bluuebunny/crossref_metadata_2025 huggingface dataset:
Interpretive criteria and quality control parameters for testing of susceptibilities of Haemophilus influenzae and Streptococcus pneumoniae to trimethoprim and trimethoprim-sulfamethoxazole. The Antimicrobial Susceptibility Testing OC Group
Authors: Fuchs P C , Barry A L , Brown S D
Published: January 2020

Two hundred twenty-eight strains of Haemophilus influenzae and 234 strains of Streptococcus pneumoniae were tested by broth microdilution and disk diffusion methods for susceptibility to trimethoprim (TMP) and TMP-sulfamethoxazole (SMX) to evaluate proposed criteria. Data are presented to support the proposed TMP MIC breakpoints of < or = 2.0 micrograms/ml for susceptibility and > or = 4.0 micrograms/ml for resistance for both species and TMP-SMX MIC breakpoints of < or = 2.0-38 micrograms/ml for susceptibility and > or = 4.0-76 micrograms/ml for resistance. Corresponding zone diameter breakpoints for H. influenzae for both drugs are proposed: < or = 10 mm = resistant; > or = 16 mm = susceptible. A 10-laboratory study documented reproducibility of such tests with standard control strains. The following control limits are proposed for tests of H. influenzae ATCC 49247 against TMP; MIC, 0.12 to 0.5 microgram/ml; zone diameter, 27 to 33 mm. The current limits for TMP-SMX were confirmed. For tests of S. pneumoniae ATCC 49619, MICs of TMP were 1.0 to 4.0 micrograms/ml and the current TMP-SMX MIC range was confirmed. Disk susceptibility tests of either drug against pneumococci were not reproducible, and consequently neither quality control limits nor interpretive criteria could be established. Endpoint interpretation and lot-to-lot variability in Mueller-Hinton agars were significant factors leading to interlaboratory variability.
Use of Gen-Probe AccuProbe Group B streptococcus test to detect group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women: comparison with traditional culture method
Authors: Bourbeau P P , Heiter B J , Figdore M
Published: January 2020

Detection of vaginal-anorectal colonization with group B streptococci (GBS) is critical to the prevention of neonatal GBS disease. The recommended method for the detection of GBS is culture of the distal vagina and anorectum in a selective broth medium followed by subculture to solid media and identification of GBS on the solid media. The purpose of this study was to compare this standard culture method with the detection of GBS directly from an enrichment broth by utilizing the Gen-Probe AccuProbe Group B Streptococcus Culture Identification Test (GPGB). A total of 502 specimens were tested in this study. Both culture and the GPGB detected 90 of 95 positive specimens (sensitivity, 94.7%). There were two false-positive GPGB results (specificity, 99.5%). An analysis of 100 consecutive specimens was performed to compare the costs associated with the use of a primary tryptic soy agar plate with 5% sheep blood (BAP) and a 3-ml tube of Todd-Hewitt broth supplemented with 10 micrograms of nalidixic acid per ml and 15 micrograms of colistin per ml (LIM broth) with subculture to another BAP and the costs associated with the GPGB. Our estimated costs were $3.68 for a negative culture including 7.0 min of labor, $5.41 for a positive culture including 8.9 min of labor, and $5.16 for the GPGB including 3.6 min of labor (based upon a test run of 10 specimens and two controls and a cost of $70.00 for a 20-test GPGB kit). Accessioning and reporting of results are not included in these costs. In conclusion, we found that the GPGB was equivalent in sensitivity to our standard culture method. While overall costs were somewhat higher for the GPGB, the GPGB has lower labor costs and offers the potential for incremental savings with higher test volumes and computer interface capability.
Molecular epidemiology of Streptococcus pneumoniae with decreased susceptibility to penicillin in a Paris children's hospital
Authors: Moissenet D , Valcin M , Marchand V , Garabédian E N , Geslin P , Garbarg-Chenon A , Vu-Thien H
Published: January 2020

Among pneumococci with decreased susceptibility or pneumococci resistant to penicillin (PRP) isolated at Armand-Trousseau children's hospital, those expressing capsular serotypes 23F, 9V, and 14 were the most frequently isolated. We compared 53 clinical isolates (14 type 9V, 26 type 23F, and 13 type 14) by analysis of chromosomal macrorestriction patterns and DNA restriction patterns of the penicillin-binding protein (PBP) genes pbp 2b, pbp 2x, and pbp 1a. All 9V isolates originated from the same clone. Five 23F clones were distinguished, the largest of which comprised 20 isolates. The main type 14 clone comprised nine isolates; three other type 14 strains were closely related to the 9V clone, probably by horizontal transfer of capsular biosynthesis genes. Most 23F and type 14 isolates shared the same PBP gene restriction patterns as the 9V clone, suggesting horizontal transfer of altered PBP genes.
Direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test
Authors: Gamboa F , Manterola J M , Viñado B , Matas L , Giménez M , Lonca J , Manzano J R , Rodrigo C , Cardona P J , Padilla E , Domínguez J , Ausina V
Published: January 2020

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) was adapted for the detection of Mycobacterium tuberculosis complex in 224 nonrespiratory specimens from 188 patients. The sensitivity and specificity of the AMTDT for such specimens, after resolution of discrepant results, were 85.7 and 100%, respectively. Pretreatment of nonrespiratory specimens with sodium dodecyl (lauryl) sulfate is mandatory to obtain consistent and reproducible AMTDT results. The use of 500 microliters of decontaminated specimen improves the sensitivity of the test. Because the AMTDT detects stable rRNA from noncultivable bacilli, it is not useful for monitoring patients receiving treatment.
Protease-defective, gp120-containing human immunodeficiency virus type 1 particles induce apoptosis more efficiently than does wild-type virus or recombinant gp120 protein in healthy donor-derived peripheral blood T cells
Authors: Kameoka M , Kimura T , Zheng Y H , Suzuki S , Fujinaga K , Luftig R B , Ikuta K
Published: January 2020

Apoptosis and syncytium formation are two mechanisms by which human immunodeficiency virus type 1 (HIV-1) impairs uninfected CD4+ T-cell function and are mainly involved in the progression of the disease to AIDS. Previously, we showed that gp120-containing, protease-deficient HIV-1 (L-2) particles generated syncytia by particle-mediated fusion with uninfected cultured CD4+ T cells. Here, we present evidence that such L-2 particles can induce apoptosis in 40 to 50% of T cells which were enriched from HIV-1-negative healthy donor-derived peripheral blood mononuclear cells (PBMC-Ts). Activation of PBMC-Ts with phytohemagglutinin, concanavalin A, or ionomycin after incubation with L-2 particles resulted in the loss of proliferative capacity and gradual induction of apoptosis over 3 days. Wild-type strain LAI particles or recombinant gp120 were markedly less efficient (< or = 15%) at inducing such apoptosis. Western blot (immunoblot) analysis revealed that L-2 particles contained a larger amount of Env gp120 than LAI particles. Either preincubation of PBMC-Ts with a Fas antagonist or preincubation of L-2 particles with soluble CD4 blocked most of the apoptosis. This suggests that L-2-like particles can play a major role in HIV-1-induced apoptosis of uninfected bystander cells.
New, ultrasensitive enzyme immunoassay for detecting vaccine- and disease-induced hepatitis A virus-specific immunoglobulin G in saliva
Authors: Ochnio J J , Scheifele D W , Ho M , Mitchell L A
Published: January 2020

Although detection of disease-induced hepatitis A virus (HAV)-specific antibodies in saliva has been successfully utilized in a few epidemiological studies, available assays fail to detect lower salivary anti-HAV levels associated with vaccine-induced immunity. We present a new capture enzyme immunoassay which employs a three-layer antibody recognition system. Evaluation of paired saliva-serum specimens from 1,025 international travellers, 134 other volunteers, and 91 hepatitis A vaccine recipients demonstrated 99.6% (95% confidence interval, 98.4 to 99.9) specificity and 98.7% (95% confidence interval, 97.7 to 99.4) sensitivity of this salivary assay in differentiating between immune and susceptible individuals, compared with serum-based methods. We conclude that this assay is sufficiently sensitive for reliable detection of both vaccine- and infection-induced HAV-specific immunoglobulin G in saliva, even when corresponding anti-HAV levels in serum are very low (< 1 IU/ml).
Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes
Authors: Martin-Casabona N , Xairó Mimó D , González T , Rossello J , Arcalis L
Published: January 2020

The increasing number of multidrug-resistant Mycobacterium tuberculosis strains has stimulated the interest of investigators in finding a rapid method for susceptibility testing. We used commercially available rRNA DNA-bioluminescence-labelled probes (Accu-Probe, Gen Probe, Inc. San Diego, Calif.) for this purpose. The study was performed in three chronological steps. (i) We studied the correlation between the photometric light units (PLUs) given by the hybridization method, the numbers of CFU per milliliter, and turbidity as nephelometric units for six different inocula of an M. tuberculosis strain over 14 days. A good correlation (c > 0.9; P < 0.05) was found from the third day for all concentrations used. (ii) Over a period of 14 days we studied the evolution of the PLUs for 20 strains growing in medium with 0.2 microl of isoniazid (H) per ml and 18 strains in medium with 1 microl of rifampin (R) per ml to standardize the method. Susceptible and resistant strains were used according to the reference proportions method in Middlebrook 7H10, and the MICs were determined in solid and liquid media. The final inoculum of a 10(-2) dilution from a McFarland no. 1 standard and reading at 3 and 5 days provided the best results. A quotient was established to find a cutoff point between resistant and susceptible strains. (iii) We used the standardized parameters in 117 tests with H and R. On day 3, the sensitivity, specificity, positive predictive value, and negative predictive value for detecting resistant strains were 86.8, 100, 100, and 90.1%, respectively, and on day 5 they were 96.2, 100, 100, and 94%, respectively. We concluded that the method is readily available, is easy to perform, and could be useful for screening resistant M. tuberculosis strains.
Limited value of PCR for detection of Toxoplasma gondii in blood from human immunodeficiency virus-infected patients
Authors: Franzen C , Altfeld M , Hegener P , Hartmann P , Arendt G , Jablonowski H , Rockstroh J , Diehl V , Salzberger B , Fätkenheuer G
Published: January 2020

Cerebral toxoplasmosis is a common, opportunistic, and often life-threatening disease in HIV-infected patients. Diagnosis is supported mainly by clinical evidence and computerized tomography or magnetic resonance imaging scans, but brain images may share features with other brain diseases occurring in HIV-infected patients. To determine the diagnostic value of PCR for the detection of Toxoplasma gondii in blood from HIV-infected patients, we examined 89 blood samples from 59 HIV-infected patients. PCR and Southern blot hybridization were done with DNA extracted from blood samples from 20 patients with confirmed cerebral toxoplasmosis and from 10 patients with suspected but not confirmed cerebral toxoplasmosis. The samples were taken before and 7 to 10 days after the beginning of antiparasitic therapy. For 9 patients who suffered from cerebral toxoplasmosis more than 6 months prior to the study and for 20 patients without any evidence for toxoplasmosis only one blood sample per patient was examined. PCR gave positive results with 5 of the 20 blood samples from patients who suffered from cerebral toxoplasmosis. After 7 to 10 days of therapy PCR results became negative in all these five cases. No amplification was seen with DNA from blood samples from the other 54 patients as the target. The results presented here show that PCR testing of blood samples from HIV-infected patients is of limited value for the diagnosis of cerebral toxoplasmosis. The sensitivity was only 25%, but the specificity was very high (100%), so this technique may be useful for discriminating between cerebral toxoplasmosis and other brain diseases which may be mistaken for toxoplasmosis.
Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources
Authors: Teixeira L M , Carvalho M G , Merquior V L , Steigerwalt A G , Brenner D J , Facklam R R
Published: January 2020

This study presents phenotypic and genotypic data for seven isolates of Vagococcus fluvialis, including four strains recovered from human clinical sources, one strain isolated from an environmental source, and two strains isolated from pigs. On the basis of phenotypic characteristics, most isolates were initially classified as "unidentified enterococci," because they resembled atypical arginine-negative enterococcal species. All seven strains as well as the type strain of V. fluvialis reacted with the AccuProbe Enterococcus genetic probe. The seven isolates had virtually indistinguishable whole-cell protein profiles that were similar to that of the V. fluvialis type strain and distinct from those of Enterococcus and Lactococcus species. DNA-DNA reassociation experiments confirmed that the strains were V. fluvialis. They were 71% or more related to the V. fluvialis type strain under optimum and stringent conditions, with 2.5% or less divergence within related sequences. All strains were susceptible to ampicillin, cefotaxime, trimethoprim-sulfamethoxazole, and vancomycin and were resistant to clindamycin, lomefloxacin, and ofloxacin. Strain-to-strain variation was observed in relation to susceptibilities to 18 other antimicrobial agents. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI. Distinctive PFGE patterns were generated, suggesting the nonclonal nature of V. fluvialis strains. Although the number of strains was small, this report provides molecular characterization of V. fluvialis and the first evidence of a possible connection of this species with human infections.
Vaginal carriage of enterotoxigenic Bacteroides fragilis in pregnant women
Authors: Leszczynski P , van Belkum A , Pituch H , Verbrugh H , Meisel-Mikolajczyk F
Published: January 2020

Bacteroides fragilis is an anaerobic bacterial species that is involved in gynecological infections and pathology. The incidence of vaginal carriage is largely unknown, and in order to study this, 120 pregnant women attending a general hospital for delivery were examined. Cultures were positive for eight of these women (6.6%). Interestingly, potential clonal relatedness could be demonstrated among several of the nonenterotoxigenic B. fragilis strains. Among the strains, only one produced metalloprotease enterotoxin. The presence of the gene for the metalloprotease, giving rise to the pathogenic effect on cultured eukaryotic HT29/C1 cells, was confirmed by a newly designed specific PCR assay. The enterotoxigenic B. fragilis (ETBF) strain was analyzed with the help of arbitrarily primed PCR (AP-PCR) and PCR-mediated ribotyping. The ETBF strain was shown to be genetically different compared to several other strains obtained from diverse sources. Our data indicate a relatively high vaginal B. fragilis carriage rate among pregnant women in Warsaw, Poland. Although neither ETBF nor B. fragilis colonization presented a clinical problem, the possible genetic relatedness among the colonizing B. fragilis strains indicates the need for additional research in the field of ETBF transmission and molecular epidemiology.
Isolation and characterization of a coronavirus from elk calves with diarrhea
Authors: Majhdi F , Minocha H C , Kapil S
Published: January 2020

This is the first report of the isolation of a coronavirus from elk calves. Two fecal samples from elk calves with diarrhea were shown to be positive for coronavirus-like particles by electron microscopy, and the particles were propagated in the human rectal tumor-18 cell line. After 24 h, syncytia were observed, and cell culture supernatants from both samples showed hemagglutinating activity with mouse erythrocytes. Cells infected with both elk coronavirus (ECV) isolates reacted with Z3A5, a monoclonal antibody against the spike protein of bovine coronavirus (BCV), on an indirect fluorescent antibody test. The protein profiles of both ECV isolates were similar to that of BCV as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. On Northern blot analysis, the transcriptional pattern of ECV was typical of coronaviruses, with a nested set of transcripts with common 3' end sequences. Based on a published nucleoprotein gene sequence for BCV (Mebus isolate), we arbitrarily designed two primers for amplification by PCR. After cloning, the nucleoprotein was sequenced and a high degree of homology (99%) between the nucleoprotein gene sequences of ECV and BCV was observed. Thus, ECV is closely related genetically and antigenically to BCV and will be a new member of antigenic group 2 of the mammalian coronaviruses, which possess hemagglutinin-esterase protein.
Tilletiopsis minor: a new etiologic agent of human subcutaneous mycosis in an immunocompromised host
Authors: Ramani R , Kahn B T , Chaturvedi V
Published: January 2020

We describe herein the isolation of Tilletiopsis minor from a subcutaneous cyst of a 70-year-old immunocompromised male. The diagnosis was based on repeated isolation of the fungus, observation of hyphal elements in tissue sections, the ability of the mold to grow at or near body temperature, and the achievement of a complete cure following surgery and antifungal therapy.
Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture
Authors: Li C , Ha T , Chi D S , Ferguson D A , Jiang C , Laffan J J , Thomas E
Published: January 2020

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.
Outbreak of Shigella sonnei in a clinical microbiology laboratory
Authors: Mermel L A , Josephson S L , Dempsey J , Parenteau S , Perry C , Magill N
Published: January 2020

Laboratory technologists (22%) developed infections with Shigella sonnei. The isolates had the same antibiogram and pulse-field gel electrophoresis pattern as an unknown isolate handled by a laboratory student. Covering faucet handles with paper towels during hand washing in the laboratory was protective. No further cases occurred after the laboratory was cleaned with a phenolic agent and a handle-free faucet was installed.
Use of the BioMerieux ID 32C yeast identification system for identification of aerobic actinomycetes of medical importance
Authors: Muir D B , Pritchard R C
Published: January 2020

The BioMerieux ID 32C Yeast Identification System was examined to determine its usefulness as a rapid method for the identification of medically important aerobic actinomycetes. More than 290 strains were tested by this method and the results were compared to those obtained by conventional methods. It was found that aerobic actinomycetes could be differentiated to species level in 7 days by the ID 32C system.
Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens
Authors: Gómez B L , Figueroa J I , Hamilton A J , Ortiz B , Robledo M A , Hay R J , Restrepo A
Published: January 2020

The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation. These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses.
Surveillance of cytomegalovirus after solid-organ transplantation: comparison of pp65 antigenemia assay with a quantitative DNA hybridization assay
Authors: Bossart W , Bienz K , Wunderli W
Published: January 2020

In a multicenter study, 113 blood samples from 19 organ transplant patients were analyzed for cytomegalovirus by the pp65 antigenemia assay and a quantitative DNA hybridization assay. Overall, there was 84% agreement among the results obtained by the two tests. Fifteen of 16 episodes of active infection were detected by both assays. One episode was missed by the pp65 assay, and one patient showed significant DNA-emia but only low-level antigenemia.
Comparison of the InPouch TV culture system and Diamond's modified medium for detection of Trichomonas vaginalis
Authors: Levi M H , Torres J , Piña C , Klein R S
Published: January 2020

This study compared the use of Diamond's modified medium to InPouch for the culture of Trichomonas vaginalis from pooled vaginal secretions. The sensitivity for InPouch was 82.4% (61/74) versus 87.8% (65/74) for Diamond's modified medium. There were no significant differences in the sensitivity and negative predictive value of InPouch compared to Diamond's modified medium.
Yersinia enterocolitica serotype O:8 septicemia in an otherwise healthy adult: analysis of chromosome DNA pattern by pulsed-field gel electrophoresis
Authors: Hosaka S , Uchiyama M , Ishikawa M , Akahoshi T , Kondo H , Shimauchi C , Sasahara T , Inoue M
Published: January 2020

We report the first case of blood culture-positive Yersinia enterocolitica serotype O:8 septicemia in Japan. Y. enterocolitica serotype O:8 infection is very rare, but chromosomal DNA analysis suggested that this bacterium may persist latently in healthy carriers throughout Japan.
Rapid susceptibility testing of Candida albicans by flow cytometry
Authors: Kirk S M , Callister S M , Lim L C , Schell R F
Published: January 2020

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.
Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions
Authors: Latouche S , Ortona E , Mazars E , Margutti P , Tamburrini E , Siracusano A , Guyot K , Nigou M , Roux P
Published: January 2020

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.
Contrast of survey results between state and a cohort of nonstate mycobacteriology laboratories: changes in laboratory practices
Authors: Denniston M M , Bird B R , Kelley K A
Published: January 2020

Based on the recommendations of a 1992 conference on tuberculosis, the Centers for Disease Control and Prevention (CDC) established programs for upgrading mycobacteriology laboratories by providing them with monies and focused training. In 1991, state public health laboratories were surveyed to determine the methods they were using for primary Mycobacterium tuberculosis testing and their turnaround times for reporting testing results. A similar survey of nonstate laboratories participating in the National Laboratory Training Network-sponsored, M. tuberculosis-focused training programs was conducted from May 1992 to June 1993. In 1994, follow-up surveys of both the state- and nonstate-laboratory cohorts were conducted with the questionnaire from the initial survey plus additional questions that asked about interventions and changes occurring in the laboratory since the original survey. Although both cohorts showed increases in the percentages of laboratories meeting the recommended turnaround times for reporting M. tuberculosis testing results and using the recommended rapid methods for testing, generally, the increases made by the state laboratories were greater. By June 1994, all state laboratories were using a rapid method for M. tuberculosis isolate identification compared with 88% of the nonstate laboratories. The percentage of laboratories identifying isolates within the recommended 21 days also increased more in the group of state laboratories than in the group of nonstate laboratories (state laboratories, 22 to 73%; nonstate laboratories, 55 to 59%). Responses from the follow-up survey showed large differences in the percentages of laboratories that received CDC funding (state laboratories, 100%; nonstate laboratories, 6%) and participated in M. tuberculosis training (state laboratories, 98%; nonstate laboratories, 45%). These results indicate that adequate funding and focused training are critical in maintaining state-of-the-art mycobacteriology laboratories.
Response of enteric gram-negative bacteria to disks containing 20 micrograms each of ampicillin and sulbactam
Authors: Isenberg H D , Alperstein P , France K
Published: January 2020

Ampicillin-sulbactam disks containing either 10 microg of each drug or 20 microg of each drug were tested against 138 recently, sequentially isolated members of the family Enterobacteriaceae. Results obtained with the higher-content disks corresponded more closely to the impressions of clinicians.
Detection and identification of two Bartonella henselae variants in domestic cats in Germany
Authors: Sander A , Bühler C , Pelz K , von Cramm E , Bredt W
Published: January 2020

To determine the prevalence of bacteremia caused by Bartonella henselae in domestic cats in the region of Freiburg, Germany, we investigated culture of blood from 100 cats from 89 different households over a 12-month period. B. henselae could be isolated from 13% (13 of 100) of these cats. In eight households with two cats each and in one household with three cats, B. henselae bacteremia was found either in all of the animals or in none of the animals. Positive cultures were more likely to be found for female, young (24 months of age or younger) cats than for male or older cats. Identification of the Bartonella isolates was made by colony morphology, by Gram staining, biochemically by RapID ANA II or Rapid ID 32 A systems, and by whole-cell fatty acid analysis. Differentiation between B. henselae and Bartonella quintana was only possible by 16S rRNA sequencing, enterobacterial repetitive intergenic consensus (ERIC)-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Genomic fingerprinting of the B. henselae isolates by ERIC-PCR yielded two different patterns based on three distinct bands.
Large-restriction-fragment analysis of Mycobacterium kansasii genomic DNA and its application in molecular typing
Authors: Iinuma Y , Ichiyama S , Hasegawa Y , Shimokata K , Kawahara S , Matsushima T
Published: January 2020

Large-restriction-fragment (LRF) polymorphisms in Mycobacterium kansasii isolates from 84 patients with bronchopulmonary infections in Japan between the 1960s and 1995 were studied by pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with VspI were most suitable for PFGE separation of 16 to 21 fragments of between 40 and 550 kbp. All 84 isolates and the type strain M. kansasii ATCC 12478 were successfully typed by LRF analysis with VspI digestion. Twenty-one distinctive LRF types were identified, and the LRF patterns tested over time were reproducible and stable. A computer-assisted dendrogram of the percent similarity demonstrated that isolates of 18 LRF types had relatively close genetic relatedness, while isolates of the remaining 3 types showed divergence. Sequence analysis of the 16S rRNA gene in the isolates showing divergent genetic relatedness revealed a sequence identical to that of a previously reported subspecies of M. kansasii. In the Chugoku district of Japan, 11 cases of M. kansasii infection which occurred in workers in a coastal industrial zone between 1982 and 1993 were caused by one particular strain tentatively named LRF type M. When both detailed demographic data for the patients and ecologic data for the M. kansasii isolates are obtained, LRF typing may be of potential use for investigating the source and transmission of M. kansasii infection.
Comparison of DNA fingerprint patterns of isolates of Mycobacterium africanum from east and west Africa
Authors: Haas W H , Bretzel G , Amthor B , Schilke K , Krommes G , Rüsch-Gerdes S , Sticht-Groh V , Bremer H J
Published: January 2020

Mycobacterium africanum is a pathogen found in tuberculosis patients in certain parts of Africa and is a member of the Mycobacterium tuberculosis complex. Biochemically, strains of M. africanum exhibit a high degree of variability, with some tendency to cluster according to their geographical origin. To investigate whether this phenotypic variability is reflected at the genetic level, we performed DNA fingerprint analysis of strains isolated from patients with pulmonary tuberculosis in Uganda and Sierra Leone. IS6110 DNA fingerprinting was carried out by the mixed-linker PCR method. A total of 138 strains of M. africanum were analyzed: 42 isolates from Uganda and 96 isolates from Sierra Leone. With few exceptions, the resulting DNA fingerprint patterns grouped together according to their country of origin. A striking lack of variability of DNA fingerprints was found for strains from Sierra Leone, where 70 of 96 isolates (61.5%) fell into clusters. The two largest clusters accounted for 41.7% of all isolates and differed by only one band, as confirmed by standard DNA fingerprinting. In contrast, only two clusters (7.1%) with two and three isolates, respectively, were found for M. africanum isolates collected in Uganda, and three of the DNA fingerprints contained fewer than seven bands. Strains of M. tuberculosis collected and processed during the same time period were highly variable in both countries. Our results support the concept of geographically defined subtypes of M. africanum. In addition, they demonstrate that natural geographic differences in the variability of IS6110 DNA fingerprints within the M. tuberculosis complex must be considered if this technique is used for epidemiologic studies.
Genetic relatedness and diversity of Cryptococcus neoformans strains in the Maltese Islands
Authors: Lo Passo C , Pernice I , Gallo M , Barbara C , Luck F T , Criseo G , Pernice A
Published: January 2020

The genetic relatedness of clinical and environmental Cryptococcus neoformans strains in the Maltese Islands was investigated by randomly amplified polymorphic DNA fingerprinting with four primers. The clinical strains isolate over the course of 1 year from AIDS patients showed identical fingerprints. The electrophoretic patterns of the two clinical strains were also the most common patterns among the environmental strains, but the patterns among the environmental strains showed a wide variability and no correlation with the site of isolation.
A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings
Authors: Jacobs M V , Snijders P J , van den Brule A J , Helmerhorst T J , Meijer C J , Walboomers J M
Published: January 2020

Two cocktails of digoxigenin-labeled human papillomavirus (HPV) type-specific oligonucleotide probes and an enzyme immunoassay (EIA) were used as a basis to developed a group-specific detection method for 14 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk (types 6, 11, 40, 42, 43, and 44) HPVs, following a general primer GP5+/bioGP6(+)-mediated PCR. The sensitivity of this high-risk/low-risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type. Comparison of HR/LR HPV PCR-EIA with radioactive Southern blot hybridization using a general probe on the same PCR products derived from 417 cytomorphologically abnormal cervical scrapings resulted in an overall agreement of 96% between the two methods. Complete concordance between group-specific HR/LR detection and individual typing results for both single and multiple infections indicate the strong specificity of this HR/LR HPV PCR-EIA. Multiple infections could be predicted by comparing PCR-EIA optical density values of the cocktail probes with one of the individual oligonucleotide probes. This novel HR/LR PCR-EIA allows accurate and rapid identification of high-risk and low-risk HPV types in cervical scrapings and will facilitate HPV detection in HPV mass-screening programs.
Impact of the human immunodeficiency virus epidemic on detection of Mycobacterium isolates in a general hospital
Authors: Bouza E , Albadalejo J , Cercenado E , Ruiz Serrano M J , Vicente T , Ortega A
Published: January 2020

The number of human samples processed in our mycobacteriology laboratory ranged from 148 per 1,000 admissions in 1988 to 263 per 1,000 admissions in 1995 (5.2% were positive). The human immunodeficiency virus (HIV)-positive population contributed 33.3% of all samples, 43.3% of all isolates, and 36% of all new patients. Given that the ratios of the total number of samples to the number of Mycobacterium-positive samples were 18.3:1 in HIV-positive patients and 28:1 in HIV-negative patients, efforts to reduce the laboratory workload should begin with the HIV-negative population.
Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis
Authors: Walls J J , Greig B , Neitzel D F , Dumler J S
Published: January 2020

The natural reservoirs for the agent of human granulocytic ehrlichiosis (HGE) are suspected to be the small mammals that host immature stages of Ixodes scapularis ticks. To determine if such small mammals are naturally infected, we collected blood and serum samples from small mammal species in rural and suburban areas of Minneapolis and St. Paul, Minn. Samples were collected from white-footed mice (Peromyscus leucopus), eastern chipmunks (Tamias striatus), southern red-backed voles (Clethrionomys gapperi), and insectivorous shrews (Blarina brevicauda and Sorex cinereus). Blood samples were tested by PCR for active infection with the HGE agent, and sera from P. leucopus mice were tested for serologic evidence of infection by indirect immunofluorescence. PCR analyses revealed the presence of HGE agent DNA in 20 of the 190 samples (10.5%) tested. Of the 119 P. leucopus mouse serum samples that were analyzed, 12 (10.1%) contained Ehrlichia equi antibodies. In 3 of 119 (2.5%) P. leucopus mice from which both blood and serum were collected. HGE agent DNA and antibodies against E. equi were present. Animals with evidence of infection with the HGE agent are widely distributed around the Minneapolis-St. Paul area in regions with known I. scapularis tick activity. Small mammals that are frequent hosts for larval I. scapularis ticks and that are found in areas where HGE occurs are likely to be a major reservoir from which infected ticks that bite humans are derived.
Comparison of four molecular typing methods for evaluating genetic diversity among Candida albicans isolates from human immunodeficiency virus-positive patients with oral candidiasis
Authors: Díaz-Guerra T M , Martínez-Suárez J V , Laguna F , Rodríguez-Tudela J L
Published: January 2020

Candida albicans strain delineation by karyotyping. NotI restriction pattern analysis, hybridization with specific probe 27A, and PCR fingerprinting with the phage M13 core sequence were performed with 30 isolates from the oral cavities of 30 human immunodeficiency virus (HIV)-infected patients and 8 reference strains. Within the panel of clinical isolates, 20 were geographically related, although 10 isolates were susceptible to fluconazole and 10 isolates were resistant to fluconazole. The remaining isolates used in this study were fluconazole resistant and geographically unrelated. A composite DNA type was defined for each of the strains as the combination of types obtained by the four molecular methods. By this procedure, a great diversity of DNA types was found among isolates from the oropharynges of HIV-infected individuals with oral candidiasis. This diversity was not reduced when isolates were evaluated on the basis of whether they came from the same geographical locale and whether they were fluconazole resistant. These data refute the idea of a clonal origin for fluconazole-resistant strains among HIV-positive patients. Karyotyping was the least discriminatory method, yielding 19 DNA types among the 38 strains analyzed. Conversely, hybridization with the 27A probe showed a unique DNA pattern for each of the strains examined in this study. Our results demonstrate that at least two different molecular methods are needed for Candida albicans typing and that there is a great deal of strain variation within the species, irrespective of place of origin or antifungal resistance patterns.
Evaluation of latex reagents for rapid identification of Candida albicans and C. krusei colonies
Authors: Freydiere A M , Buchaille L , Guinet R , Gille Y
Published: January 2020

A total of 322 yeast strains and yeastlike organisms belonging to the genera Candida, Cryptococcus, Geotrichum, Saccharomyces, and Trichosporon were tested with the new monoclonal antibody-based Bichro-latex albicans and Krusei color latex tests. Comparison of results with those obtained by conventional identification methods showed 100% sensitivity for both latex tests and 100% and 95% specificity for the Bichro-latex albicans and Krusei color tests, respectively. Because the test is easy to read and quick to perform, the Bichro-latex albicans test may be useful for rapid identification of Candida albicans colonies in the clinical laboratory.
Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay
Authors: Smith M B , Bergmann J S , Woods G L
Published: January 2020

To evaluate the ability of the Amplicor MTB Assay to detect Mycobacterium tuberculosis complex (MTBC) organisms in BACTEC 12B broth cultures, 249 cultures with a growth index (GI) of > or = 20 from 160 patients were tested retrospectively. Specimens were processed by standard methods, and then BACTEC 12B vials and Middlebrook 7H11/7H115 plates were inoculated, incubated, and interpreted in accordance with the manufacturer's instructions and laboratory protocol. From 12B vials with a GI of > or = 20, and aliquot of broth was removed and frozen at -20 degrees C until assayed by PCR. PCR results were compared to those obtained by the usual laboratory protocol, whereby MTBC organisms were identified by a DNA probe assay performed on broth from 12B vials with a GI or > or = 300 or on colonies from solid medium. Of the 249 broth cultures evaluated, 142 contained mycobacteria, including 44 that contained MTBC organisms. Of these 44 cultures, 41 were PCR positive; the 3 that were PCR negative were blood specimens collected in an Isolator tube. All 98 cultures with nontuberculous mycobacteria and the 107 that did not contain mycobacteria were PCR negative. Thus, the sensitivity and specificity of PCR were 93 and 100%, respectively. For those culture sin which MTBC organisms were identified by both the DNA probe and PCR assays, the mean time from specimen inoculation to detection and identification of MTBC organisms was 16 (range, 4 to 26) days for the PCR and 28 (range, 13 to 43) days for the DNA probe assay (P < 0.0001). In summary, PCR is a rapid, reliable method for detection of MTBC organisms in BACTEC 12B broth cultures with a GI of > or = 20.
The effect of Tn916 insertions on contour-clamped homogeneous electrophoresis patterns of Enterococcus faecalis
Authors: Thal L A , Silverman J , Donabedian S , Zervos M J
Published: January 2020

Contour-clamped homogeneous gel electrophoresis has increasingly been used and has generally been considered the method of choice for the delineation of enterococcial strains. It has been suggested that the contour-clamped homogeneous electric field (CHEF) electrophoresis digestion patterns of genomic DNA indicate the relatedness of strains when SmaI patterns differ by six or fewer bands. To evaluate the potential reasons for the diversity of bands among clonally related isolates, we studied plasmid-free Enterococcus faecalis FA2-2 and derivatives with transposon Tn916 (encoding for tetracycline resistance) insertions on the chromosome. We obtained derivatives with seven different Tn916 insertion sites and up to two copies of Tn916 on the chromosome, resulting in differences of one to seven bands by CHEF electrophoresis; eight different patterns were observed. With Tn916 insertions, there was either (i) loss of a band(s) with the generation of a new band(s), (ii) the generation of a new band(s) only, or (iii) the loss of a band(s) with no new visible band(s). These results indicate that strains can have up to seven different SmaI bands by CHEF electrophoresis and still be closely related.
Detection of hepatitis G virus (GB virus C) RNA in human saliva
Authors: Chen M , Sönnerborg A , Johansson B , Sällberg M
Published: January 2020

Using PCR and genomic sequencing, we confirmed the presence of and homology between hepatitis G virus (HGV) (also called GB virus C) RNA in six serum samples and that in two saliva samples obtained from 34 patients with chronic hepatitis C virus infections. Thus, HGV may be found outside the circulatory system.
Dissemination of a single clone of methicillin-resistant Staphylococcus aureus among Turkish hospitals
Authors: van Belkum A , van Leeuwen W , Verkooyen R , Saçilik S C , Cokmus C , Verbrugh H
Published: January 2020

A collection of 39 methicillin-resistant Staphylococcus aureus (MRSA) stains derived from six different hospitals in Ankara and one hospital in Barsa, Turkey, were analyzed by multiple genotyping. In agreement with the other genotyping assays, pulsed-field gel electrophoresis of DNA macrorestriction fragments identified genetic homogeneity among all MRSA isolates studies. It is concluded that a major clone of MRSA has spread through a large part of Turkey, causing longitudinally persistent colonization in all of the institutions surveyed.
False-positive results from cultures of Mycobacterium tuberculosis due to laboratory cross-contamination confirmed by restriction fragment length polymorphism
Authors: Bauer J , Thomsen V O , Poulsen S , Andersen A B
Published: January 2020

During 1994, a cross-contamination problem leading to false-positive cultures of Mycobacterium tuberculosis was revealed in a mycobacteriology laboratory processing 30,000 to 35,000 samples per year. Molecular strain typing based on restriction fragment length polymorphism confirmed the contaminations. Out of 1,439 positive cultures of Mycobacterium tuberculosis, 49 samples from 48 patients were suspected to be cross-contaminated. In 37 cases, growth was observed both in BACTEC vials and on solid media, indicating that the contamination took place during the processing of the samples. The majority of the contaminated samples had been handled by the same technician.