors: Wendt C , Dietze B , Dietz E , Rüden H
Published: January 2020

Acinetobacter spp. have frequently been reported to be the causative agents of hospital outbreaks. The circumstances of some outbreaks demonstrated the long survival of Acinetobacter in a dry, inanimate environment. In laboratory experiments, we compared the abilities of five Acinetobacter baumannii strains, three Acinetobacter sp. strains from the American Type Culture Collection (ATCC), one Escherichia coli ATCC strain, and one Enterococcus faecium ATCC strain to survive under dry conditions. Bacterial solutions of the 10 strains were inoculated onto four different material samples (ceramic, polyvinyl chloride, rubber, and stainless steel) and stored under defined conditions. We investigated the bacterial counts of the material samples immediately after inoculation, after drying, and after 4 h, 1 day, and 1, 2, 4, 8, and 16 weeks of storage. A statistical model was used to distribute the 40 resulting curves among four types of survival curves. The type of survival curve was significantly associated with the bacterial strain but not with the material. The ability of the A. baumannii strains to survive under dry conditions varied greatly and correlated well with the source of the strain. Strains isolated from dry sources survived better than those isolated from wet sources. An outbreak strain that had caused hospital-acquired respiratory tract infections survived better than the strains from wet sources, but not as well as strains from dry sources. Resistance to dry conditions may promote the transmissibility of a strain, but it is not sufficient to make a strain an epidemic one. However, in the case of an outbreak, sources of Acinetobacter must be expected in the dry environment.
Rapid detection of oxacillin-resistant Staphylococcus aureus in blood cultures by an impedance method
Authors: Wu J J , Huang A H , Dai J H , Chang T C
Published: January 2020

The feasibility of using an impedance method for direct detection of oxacillin-resistant Staphylococcus aureus (ORSA) in blood cultures was evaluated. An aliquot (0.1 ml) of the positive blood culture, which showed growth of gram-positive cocci and demonstrated thermonuclease activity, was inoculated into the module well of a Bactometer incubator (bioMerieux Vitek, Hazelwood, Mo.) containing 0.6 ml of Mueller-Hinton agar supplemented with oxacillin (2 microg/ml). The modules were incubated at 37 degrees C, and the change in impedance in each well was continuously monitored by the instrument at 6-min intervals for 24 h. ORSA strains from blood cultures could multiply in the oxacillin-containing medium, and a time point (detection time [DT]) at which an accelerating change of impedance occurred in the medium was obtained, with an average of 5.5 h. The growth of oxacillin-sensitive S. aureus (OSSA) strains was largely inhibited, and no DT was obtained for these strains within an incubation period of 24 h. For 96 positive blood cultures (38 ORSA and 58 OSSA) tested, 36 and 57 were found to be oxacillin resistant and oxacillin sensitive, respectively, by the impedance method. The impedance method had a sensitivity and specificity of 94.7 and 98.3%, respectively, for the detection of ORSA and had an agreement of 96.9% with the disc diffusion method. Comparable results were obtained by the testing of 235 clinical stock cultures of S. aureus (149 ORSA and 86 OSSA). The impedance test is simple for detecting ORSA in blood cultures and may allow proper antimicrobial treatment almost 36 h before the results of the conventional culture methods are available.
Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans
Authors: Tornatore M A , Noskin G A , Hacek D M , Obias A A , Peterson L R
Published: January 2020

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, < or = 10 microg/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.
Relationship between adhesion to intestinal Caco-2 cells and multidrug resistance in Klebsiella pneumoniae clinical isolates
Authors: Di Martino P , Sirot D , Joly B , Rich C , Darfeuille-Michaud A
Published: January 2020

Klebsiella pneumoniae is an opportunistic gram-negative pathogen involved in outbreaks of nosocomial infections in intensive care units. Strains are resistant to multiple antibiotics, and 15 to 30% of them are also resistant to the broad-spectrum cephalosporins by the production of R plasmid-encoded extended-spectrum beta-lactamases. Because the gastrointestinal tracts of patients have been shown to be the reservoir for nosocomial strains of K. pneumoniae, we looked for a correlation between antibiotic resistance and adhesion of K. pneumoniae strains to intestinal cells. We investigated adhesion to the human intestinal epithelial Caco-2 cell line of 61 clinical K. pneumoniae strains isolated in hospitals in Clermont-Ferrand, France. None of the strains tested expressed the previously described adhesive factors CF29K and KPF-28. Adhesive properties were found for 42.6% of the strains tested (26 strains). Just 7.7% (2 strains) of the 26 strains producing only the chromosomally encoded SHV-1 beta-lactamase adhered to the Caco-2 cell line, whereas 68.5% (24 strains) of the 35 strains producing a plasmid-encoded beta-lactamase were adherent. All the adherent strains, and even the two strains producing only the SHV-1 enzyme, harbored at least one self-transmissible R plasmid. At variance for CAZ-1/TEM-5 or CAZ-5/SHV-4 beta-lactamase-producing K. pneumoniae strains, curing and mating experiments demonstrated that the self-transmissible R plasmids encoding the TEM-1, CTX-1/TEM-3, CAZ-2/TEM-8, CAZ-6/TEM-24, or CAZ-7/TEM-16 beta-lactamase were not involved in the adhesion of K. pneumoniae strains to intestinal epithelial cells. Nevertheless, there was an association of multiple antibiotic resistance, including resistance to extended-spectrum cephalosporins, and adhesive properties in K. pneumoniae clinical isolates.
An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis
Authors: Nicholson W L , Comer J A , Sumner J W , Gingrich-Baker C , Coughlin R T , Magnarelli L A , Olson J G , Childs J E
Published: January 2020

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.
Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay
Authors: Crotchfelt K A , Welsh L E , DeBonville D , Rosenstraus M , Quinn T C
Published: January 2020

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.
Detection of hydatid antigen in urine by countercurrent immunoelectrophoresis
Authors: Parija S C , Ravinder P T , Rao K S
Published: January 2020

Hydatid antigen was demonstrated for the first time in the urine of patients with hydatid disease by countercurrent immunoelectrophoresis (CIEP). The antigen was detected in the concentrated urine of 7 of 16 (43.75% positive) patients with surgically confirmed hydatid disease, 4 of 10 (40% positive) patients with ultrasound-proven hydatid disease (daughter cysts or prominent septation and hydatid sands demonstrated by ultrasound), and 8 of 14 (57.14% positive) patients with clinically diagnosed (presumptive) hydatid disease. No antigen was detected in the concentrated urine from 24 patients with parasitic diseases other than hydatid disease. However, antigen was detected in 2 (8% false positive) of 25 concentrated urine samples collected from healthy control subjects (blood donors and students). These result suggest that the detection of hydatid antigen in the urine by CIEP is a simple, rapid, and noninvasive method of diagnosis of hydatid disease.
Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format
Authors: Kessler H H , Dodge D E , Pierer K , Young K K , Liao Y , Santner B I , Eber E , Roeger M G , Stuenzner D , Sixl-Voigt B , Marth E
Published: January 2020

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.
Demonstration of vertical transmission of Streptococcus suis in swine by genomic fingerprinting
Authors: Amass S F , SanMiguel P , Clark L K
Published: January 2020

Isolates of Streptococcus suis serotype 5 collected from three sows and nine of their pigs at birth were analyzed by genomic DNA fingerprinting. The cleavage patterns of DNA from S. suis isolated from the sows matched the cleavage patterns of DNA from S. suis isolated from their respective pigs.
A single-tube nested PCR for Pneumocystis carinii f. sp. hominis
Authors: Tang X , Bartlett M S , Smith J W , Lee C H
Published: January 2020

A single-tube nested PCR which amplifies the internal transcribed spacer (ITS) regions of the rRNA genes of human Pneumocystis carinii was developed. The outer primers for the first PCR, which anneal to the 18S and the 26S rRNA genes of P. carinii, were made to have a midpoint temperature (Tm) of 74 degrees C. The inner primers for the second PCR have a Tm of 56 to 58 degrees C and are specific for human P. carinii; they anneal to an area close to the beginning of ITS1 and the junction of ITS2 and the 26S rRNA genes. The reaction mixture contained 2.5 pmol of the first-PCR primers and 25 pmol of the second-PCR primers. The first PCR was performed at an annealing temperature of 68 degrees C, which did not allow the second-PCR primers to function. Since very small amounts (2.5 pmol) of the first-PCR primers were used, they were exhausted when the first PCR was completed. The single-tube nested PCR did not amplify P. carinii isolated from rats, mice, or ferrets. All 10 bronchoalveolar lavage (BAL) specimens from patients with P. carinii pneumonia were positive, whereas all 10 BAL specimens from patients with other diseases or patients infected with several commonly found fungi were negative by PCR.
Endocarditis caused by nonhemolytic group B streptococcus
Authors: Miranda C , Gámez M I , Navarro J M , Rosa-Fraile M
Published: January 2020

We report a case of bacterial endocarditis caused by nonhemolytic group B streptococcus (GBS) in a 67-year-old man with no predisposing risk factors. Nonhemolytic GBS strains rarely cause illness and are usually detected in perinatal infections. We believe this to be the first reported case of endocarditis caused by a nonhemolytic strain of GBS.
Comparison of radioimmunoassay and enzyme immunoassay kits for detection of Legionella pneumophila serogroup 1 antigen in both concentrated and nonconcentrated urine samples
Authors: Domínguez J A , Matas L , Manterola J M , Blavia R , Sopena N , Belda F J , Padilla E , Giménez M , Sabrià M , Morera J , Ausina V
Published: January 2020

We evaluated a commercial enzyme immunoassay (EIA) kit for detection of Legionella pneumophila serogroup 1 soluble antigen by comparing it to radioimmunoassay (RIA), using both concentrated and nonconcentrated urine samples. The sensitivity of EIA was 67.4% in nonconcentrated urine samples and 82.6% in concentrated urine samples. The sensitivity of RIA was 60.9% and 84.8% in nonconcentrated and concentrated urine samples, respectively. Our study indicates that the sensitivity and specificity of EIA are comparable to those of RIA, and that concentrating the antigen by selective ultrafiltration increases sensitivity for both EIA and RIA, with no significant decrease in specificity.
A subtype-specific peptide-based enzyme immunoassay for detection of antibodies to the G protein of human respiratory syncytial virus is more sensitive than routine serological tests
Authors: Langedijk J P , Brandenburg A H , Middel W G , Osterhaus A , Meloen R H , van Oirschot J T
Published: January 2020

Peptides deduced from the central conserved region (residues 158 to 189) of protein G of human respiratory syncytial virus (HRSV) subtypes A and B were used as antigens in subtype-specific enzyme-linked immunosorbent assays (G-peptide ELISAs). These G-peptide ELISAs were compared with seven other serological assays to detect HRSV infection: ELISAs based on complete protein G, on fusion protein F, and on nucleoprotein N; a complement fixation assay; a virus neutralization test; and ELISAs for the detection of immunoglobulin A (IgA) or IgM antibodies specific for HRSV. In paired serum samples from patients with HRSV infection, more infections were diagnosed by the G-peptide ELISA (67%) than by all other serological tests combined (48%). Furthermore, for 16 of 18 patients (89%), the G-peptide ELISAs were able to differentiate between antibodies against HRSV subtypes A and B. This study shows that peptides corresponding to the central conserved region of the attachment protein G of HRSV can successfully be used as antigens in immunoassays. The G-peptide ELISA appeared to be more sensitive than conventional tests for the detection of HRSV antibody titer rises.
Serial surveillance cultures of skin and catheter hub specimens from critically ill patients with central venous catheters: molecular epidemiology of infection and implications for clinical management and research
Authors: Atela I , Coll P , Rello J , Quintana E , Barrio J , March F , Sanchez F , Barraquer P , Ballus J , Cotura A , Prats G
Published: January 2020

A prospective study of 45 central venous catheters was conducted to assess, by strain delineation, the turnover of skin and catheter hub (superficial) colonization and the relative contributions of catheter hub and skin colonization to catheter tip colonization. Serial quantitative cultures of skin and catheter hub were performed. Catheter tip, blood, and specimens for culture from targeted superficial sites (TSSs) were also collected at the time of catheter removal. Strains from 17 tip-positive catheters were delineated by pulsed-field gel electrophoresis. Only 12 (28.6%) of 42 skin strains and 14 (31.1%) of 45 catheter hub strains were found to be present at the time of catheter removal. In addition, only 9 (29.0%) of the 31 tip-colonizing strains were present on TSSs. Moreover, 15 (48.4%) of the 31 tip-colonizing strains had a superficial origin, and the other 16 (51.6%) were of unknown origin. In catheters suspected of infection, cultures of TSSs had a negative predictive value for catheter-related bacteremia of 94.4% but a positive predictive value of 44.4%. When the causative agent was identified (to the strain level) these values dropped to 80.9 and 18.7%, respectively. The study shows that skin and catheter hub colonization is a common, dynamic phenomenon. Strains recovered from TSSs showed a low level of correlation with strains from previous cultures of specimens from superficial sites and catheter tip isolates. Consequently, TSSs cannot be recommended for use in determining the therapy. However, catheter-related bacteremia is uncommon when cultures of TSSs are negative.
PCR-single-stranded confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens
Authors: Newcombe J , Dyer S , Blackman L , Cartwright K , Palmer W H , McFadden J , Blackwell L
Published: January 2020

Subspecific typing of clinical meningococcal strains is important in the investigation of outbreaks and for disease surveillance. Serogrouping, typing, and subtyping of strains currently require isolation of a meningococcus from one or more clinical specimens. However, the increasing widespread practice of preadmission administration of parenteral antibiotics has resulted in a decrease in the frequency of positive cultures obtained from blood and cerebrospinal fluid. Confirmation of meningococcal disease can be obtained by meningococcus-specific PCR from both cerebrospinal fluid (H. Ni et al., Lancet 340:1432-1434, 1992) and peripheral blood (J. Newcombe et al., J. Clin. Microbiol. 34:1637-1640, 1996) specimens. However, current PCR protocols do not yield epidemiologically useful typing information. We report here the use of PCR-single-stranded confirmational polymorphism (PCR-SSCP) analysis to amplify and type meningococcal DNA present in clinical specimens. PCR-SSCP analysis with the VR1 region of the Neisseria meningitidis porA gene as the target produced unique banding patterns for each serosubtype. Direct PCR-SSCP of clinical specimens can therefore provide typing data that can be used to investigate the epidemiology of clusters of cases and outbreaks and for disease surveillance in situations in which culture of patient specimens proves negative.
Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis
Authors: del Aguila C , Lopez-Velez R , Fenoy S , Turrientes C , Cobo J , Navajas R , Visvesvara G S , Croppo G P , Da Silva A J , Pieniazek N J
Published: January 2020

Enterocytozoon bieneusi, a microsporidian parasite, has been recognized since 1985 as an agent of intestinal microsporidiosis leading to malabsorption syndrome, diarrhea, and weight loss in AIDS patients. Recently, however, we have identified E. bieneusi spores in the sputum, bronchoalveolar lavage, and stool samples of an AIDS patient with a 2-year history of intestinal microsporidiosis. The spores were characterized by Weber's chromotrope-based staining, immunofluorescence tests, and PCR. No microsporidia were detected in urine samples by the same techniques. PCR was performed with DNAs purified from specimens with E. bieneusi-, Encephalitozoon cuniculi-, Encephalitozoon hellem-, and Encephalitozoon (Septata) intestinalis-specific primers. Treatment with albendazole and loperamide resulted in an improvement of intestinal symptoms, without eradication of the parasite. To our knowledge, this is the second report of the identification of E. bieneusi spores in respiratory and enteric samples obtained from an AIDS patient. Although no pulmonary pathology could be established in either of these cases, it is now clear that E. bieneusi is capable of colonizing the respiratory tract and it is suggested that investigators should be aware of the possibility of finding E. bieneusi spores in respiratory secretions.
Cross-reactions of reagents from streptococcal grouping kits with Streptococcus porcinus
Authors: Thompson T , Facklam R
Published: January 2020

Streptococcus porcinus is usually associated with swine. Because we have received several isolates from human sources that had cross-reacted with commercial group B streptococcal reagents, we examined several commercial kits to determine the extent of this cross-reaction. Fifteen reference and 15 clinical strains of S. porcinus were tested for cross-reactions with group B streptococcal reagents from 12 different commercial kits. Cross-reactions were detected with all group B reagents, but the number of cross-reactions varied with each kit. We recommend that manufacturers of reagents designed to identify group B streptococci by serologic methods test their reagents for cross-reactions with selected S. porcinus cultures or antigens.
Comparison of methods for extraction of nucleic acid from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences
Authors: Klein A , Barsuk R , Dagan S , Nusbaum O , Shouval D , Galun E
Published: January 2020

The sensitivity of PCR for the amplification of target nucleic acid sequences in clinical diagnostics may often be reduced due to the presence of inhibitory factors. Hemolytic serum contains a number of PCR inhibitors, one of which is hemin. In this study we have found that conventional methods of DNA extraction were not sufficient for the removal of PCR-inhibitory compounds in hemolytic serum. We have therefore compared the efficiency of several commercial and noncommercial methods of nucleic acid purification from hemolytic serum samples prior to PCR amplification. Separation with the QIAamp HCV kit, dialysis with Millipore filters, and bovine serum albumin absorption were all found to be suitable extraction methods for eliminating inhibitors from hemolytic serum for PCR amplification. Using these methods we were able to detect very low levels of hepatitis B virus DNA in hemolytic serum.
Evaluation of the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens
Authors: Ausina V , Gamboa F , Gazapo E , Manterola J M , Lonca J , Matas L , Manzano J R , Rodrigo C , Cardona P J , Padilla E
Published: January 2020

Five hundred twenty processed respiratory specimens from 326 patients received for the diagnosis of tuberculosis or other mycobacterial infections were tested by means of the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, which uses ligase chain reaction technology for the direct detection of M. tuberculosis complex in respiratory specimens. The results of the LCx M. tuberculosis Assay were compared with the results of culture and staining techniques. After a combination of culture results and the patient's clinical data, a total of 195 specimens were collected from 110 patients who were positively diagnosed as having pulmonary tuberculosis. Twenty-three of these 195 specimens which corresponded to 10 patients with a history of pulmonary tuberculosis (TB) and anti-TB treatment ranging from 1 to 6 months were culture negative. The other 172 specimens were culture positive for M. tuberculosis. With an overall positivity rate of 37.5% (195 of 520 specimens), the sensitivity, specificity, and positive and negative predictive values were 90.8, 100, 100, and 94.7%, respectively, for the LCx M. tuberculosis Assay; 88.2, 100, 100, and 93.4%, respectively, for culture; and 82.6, 92, 72.9, and 97.6%, respectively, for acid-fast staining. For 161 specimens (82.6%) from patients smear positive for the disease and 34 specimens (17.4%) from patients smear negative for the disease, the sensitivity values for the LCx M. tuberculosis Assay were 98.8 and 53%, respectively. There were no statistically significant differences in the sensitivities and specificities between the LCx M. tuberculosis Assay and culture (P > 0.05). Conclusively, the LCx M. tuberculosis Assay has proved to have an acceptable sensitivity and a high specificity in detecting M. tuberculosis and has the potential of reducing the diagnosis time to an 8-h working day.
Flow cytometric detection of Cryptosporidium oocysts in human stool samples
Authors: Valdez L M , Dang H , Okhuysen P C , Chappell C L
Published: January 2020

Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.
Detection and clinical significance of extended-spectrum beta-lactamases in a tertiary-care medical center
Authors: Emery C L , Weymouth L A
Published: January 2020

The prevalence of extended-spectrum beta-lactamase (ESBL)-mediated resistance remains unknown for most hospitals, and national guidelines for testing and reporting ESBL-mediated resistance have not yet been developed. We undertook a study to determine the prevalence of ESBLs and the clinical need for testing in our tertiary-care medical center. Members of the family Enterobacteriaceae isolated over a 6-month period for which ceftazidime or ceftriaxone MICs were greater than 1 microg/ml were tested for production of ESBLs by the double-disk synergy method. Approximately 1.5% of isolates of the family Enterobacteriaceae (50 of 3,273), which were isolated from 1.2% of patients (23 of 1,844), were found to express ESBLs. ESBL-producing strains included eight different species and were isolated from patients located throughout the hospital, including outpatient clinics. By using the interpretive guidelines of the National Committee for Clinical Laboratory Standards, 26 to 39% of the isolates would have been reported to be susceptible to ceftazidime, depending upon the routine susceptibility method used. However, tests with cefpodoxime found all of the ESBL-producing strains to be resistant or intermediate. Nine patients infected with ESBL-producing isolates were treated with therapy which included an expanded-spectrum cephalosporin. Seven were cured. The deaths of the other two patients were not attributed to bacterial resistance missed by routine susceptibility testing. These observations suggest that in our tertiary-care medical center, it may not be clinically necessary or cost-effective at this time to institute additional testing on a routine basis to detect ESBL production in all clinical isolates of the family Enterobacteriaceae.
Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems
Authors: Cornfield D B , Beavis K G , Greene J A , Bojak M , Bondi J
Published: January 2020

The BACTEC 460 system currently provides the most rapid detection of mycobacterial growth, but the system is radiometric and requires needles to inoculate specimens through the bottle's septum. The Mycobacteria Growth Indicator Tube (MGIT) system has a liquid medium, like the BACTEC system, and does not require needles when inoculating specimens. We compared mycobacterial growth from 510 specimens in the two systems. Average time to acid-fast bacillus (AFB) detection and identification to the species level was less with the BACTEC system, but this result was statistically significant only for AFB detection in specimens containing Mycobacterium avium-M. intracellulare complex. The contamination rate with MGIT was 29%; the BACTEC rate was 5%. To investigate MGIT contamination, we initiated a second study with changes in specimen processing. The MGIT contamination rate was reduced to 12%; the BACTEC rate was not significantly affected (5.5%). The most likely explanation for the contamination in MGIT is the richness of its medium compared to the BACTEC medium. Cost analysis for the two systems in a laboratory that processes 4,500 specimens a year is presented. The data suggest that the BACTEC 460 and the MGIT systems are approximately equivalent in cost and ability to support the growth of AFB. The MGIT system appears safer and easier to use and was preferred by laboratory personnel, but it cannot currently be used for blood specimens or antituberculosis susceptibility testing.
Evaluation of the Vidas Chlamydia test to detect and verify Chlamydia trachomatis in urogenital specimens
Authors: Schachter J , Jones R B , Butler R C , Rice B , Brooks D , Van der Pol B , Gray M , Moncada J
Published: January 2020

The Vidas Chlamydia test (CHL) is an automated enzyme-linked immunofluorescence assay for the detection of Chlamydia trachomatis. Positive and equivocal results are confirmed with a blocking assay. A mouse monoclonal antibody directed against the chlamydial lipopolysaccharides was used for the test. The CHL assay is widely used in Europe, but U.S. experience with it is limited. Three clinical test sites (The Arlington Hospital, Arlington, Va., Indiana University, Indianapolis, and the University of California, San Francisco) compared CHL with tissue culture (TC) for the identification of chlamydia in urogenital specimens (2,453 females and 850 males). True positives (TP) were defined as either TC positive or TC negative and CHL positive by a positive direct fluorescent-antibody assay or PCR test. Overall prevalence was 5.5% for females, 10.3% for male urethral swabs, and 10.7% for combined male TC urethral swabs and CHL with first catch urine (FCU) specimens. Compared to TP, CHL and TC had sensitivities of 89.6 and 94.1% with female cervical swabs and 90.9 and 86.4% with male urethral swabs, respectively. CHL sensitivity was 81.2 for male FCU specimens and 77.7% for matching male TC swabs. There were relatively few false-positive results, with all specificities being >99.4%. With the blocking assay, Vidas CHL specificity was >99.7%. However, male FCU specimen sensitivity was compromised because 9.2% (7 of 76) of the TP were initially positive but were not confirmed. An improvement in the Vidas blocking assay is needed before we can recommend its use with male urine. Alternatively, one could argue that the specificity of the test is so high that a confirmatory assay is not needed. For male and female swabs, the Vidas CHL assay has a performance that is similar to that of TC.
A case of peritonitis caused by Roseomonas gilardii in a patient undergoing continuous ambulatory peritoneal dialysis
Authors: Sandoe J A , Malnick H , Loudon K W
Published: January 2020

A case of peritonitis caused by Roseomonas gilardii in a patient receiving continuous ambulatory peritoneal dialysis is presented. The patient's domestic water supply was implicated as the probable source of infection. This is the first report of R. gilardii causing such an infection.
Laboratory diagnosis of respiratory virus infections in 24 hours by utilizing shell vial cultures
Authors: Engler H D , Preuss J
Published: January 2020

Immunofluorescence staining of centrifugation-enhanced shell vial (SV) cultures for respiratory viruses (RV) after 24 h of incubation, rather than the more commonly prescribed times of 48 h and 5 days, allowed for the detection of 77% of the RV-positive specimens that would ordinarily not have been detected as positive until 48 h. Staining SVs at 24 h also permitted earlier detection of viruses that were missed by rapid antigen detection methods.
Effects of storage temperature and time on qualitative and quantitative detection of cytomegalovirus in blood specimens by shell vial culture and PCR
Authors: Roberts T C , Buller R S , Gaudreault-Keener M , Sternhell K E , Garlock K , Singer G G , Brennan D C , Storch G A
Published: January 2020

Cytomegalovirus (CMV) infectious titers and DNA levels were determined by quantitative shell vial culture and quantitative-competitive PCR with blood samples from 10 renal transplant recipients with active CMV infection. Blood samples were stored at either room temperature or 4 degrees C and were processed at intervals of 0, 6, 24, 48, and 72 h. All samples were culture and PCR positive at baseline. Whereas the sensitivity of shell vial culture progressively declined, with only 55% positive at 24 h and 10% positive at 48 h, all samples remained PCR positive at all time points. Furthermore, the infectious titer diminished by 83 to 91% by 24 h compared to that at baseline (P < 0.0001), but quantitative DNA levels did not decline over time. Storage temperature had no significant effect on either infectious titer or DNA levels.
Rapid detection and typing of herpes simplex virus DNA in clinical specimens by the hybrid capture II signal amplification probe test
Authors: Cullen A P , Long C D , Lörincz A T
Published: January 2020

A second-generation signal amplification, nucleic acid-based test for the rapid detection and typing of herpes simplex virus (HSV) DNA was developed and evaluated with artificial and clinical specimens. The analytical sensitivity of the Hybrid Capture II (HC II) HSV DNA assay was determined by testing either cloned HSV DNA or total genomic HSV DNA titrations and resulted in detection thresholds of between 5 x 10(3) and 1 x 10(4) copies per assay. Specificity was assessed by testing a panel of bacteria and viruses commonly found in the female genital tract. Sensitivity was assessed by testing 112 ulcerative genital lesions by the HC II assay and comparing the results to those obtained by routine cell culture. Discrepant results were resolved by PCR testing. After resolution of the discrepant results, the sensitivity of the HC II assay compared to the consensus result (the results of two of three tests, the HC II assay, culture, and PCR, were in agreement) was 93.2% (41 of 44 specimens), and the specificity was 100% (60 of 60 specimens). Culture gave a sensitivity of 84.1% (37 of 44 specimens) and a specificity of 100% (60 of 60 specimens) compared to the consensus result. The results of HSV typing by the HC II assay and culture agreed in all cases. The HC II assay is a rapid and accurate assay for detecting and typing HSV types 1 and 2, with a sensitivity comparable to that of culture and greater ease of use than culture.