bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic diversity of Neisseria gonorrhoeae IB-2 and IB-6 isolates revealed by whole-cell repetitive element sequence-based PCR
Authors: Poh C L , Ramachandran V , Tapsall J W
Published: January 2020

Phenotypic characterization of 19 Neisseria gonorrhoeae serovar IB-2 and 8 serovar IB-6 isolates by the combined use of auxotypes, serological characterization, and penicillin susceptibility testing indicated intraserovar genetic diversity. In the present study, we applied whole-cell repetitive element sequence-based PCR (rep-PCR) analysis which allows a rapid assessment of the clonal relationships of IB-2 and IB-6 isolates. DNA templates were prepared by boiling cells harvested directly from plate cultures, eliminating the need for time-consuming phenol extraction. Six different rep-PCR profiles were established among the 19 IB-2 isolates. Rep-PCR typing results had a good correlation with pulsed-field gel electrophoresis patterns. It is slightly less discriminatory than BglII-generated macrorestriction pattern analysis by pulsed-field gel electrophoresis. It is capable of discriminating epidemiologically related from epidemiologically unrelated IB-2 isolates. It should serve as a rapid and useful subtyping tool for epidemiologic investigations in which there is a predominance of major serovar groups.
Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods
Authors: Springer B , Stockman L , Teschner K , Roberts G D , Böttger E C
Published: January 2020

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-to-day routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.
Cost and time savings following introduction of rejection criteria for clinical specimens
Authors: Morris A J , Smith L K , Mirrett S , Reller L B
Published: January 2020

We have evaluated the yield of several tests and have instituted specimen rejection criteria to reduce costs and save time. For a 12-month period, we recorded the reduction of these tests and calculated the resultant cost and time savings. Seven changes were analyzed: not performing fungal or mycobacterial (acid-fast bacillus) cultures on cerebrospinal fluid (CSF) specimens from patients without known immunosuppression when chemistry and cell count are normal; not performing routine stool culture or ovum and parasite examination on specimens from patients in the hospital for > 3 days; not culturing endotracheal suction aspirates when no organisms or > 10 squamous epithelial cells are present; discontinuing broth cultures on all specimens except for tissue, continuous ambulatory peritoneal dialysis fluid, and CSF from patients with shunts; and eliminating bacterial antigen tests. For each test, the number not performed (n), reagent savings, and technologist time saved, respectively, were as follows: CSF fungal culture, 267, $999, and 67 h; CSF acid-fast bacillus culture, 275, $1,662, and 124 h; stool cultures, 320, $2,991, and 98 h; ovum and parasite examinations, 216, $525, and 108 h; endotracheal suction aspirate cultures, 1,505, $4,447, and 306 h; broth cultures, 5,218, $4,931, and 80 h; and bacterial antigen tests, 2,598, $2,293, and 299 h. Overall, 5,181 tests were rejected and 5,218 broth cultures were omitted. Achievable savings were $28,000 in reagent costs and 1,082 h of technologist time. In conclusion, rejecting specimens of proven low yield saves reagent costs and, more importantly, saves technologist time. This time can be spent on specimens having greater diagnostic utility.
Detection of human papillomavirus (HPV) type 47 DNA in malignant lesions from epidermodysplasia verruciformis by protocols for precise typing of related HPV DNAs
Authors: Adachi A , Kiyono T , Hayashi Y , Ohashi M , Ishibashi M
Published: January 2020

Our discovery of human papillomavirus type 47 (HPV47) in benign lesions from a patient suffering from epidermodysplasia verruciformis prompted us to examine whether the viral DNA also resided in malignant lesions from the same patient. By using newly devised protocols for amplifying a group of epidermodysplasia verruciformis-associated HPV DNAs by PCR and differentially identifying them by reverse-phase dot blot hybridization, we demonstrated that HPV47 DNA, but not other HPV DNAs of the group, was abundant (about 10(3) copies per diploid amount of cell DNA) in DNAs prepared from three carcinomas. Using DNA from one of these carcinomas, we also confirmed that DNA of HPV5, HPV14, or HPV21, detected in significant amounts in DNAs from benign lesions from the patient, were present only in negligible amounts or not at all. The results suggest the involvement of HPV47 DNA in tumorigenesis. Furthermore, we demonstrated by the Southern technique that most, if not all, of the HPV47 DNA consists of either a unit (or a nongrossly deleted unit) length of the viral genome carrying no (or no gross) internal rearrangements or tandem repeats. This and other results obtained by this technique indicated that a considerable amount of the viral DNA resides as a circular monomer a unit length of the viral genome in carcinoma cells, while the remainder reside as catenanes, concatemers, or both. The concatemers were considered more likely to be replicated without integration into cellular DNA than to be integrated, because no bands for the corresponding fragments including integration sites were detected by treatment with restriction enzymes that would have produced such fragments.
Acute Bordetella pertussis infection in an adult
Authors: Smith S , Tilton R C
Published: January 2020

Bordetella pertussis was transmitted from an immunized boy to his father and possibly to other family members. This case report demonstrates that although unusual, acute adult B. pertussis infection can occur. B. pertussis immunization may not prevent infection but can reduce the severity. B. pertussis was detected in sputum from the adult by direct-fluorescent antibody staining and was grown on Regan-Lowe medium. Serology tests confirmed the infection in the adult.
Evaluation of a commercial enzyme-linked immunosorbent assay for detection of serum immunoglobulin G response to human herpesvirus 6
Authors: Sloots T P , Kapeleris J P , Mackay I M , Batham M , Devine P L
Published: January 2020

A rapid (60-min) commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) class antibodies to human herpesvirus 6 (HHV-6) was evaluated. The specificity of the ELISA for HHV-6 was confirmed by absorption studies, with the reactivities of HHV-6-positive sera being unaffected by other herpesviruses (cytomegalovirus, herpes simplex virus, and varicella-zoster virus) or the HSB2 cell line used to culture HHV-6. HHV-6 IgG antibody levels in a panel of 502 serum samples were determined by ELISA and an indirect immunofluorescence assay (IFA). Results obtained by the two methods were in close agreement, suggesting that the ELISA provides a suitable test method for the determination of HHV-6 IgG antibodies in a routine clinical laboratory. Both tests were positive in 398 cases (79%), and both were negative in 71 cases (14%), with a different result obtained by IFA and ELISA in only 33 cases (7%). Furthermore, absorption of sera with HHV-6 prior to assay revealed that the majority of these results were false positive (n = 8) or false negative (n = 23) in the IFA (true positives or negatives in the ELISA). Subsequently, the ELISA showed a sensitivity of 99.76% and a specificity of 98.75%. HHV-6-specific IgG levels were also determined in paired serum samples collected from 49 donors--14 with exanthem subitum (ES), 15 with ES which was complicated with central nervous system involvement, and 20 undergoing bone marrow transplantation--in whom HHV-6 infection had been demonstrated by virus isolation and/or PCR. All patients with ES or central nervous system complications showed an increase in HHV-6-specific IgG, indicating that this ELISA may be a useful aid in the diagnosis of these conditions. Furthermore, 14 of 20 patients undergoing bone marrow transplantation showed an increase in HHV-6-specific IgG levels, possibly reflecting a reactivation of HHV-6 in these patients.
Mycobacterium malmoense bacteremia in two AIDS patients
Authors: Fakih M , Chapalamadugu S , Ricart A , Corriere N , Amsterdam D
Published: January 2020

We report two cases of Mycobacterium malmoense bacteremia in two patients with AIDS. These are the first reported cases of disseminated M. malmoense in human immunodeficiency virus patients occurring in the United States. This slow-growing organism can cause invasive disease mimicking Mycobacterium avium complex infection; recognition and identification of this organism by mycobacteriology laboratories are essential for appropriate diagnosis and therapy of disseminated disease.
Immunoglobulin A-specific serodiagnosis of acute human cytomegalovirus infection by using recombinant viral antigens
Authors: Vornhagen R , Hinderer W , Sonneborn H H , Bein G , Matter L , The T H , Jahn G , Plachter B
Published: January 2020

The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for immunoglobulin A serodiagnosis. Application of autologous fusion proteins which combine these polypeptides may be useful especially for the early detection of acute secondary human cytomegalovirus infection.
Susceptibility testing of Propionibacterium acnes comparing agar dilution with E test
Authors: Smith M A , Alperstein P , France K , Vellozzi E M , Isenberg H D
Published: January 2020

Propionibacterium acnes has been identified as a significant agent of nosocomial infections, including endophthalmitis. Data concerning susceptibility of P. acnes to newer beta-lactam antibiotics and fluoroquinolones are limited. Recent reports suggest that quinolones have activity against these organisms sufficient to warrant further study. We undertook a study to select appropriate antimicrobial agents for use in a rabbit model of P. acnes endophthalmitis. We compared the antibiotic susceptibilities of P. acnes by using the National Committee for Clinical Laboratory Standards method of agar dilution with the E test. Thirteen clinical isolates obtained from eye specimens and three American Type Culture Collection control strains were tested against 14 antibiotics. All the clinical isolates were susceptible by both methods to piperacillin, piperacillin-tazobactam, ampicillin-sulbactam, ticarcillin-clavulanate, cefotaxime, cefotetan, ceftriaxone, cefoxitin, and imipenem in addition to clindamycin but were resistant to metronidazole. The clinical P. acnes isolates also displayed high-level susceptibility to ciprofloxacin, sparfloxacin, and ofloxacin. Almost all the P. acnes strains demonstrated E-test MICs within 2 dilutions of the MICs observed by the agar dilution method. Those few strains for which discrepancies were noted exhibited E-test susceptibilities three- to fivefold dilutions lower than the agar dilution method susceptibilities but only with ampicillin-sulbactam, ticarcillin-clavulanate, and/or clindamycin. On the basis of our study, all of clinical eye isolates were susceptible to these newer antimicrobial agents and the two methods demonstrated similar susceptibility patterns.
Immunomagnetic separation and solid-phase detection of Bordetella pertussis
Authors: Stark M , Reizenstein E , Uhlén M , Lundeberg J
Published: January 2020

In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.
Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole
Authors: Rex J H , Pfaller M A , Lancaster M , Odds F C , Bolmström A , Rinaldi M G
Published: January 2020

Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines. Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism. Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established. These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C. parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C. krusei ATCC 6258.
Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA
Authors: Down J A , O'Connell M A , Dey M S , Walters A H , Howard D R , Little M C , Keating W E , Zwadyk P , Haaland P D , McLaurin D A , Cole G
Published: January 2020

A total of 294 clinical respiratory specimens, including 75 with culture-positive results, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification (SDA) of DNA. A region of the IS6110 insertion element and an internal control sequence were amplified and then detected by a chemiluminescence assay. Receiver operator-characteristic curves were used to evaluate three methods for declaring specimens positive for M. tuberculosis. By the preferred method, SDA chemiluminescence results were converted to theoretical numbers of M. tuberculosis organisms. A positive threshold (PT) value, above which 95% of the SDA results were judged to be M. tuberculosis positive (sensitivity = 95%), was found to be 2.4 M. tuberculosis organisms per SDA reaction. The analogous PT value for 95% sensitivity on smear-positive specimens was 3.6 M. tuberculosis organisms per reaction. The PT of 2.4 M. tuberculosis organisms per reaction detected 100% of culture-positive, smear-positive specimens (sensitivity = 100%), while 95% sensitivity was achieved with a PT of 15.5 M. tuberculosis organisms per reaction. Specificities, which were calculated with respect to culture- and smear-negative specimens, ranged from 96% at a PT of 15.5 M. tuberculosis organisms to 84% at a PT of 2.4 M. tuberculosis organisms per reaction. The M. tuberculosis-negative specimens were also segregated according to whether the patients received antituberculosis chemotherapy. SDA specificity ranged from 90% (PT = 2.4 M. tuberculosis organisms) to 98% (PT = 15.5 M. tuberculosis organisms) for the M. tuberculosis-negative specimens from patients who had not received chemotherapy. SDA specificity in the M. tuberculosis-negative specimens from patients who received chemotherapy was lower (85 to 94%). This study represents the first large-scale demonstration of M. tuberculosis detection in clinical sputum specimens by isothermal DNA amplification with SDA.
Identification of murine helicobacters by PCR and restriction enzyme analyses
Authors: Riley L K , Franklin C L , Hook R R , Besch-Williford C
Published: January 2020

Three murine helicobacter species have recently been identified: Helicobacter hepaticus, Helicobacter muridarum, and Helicobacter bilis. Infections with H. hepaticus and H. bilis have been associated with hepatitis and hepatic neoplasia. In this study, oligonucleotide primers were designed from regions of the 16S rRNA gene that are conserved among members of the Helicobacter genus. The assay amplified the expected 374-bp product from all three rodent Helicobacter species and was able to detect as little as 5 pg of H. hepaticus, H. bilis, or H. muridarum DNA. The specificity of the reaction was determined by testing cecal DNA from uninfected mice and mice with documented Helicobacter infections and by testing DNA from other bacterial genera. A product of the expected size was generated with cecal DNA from Helicobacter-infected mice but not with DNA from uninfected mice. With the exception of that of "Flexispira rappini, " which is closely related to the Helicobacter genus, DNA from other bacterial genera was not amplified with the Helicobacter genus-specific primers. MboI, MaeI, and HhaI restriction enzyme analyses of the amplified product were able to differentiate among the murine Helicobacter species but could not differentiate H. bilis from "F. rappini." To distinguish H. bilis, a reverse primer based on H. bilis 16S rRNA sequence was designed. PCR with the H. bilis-specific reverse primer (Hbr) and the Helicobacter genus-specific forward primer (H276f) amplified H. bilis DNA but not DNA from "F. rappini" or other rodent helicobacters. Examination of a large number of murine cecal tissues with this combination of PCR assays and restriction enzyme analyses indicated that H. hepaticus and H. bilis infections are widespread in laboratory mouse and rat colonies.
Evaluation of Amplicor MTB test as adjunct to smears and culture for direct detection of Mycobacterium tuberculosis in the French Caribbean
Authors: Devallois A , Legrand E , Rastogi N
Published: January 2020

A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.
Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens
Authors: Herrera E A , Segovia M
Published: January 2020

A PCR assay based on the species-specific mtp40 genomic fragment was developed for the specific detection and identification of Mycobacterium tuberculosis in different uncultured clinical specimens. The aim of the study was to evaluate the clinical applicability of this target DNA in comparison with those of conventional microbiological methods and to compare the results obtained with those obtained after amplification with the IS6110 repetitive element. Discrepant results were interpreted in conjunction with the patients' clinical data, medical histories, and response to therapy. A total of 172 specimens from 162 patients with respiratory symptoms were tested, 101 specimens were obtained from 92 patients clinically suspected of having tuberculosis, and 71 specimens were obtained from 70 patients without known mycobacterial infection. The results of our study suggest that PCR amplification with the mtp40 genomic fragment provides a highly sensitive and specific technique for the detection of M. tuberculosis strains in clinical samples. It allows for the differentiation between M. tuberculosis and other related mycobacteria, including M. bovis, and is more specific than the IS6110 target. For these and other reasons, we propose that the mtp40 assay is a possible alternative for the specific direct detection of M. tuberculosis in clinical laboratories.
A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood
Authors: Grundy J E , Ehrnst A , Einsele H , Emery V C , Hebart H , Prentice H G , Ljungman P
Published: January 2020

The presence of cytomegalovirus (CMV) in the blood has important consequences for patient management, and an external quality control study of its detection by the PCR was conducted by the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation. Forty-eight coded peripheral blood samples from bone marrow transplant recipients were processed in parallel in three European centers by using the routine in-house PCR assay. Protocols varied in choice of primers, specificity and amplificability controls, and sample processing. Results for 38 of 47 samples agreed, 35 being negative and 3 positive. Of the 12 samples reported as positive by a least one center, only 3 were found to be positive by all three centers, 1 was found to be positive by two centers, and the remaining 8 were found to be positive by one center only. The nine discrepant samples appeared to contain around 1,000-fold less viral DNA than the three concordant positive samples. CMV detection was affected both by the number of leukocytes from which DNA was extracted and by the number of cell equivalents added per PCR. External quality control schemes for CMV PCR are clearly necessary in order to compare data from different centers, and recommendation for standardizing the PCR detection of CMV in blood leukocytes are made.
Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi
Authors: Kirchhoff L V , Votava J R , Ochs D E , Moser D R
Published: January 2020

The diagnosis of acute infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is generally made by detecting parasites by microscopic examination of fresh blood. Although highly specific, this approach often lacks sensitivity. Several years ago, PCR assays for the detection of T. cruzi were described, but the sensitivities and specificities of these tests have not yet been defined precisely. In the present study, we first compared the sensitivities of PCR methods that differ in sample processing as well as in the target sequences that are amplified. Then, we challenged eight mice with T. cruzi, and on 31 days over a 380-day period, we compared the ability of the PCR method with the highest sensitivity to detect parasites in blood with that of microscopic examination. During the acute phase of the infections, parasites were detected on average 3.9 days earlier by the PCR method than by microscopy. Furthermore, the infected mice were consistently positive by the PCR method during the chronic phase, while parasites were intermittently detected by microscopic examination during that period. Overall, among the 248 comparisons, in 84 the PCR method was positive and no parasites were seen by microscopic examination, whereas the reverse was true in only 1 case, a difference that is highly significant. These findings suggest that this approach should be in patients suspected of having acute Chagas' disease. Moreover, the higher sensitivity of the PCR method observed in both the acute and chronic phases of the T. cruzi infections in the mice that we studied indicates that this approach should be useful in evaluating experimental drugs in T. cruzi-infected laboratory animals.
Detection of Yersinia enterocolitica serogroup O:3 by a PCR method
Authors: Weynants V , Jadot V , Denoel P A , Tibor A , Letesson J J
Published: January 2020

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.
Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis
Authors: Liveris D , Wormser G P , Nowakowski J , Nadelman R , Bittker S , Cooper D , Varde S , Moy F H , Forseter G , Pavia C S , Schwartz I
Published: January 2020

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.
New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay
Authors: Miyamoto J , Koga H , Kohno S , Tashiro T , Hara K
Published: January 2020

We developed a novel method for early detection of drug-resistant strains of Mycobacterium tuberculosis by using the hybridization protection assay (HPA). The number of viable bacteria during the incubation period correlated well with the number of relative light units measured by the HPA. In addition, the relative light unit values of susceptible strains on the first, third and fifth days of incubation were significantly different from those of resistant strains for both isoniazid and rifampin. Our results suggest that after isolation of the organism from clinical specimens, drug-resistant strains of M. tuberculosis are accurately detected by the HPA even after 1 day of incubation with the drug.
Demonstration of a lack of synergistic effect of rotavirus with other diarrheal pathogens on severity of diarrhea in children
Authors: Unicomb L E , Faruque S M , Malek M A , Faruque A S , Albert M J
Published: January 2020

The severity of group A rotavirus (RV) diarrhea was compared with that of mixed infections of RV with diarrheagenic Escherichia coli, Vibrio cholerae O1, and Shigella species by a scoring system. The severity of mixed infections of RV and E. coli was the same as that of infections with RV alone. RV infections mixed with V. cholerae and Shigella species mimicked cholera and shigellosis, respectively.
Improved detection of JC virus DNA in cerebrospinal fluid for diagnosis of AIDS-related progressive multifocal leukoencephalopathy
Authors: de Luca A , Cingolani A , Linzalone A , Ammassari A , Murri R , Giancola M L , Maiuro G , Antinori A
Published: January 2020

Several methods to increase the sensitivity of JC virus (JCV) DNA detection in cerebrospinal fluid (CSF) for a noninvasive diagnosis of AIDS-related progressive multifocal leukoencephalopathy (PML) were investigated. When CSF collected at clinical presentation was tested, JCV DNA was detected in 8 of 19 patients with PML by standard PCR (sensitivity, 42%; 95% confidence interval [CI], 21 to 66%) and in 14 of 19 by nested PCR (sensitivity, 74% [95% CI, 49 to 90%]; P = 0.014 [McNemar's test]. For multiple serial CSF samples, standard PCR yielded JCV DNA for 11 of 19 PML patients (sensitivity, 58% [95% CI, 34 to 79%]) and nested PCR yielded JCV DNA for 17 of 19 patients (sensitivity, 90% [95% CI, 66 to 98%]; P = 0.014). The majority of the false-negative samples were found to contain PCR inhibitors. Standard PCR did not detect JCV DNA in CSF from any of the 83 AIDS patients with other diagnosis (100% specificity [95% CI, 95 to 100%]); JCV DNA was found in CSF from one control patient by nested PCR (99% specificity [95% CI, 93 to 100%]).
The porA alleles are identical in subgroup III serogroup A Neisseria meningitidis strains isolated in China in the 1960s and 1980s
Authors: Malorny B , Maiden M C , Achtman M
Published: January 2020

The porA genes from serogroup A, subgroup III strains isolated in the People's Republic of China in 1966 and in 1984 and 1985 were amplified by PCR at an annealing temperature of 75 degrees C. The DNA sequences (5 strains) and the restriction patterns generated by MspI (14 strains) were identical, unlike the results reported by Peixuan et al. (Z. Peixuan, H. Xujing, and X. Li, J. Clin. Microbiol. 33:458-462, 1995). Furthermore, PCR products which were amplified at an annealing temperature of 60 degrees C, as described by Peixuan et al., were heterogeneous in our study.
Enhanced amplification of hepatitis C virus (HCV) cDNA by PCR: detection of HCV RNA in archival sera
Authors: Beardsley A M , Gowans E J , Burrell C J , Marmion B P
Published: January 2020

A reverse transcription-PCR assay which successfully amplified hepatitis C virus RNA from poorly stored archival sera was optimized. Maximum sensitivity was achieved with Moloney murine leukemia virus RNase H- reverse transcriptase and by a single round of PCR amplification of a short (112-bp) fragment of the 5' untranslated region of the viral genome.
Classification of rotavirus into G and P types with specimens from children with acute diarrhea in New Delhi, India
Authors: Husain M , Seth P , Dar L , Broor S
Published: January 2020

Sixty rotavirus-positive stool specimens from children with diarrhea were classified into G and P genotypes. G typing was done by PCR and then by hybridization with G type-specific (G1 to G4) oligonucleotide probes, whereas nested PCR was performed for P typing. Thirty-nine samples could be classified into both G and P types, of which P8G1 and P4G2 (33% each) genotypes were predominant. The P6 genotype was detected in four children with diarrhea.
Evaluation of Vitek GPS-SA card for testing of oxacillin against borderline-susceptible staphylococci that lack mec
Authors: Knapp C C , Ludwig M D , Washington J A , Chambers H F
Published: January 2020

Fifty-one Staphylococcus aureus strains lacking mec for which oxacillin MICs were 1 to 8 micrograms/ml were tested against oxacillin and the combination of oxacillin and clavulanic acid with the Vitek GPS-SA card, the reference broth microdilution method, and the oxacillin agar screen plate. Of the 51 strains, 44 (86%) did not grow on the oxacillin agar screen plate, broth microdilution MICs were 1 to 2 micrograms/ml, and GPS-SA card MICs were < or = 2 micrograms/ml, with the exception of 3 strains that failed to grow in the card on repeated attempts. Another seven strains did grow on the oxacillin agar screen plate. For four of the latter group of strains, oxacillin broth microdilution MICs were > 4 micrograms/ml and GPS-SA card MICs were > or = 4 micrograms/ml; for the other 3 strains, corresponding MICs were 4 and < or = 2 micrograms/ml, respectively. The GPS-SA card classified 86% of strains as oxacillin susceptible.
Evolutionary origin of a monophasic Salmonella serovar, 9,12:l,v:-, revealed by IS200 profiles and restriction fragment polymorphisms of the fljB gene
Authors: Burnens A P , Stanley J , Sechter I , Nicolet J
Published: January 2020

The emergence in several countries of the monophasic serogroup D1 serovar Salmonella 9,12:l,v:- provided the opportunity to study its evolutionary origin. According to current models, such a variant serovar could have arisen by horizontal transfer of a new flagellar gene to a preexisting monophasic Salmonella strain or, alternatively, by the loss of the phase 2 flagellar gene of an originally biphasic Salmonella strain. Five known serovars of Salmonella, S. panama, S. kapemba, S. goettingen, S. zaiman, and S. mendoza, could have been possible ancestors of the new variant. The profiles of the insertion element IS200, which has been shown to provide phylogenetic markers for serogroup D1 salmonellae, were analyzed in relation to the restriction fragment length polymorphisms of the phase 2 flagellar gene. Together they provide unequivocal evidence that Salmonella 9,12:l,v:- arose from a strain of S. goettingen. Analysis of the flj operon of the variant indicated that loss of phase 2 flagellar antigen expression occurred through deletion of the hin gene and adjacent DNA, thereby blocking the phase 2 flagellar gene in the off position.
Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis
Authors: Deguchi T , Yasuda M , Uno M , Tada K , Iwata H , Komeda H , Maeda S , Latila V , Saito I , Kawada Y
Published: January 2020

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis.
Rapid species identification of "Streptococcus milleri" strains by line blot hybridization: identification of a distinct 16S rRNA population closely related to Streptococcus constellatus
Authors: Jacobs J A , Schot C S , Bunschoten A E , Schouls L M
Published: January 2020

A collection of 399 "Streptococcus milleri" strains were identified to the species level by the use of a line blot assay. Their PCR-amplified partial 16S rRNA gene sequences were hybridized with species-specific 5'-biotinylated oligonucleotide probes homologous to the bp 213 to 231 regions of the 16S rRNA gene sequences of the type strains Streptococcus anginosus ATCC 33397, Streptococcus constellatus ATCC 27823, and Streptococcus intermedius ATCC 27335. The hybridization results were compared with the reference phenotypic identification method data (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). Most strains (357 of 399 [89.5%]) reacted unambiguously with only one probe. However, 42 of the 399 strains (10.5%) reacted with both the S. constellatus- and S. intermedius-specific probes; 41 of them were phenotypically identified as S. constellatus. These dually reactive strains hybridized with a 5'-biotinylated probe based on the bp 213 to 231 region of the 16S rRNA gene sequence of one of two species. Analysis of the 5' ends of the 16S rRNA gene sequences (487 bp) demonstrated that the dually reactive strains represent a distinct rRNA population sharing 98.1% sequence similarity with S. constellatus. Phenotypic consistency between the dually reactive strains and the S. constellatus strains was not demonstrated. Line blot hybridization proved to be a simple and inexpensive method to screen large numbers of strains for genetic relatedness, and it allowed the detection of a distinct 16S rRNA type within the "S. milleri" group.
Analysis of hepatitis C virus isolates by serotyping and genotyping
Authors: van Doorn L J , Kleter B , Pike I , Quint W
Published: January 2020

Hepatitis C virus (HCV)-positive sera from 106 chronically infected patients which had previously been genotyped were characterized by serotyping. Genotypes were determined by a first-generation line probe assay (INNO-LiPA HCV) and by sequence analyses of the core, core-E1, and NS5B regions. HCV serotypes were determined by measuring type-specific antibodies to NS4-derived peptide antigens (Murex HCV serotyping 1-6 assay). Of 106 serum samples, serotype-specific antibodies were detected in 88 (sensitivity, 83.0%), and 77 (specificity, 87.5%) of these serotypeable samples revealed a corresponding serotype (total concordance, 72.6%). Eleven samples revealed discrepant results as follows. (i) Five serum samples in which only a single genotype was detected contained an additional serotype. (ii) In one sample with two genotypes only one serotype was detected. (iii) In five isolates the serotype (all serotype 1) was completely different from the genotype. Double infections, as determined by genotyping, were confirmed by serotyping in two of four cases. Of 11 serum samples from chronically infected hemodialysis patients, 7 (64%) were reactive in the serotyping assay. In conclusion, genotyping allows discrimination between (sub)types but requires the relatively complex reverse transcriptase PCR. The novel serotyping assay offers an alternative method to distinguish the major types of HCV, although the sensitivity of the assay may be limited by the immunocompetence of the infected host.
Molecular epidemiology of Mycobacterium tuberculosis strains isolated from different regions of Italy and Pakistan
Authors: Sechi L A , Zanetti S , Delogu G , Montinaro B , Sanna A , Fadda G
Published: January 2020

The use of the (GTG)5 oligonucleotide, a repetitive marker in the Mycobacterium tuberculosis chromosome, as a primer in association with an IS6110 outlooking primer has been successfully applied to a PCR-based fingerprinting method. This method classified 62 strains of M. tuberculosis, isolated from human immunodeficiency virus-seropositive and -seronegative patients in different regions of Italy and Pakistan, as having 53 different patterns. The results were compared with traditional IS6110 fingerprinting, by which 47 distinct patterns were observed.
Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species
Authors: Fricker-Hidalgo H , Vandapel O , Duchesne M A , Mazoyer M A , Monget D , Lardy B , Lebeau B , Freney J , Ambroise-Thomas P , Grillot R
Published: January 2020

API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates. The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively. The API Candida system is easy to use and rapid (result in 18 to 24 h).
Interlaboratory comparison of sequence-specific PCR and ligase detection reaction to detect a human immunodeficiency virus type 1 drug resistance mutation. The AIDS Clinical Trials Group Virology Committee Drug Resistance Working Group
Authors: Shafer R W , Winters M A , Mayers D L , Japour A J , Kuritzkes D R , Weislow O S , White F , Erice A , Sannerud K J , Iversen A , Pena F , Dimitrov D , Frenkel L M , Reichelderfer P S
Published: January 2020

Sequence-specific PCR was used in six laboratories and a ligase detection reaction was used in one laboratory to detect the zidovudine-resistance mutation at codon 215 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase DNA. The genotypes of 27 different clinical samples, including cultured HIV-1 isolates, peripheral blood mononuclear cells, and plasma, were correctly identified by 140 of 154 (91%) assays. The sensitivity for detecting a mutation was 96% for HIV-1 reverse transcriptase DNA clone mixtures containing 30% mutant DNA and 62% for mixtures containing 6% mutant DNA.
Further studies on biochemical characteristics and serologic properties of the genus Aeromonas
Authors: Janda J M , Abbott S L , Khashe S , Kellogg G H , Shimada T
Published: January 2020

We characterized a collection of 268 Aeromonas isolates from diverse sources (clinical, animal, and environmental sources) for their species and serogroup designations. Overall, 97% of these strains could be identified to the genomospecies level by using an expanded battery of biochemical tests. Members of the Aeromonas hydrophila complex (A. hydrophila, HG2, and A. salmonicida), a group that has previously been difficult to separate biochemically, could easily be distinguished from one another by using a number of recently described phenotypic properties which included utilization of DL-lactate and urocanic acid. Differences in species distributions on the basis of the source of isolation were noted. Serogroup analysis of these 268 isolates plus a number of reference cultures indicated that (i) each genomospecies is serologically heterogeneous and individual serogroups can be found in more than one species, (ii) most type or reference strains for each hybridization group are not serologically representative of the genomospecies at large, (iii) serogroups O:11, O:34, and O:16 predominate clinically (48%), supporting previous studies indicating their importance in human infections, and (iv) most A. trota strains do not express the O139 antigen of Vibrio cholerae. The collective results suggest that both species and serogroup designations are important factors in establishing which isolates can cause human infections when they are acquired from nonclinical sources (foods, animals, and the environment).
Amplification of residual DNA sequences in sterile bronchoscopes leading to false-positive PCR results
Authors: Kaul K , Luke S , McGurn C , Snowden N , Monti C , Fry W A
Published: January 2020

PCR has been used successfully for the direct detection of Mycobacterium tuberculosis in uncultured patient samples. Its potential is hindered by the risk of false-positive results as a result of either amplicon carryover of cross-contamination between patient samples. In the present study, we investigated whether residual amplifiable human or M. tuberculosis DNA could remain in sterile bronchoscopes and potentially be a cause of false-positive PCR results in subsequent patient samples. Sterilized bronchoscopes were flushed with sterile saline, and the collected eluate was submitted for PCR amplification of IS6110 sequences and exon 8 of the human p53 gene. Of a total of 55 washes of sterile bronchoscopes from two institutions, 2 (3.6%) contained amplifiable M. tuberculosis DNA and 11 (20%) contained residual human DNA. These findings indicate that residual DNA can remain in sterilized bronchoscopes and can be a source of false-positive PCR results.