bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region
Authors: Dobner P , Feldmann K , Rifai M , Löscher T , Rinder H
Published: January 2020

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.
Rapid Immunoassay for detection of Escherichia coli O157 directly from stool specimens
Authors: Park C H , Vandel N M , Hixon D L
Published: January 2020

A new and rapid ( < 1 h) enzyme-linked immunosorbent assay (ELISA) was compared with conventional sorbitol-MacConkey agar (SMAC) culture for the detection of Escherichia coli O157 from stool specimens. Among 34 positive specimens, confirmed by colony-sweeping and immunofluorescence stain methods, 6 did not exhibit visible sorbitol-negative colonies on SMAC. These six specimens would have been considered to be negative if SMAC alone had been used. The ELISA detected 31 of the 34 positive samples, including 5 of the above-mentioned 6 false-negative samples, resulting in a sensitivity and specificity of 91.2 and 99.5%, respectively. Cross-reactivity with other enteric pathogens was not noted by ELISA. The SMAC method had a sensitivity and specificity of 82.4 and 100%, respectively. The ELISA-negative specimens do not require culture confirmation, whereas positive results must be considered to be presumptive until confirmed by culture. The test is accurate and is easy to perform, making it a very efficient method for screening stool specimens for E. coli O157.
Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting
Authors: Boerlin P , Boerlin-Petzold F , Goudet J , Durussel C , Pagani J L , Chave J P , Bille J
Published: January 2020

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.
Misidentification of toxigenic Corynebacterium diphtheriae as a Corynebacterium species with low virulence in a child with endocarditis
Authors: Pennie R A , Malik A S , Wilcox L
Published: January 2020

A 6-year-old boy presented to a university hospital in Malaysia with infective endocarditis complicating cyanotic congenital heart disease. Blood cultures showed a gram-positive, aerobic, coryneform-like bacillus identified by the hospital laboratory as Corynebacterium xerosis, but a reference laboratory identified the organism as a toxigenic strain of Corynebacterium diphtheriae. The two laboratories concurred on all biochemical test results except for sucrose fermentation.
Veillonella infections in children
Authors: Brook I
Published: January 2020

From 1974 to 1994, 2,033 specimens from children were submitted for cultures for anaerobic bacteria. Eighty-three Veillonella spp. were recovered from 83 children (4%). Most Veillonella species were recovered from abscesses, aspiration pneumonias, burns, bites, and sinuses. The infections were polymicrobial in 79 (95%) patients, but in 4 (5%) patients, Veillonella species were recovered in pure culture. The predisposing conditions associated with the recovery of these organisms were previous surgery, malignancy, steroid therapy, foreign body, and immunodeficiency. These data illustrate that Veillonella spp. are found infrequently in children, mostly in association with mixed infections, and are recovered mixed with mouth and bowel flora.
Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli
Authors: Sowers E G , Wells J G , Strockbine N A
Published: January 2020

Agglutination reactions obtained with three commercial latex reagents for detecting Escherichia coli O157 antigen (Oxoid Diagnostic Reagents, Hampshire, England; Pro-Lab Inc., Richmond Hill, Ontario, Canada; and Remel Microbiology Products, Lenexa, Kans.) and one for detecting H7 antigen (Remel) were compared with those obtained by standard serologic methods by using the Centers for Disease Control and Prevention (CDC) reference antisera for O157 and H7 antigens. For 159 strains of E. coli and related organisms, the Oxoid, Pro-Lab, and Remel O157 latex reagents each had a sensitivity and specificity of 100% compared with the CDC reference antiserum. For 106 strains of E. coli and related organisms that were not enhanced for motility through semisolid medium, the Remel H7 latex reagent had a sensitivity of 96% and a specificity of 100% compared with the standard tube agglutination method with CDC H7 antiserum. Measures to enhance motility were needed for some strains to detect the H7 antigen. Our findings demonstrate that the commercial latex reagents are good alternatives to standard serologic methods for identifying the O157 and H7 antigens of E. coli.
Direct identification of Mycobacterium species in Bactec 7H12B medium by gas-liquid chromatography
Authors: Chou S , Chedore P , Haddad A , Paul N R , Kasatiya S
Published: January 2020

Twenty-nine Mycobacterium reference strains representing 10 species and 60 mycobacterial cultures isolated from sputum specimens were studied. These cultures were grown in Bactec 7H12B medium (Becton Dickinson and Co., Paramus, N.J.) supplemented with oleic acid-albumin-dextrose-catalase enrichment broth (Becton Dickinson and Co., Cockeysville, Md.). The cultures were analyzed by gas-liquid chromatography for their fatty acids, secondary alcohols, and mycolic acid cleavage products. All of the clinical isolates could be identified by comparing their gas-liquid chromatography profiles with those of the reference strains. The data indicate that this method significantly shortens the turnaround time and could be used for the early detection and identification of mycobacterial species.
Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease
Authors: Hilton E , Devoti J , Sood S
Published: January 2020

In October 1994, the Second National Conference on the Serologic Diagnosis of Lyme Disease recommended a two-step approach to serological testing. The first step was the performance of an enzyme-linked immunosorbent assay (ELISA); the second step was a confirmatory immunoblot. New criteria for the interpretation of a positive immunoblot were also recommended. The committee decided to omit the 31- and 34-kDa bands (OspA and OspB, respectively) from the choice of bands considered diagnostic for a positive immunoblot. Since we had previously included these in our diagnostic criteria for Lyme disease-positive immunoblots, we reviewed data for all patients attending a Lyme disease center with positive ELISAs and immunoblot assays for Lyme disease from 1 September 1992 to 31 December 1993. The criteria for a positive Western blot (immunoblot) were the presence of 5 or 12 bands, including the 10 recommended by the conference, and the presence of the 31- and 34-kDa protein bands. Of the 136 patients evaluated, 50 were considered to have Lyme disease. Of these 50, 4 (8%) would not have met immunoblot criteria for the diagnosis if the new recommendations were used. Had the 31- and 34-kDa bands been included as part of the diagnostic requirements for immunoblot, these patients would have been included. Although overdiagnosis of Lyme disease appears to be the more frequent problem, our concern is that the exclusion of the 31- and 34-kDa protein bands from the diagnostic criteria may result in the underdiagnosis of Lyme disease by those who would rely too heavily on serological confirmation. The addition of the 31- and 34-kDa bands to those recommended for confirmatory immunoblot should be reconsidered.
Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization
Authors: Buck G E
Published: January 2020

The use of rapid-cycle PCR combined with colorimetric microwell hybridization for detecting Bordetella pertussis was investigated. Rapid cycling was performed with an air thermocycler (model 1605; Idaho Technology, Idaho Falls, Idaho). Although the instrument was originally designed to be used with capillary tubes, an adapter that allows this instrument to be used with PCR tubes has recently been introduced. Because of the low heat capacity of air, the thermocycler has rapid transition rates between temperatures. The combination of a rapid temperature transition rate, small sample volume (10 microliters), and overshooting or undershooting of the temperature set points allowed the cycles to be reduced to 5 s for denaturation and 10 s for extension and annealing. Thus, the amplification could be completed in a total of approximately 35 min. Amplified DNA was detected with biotin-labeled primers and by hybridization to a capture probe immobilized in microwell plates. When simulated clinical specimens consisting of pooled nasopharyngeal washes with known numbers of B. pertussis organisms were examined by this procedure, as little as one organism per 5 microliters of sample could be detected. Six nasopharyngeal aspirates or washes from culture-positive patients were positive by PCR, as were two of seven specimens obtained from patients that were negative by culture and direct fluorescent-antibody assay. The two patients who were PCR positive but culture and direct fluorescent-antibody assay negative had clinical disease compatible with pertussis. This method appears to be a sensitive, convenient means of detecting B. pertussis in clinical specimens. The total time required for specimen processing, amplification, and detection is about 2.5 h.
Molecular subtyping of Neisseria meningitidis serogroup B: comparison of five methods
Authors: Swaminathan B , Matar G M , Reeves M W , Graves L M , Ajello G , Bibb W F , Helsel L O , Morales M , Dronavalli H , el-Swify M , DeWitt W , Hunter S B
Published: January 2020

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.
Molecular epidemiology of Enterobacter aerogenes acquisition: one-year prospective study in two intensive care units
Authors: Davin-Regli A , Monnet D , Saux P , Bosi C , Charrel R , Barthelemy A , Bollet C
Published: January 2020

To evaluate the respective contributions of patient-to-patient transmission and endogenous acquisition of Enterobacter aerogenes isolates, we conducted a prospective epidemiologic study in two intensive care units (ICUs) between May 1994 and April 1995. We collected a total of 185 E. aerogenes isolates: 130 from 51 patients in a surgical ICU (SICU), 45 from 26 patients in a medical ICU (MICU), and 10 from the environments in these two ICUs. All isolates were typed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus PCR. Among the 175 clinical isolate, we observed 40 different profiles by random amplification of polymorphic DNA and 36 different profiles by enterobacterial repetitive intergenic consensus PCR. We identified a ubiquitous and prevalent clone, corresponding to 58% of SICU and 41% of MICU clinical isolates. Three epidemiologically related strains were specific to each ICU and represented 17% of SICU and 24% of MICU clinical isolates; unique type strains represented 17 and 29% of SICU and MICU clinical isolates, respectively, and E. aerogenes strains which were spread to a limited degree and which were isolated less than five times during the 1-year study period represented 8 and 6% of SICU and MICU clinical isolates, respectively. Our results show that E. aerogenes is acquired in the ICU in three different ways: patient-to-patient spread of a prevalent or an epidemiologically related strain, acquisition de novo of a strain from patients' own flora, and acquisition of a nonendemic strain followed by occasional patient-to-patient transmission. The findings point out the importance of patient-to-patient transmission in E. aerogenes acquisition and suggest that changes in E. aerogenes ecology in the hospital have taken place during the past decade.
Comparison of cell culture, mouse inoculation, and PCR for detection of Toxoplasma gondii: effects of storage conditions on sensitivity
Authors: James G S , Sintchenko V G , Dickeson D J , Gilbert G L
Published: January 2020

The sensitivity of detection of a wild-type strain of Toxoplasma gondii by cell culture, mouse inoculation, and PCR was determined following sample storage under conditions to which clinical specimens may be subjected during transport to the testing laboratory. Sample storage at -20 degrees C significantly decreased the sensitivity of mouse inoculation. The sensitivity of cell culture decreased with sample storage at 4 and -20 degrees C. The sensitivity of PCR was reduced by storage at 4 degrees C for 48 h, freezing, and heating. These findings have implications for the selection of appropriate methods for the direct detection of T. gondii organisms in suboptimally transported clinical samples.
Identification of extrapulmonary Pneumocystis carinii in immunocompromised rats by PCR
Authors: Chary-Reddy S , Graves D C
Published: January 2020

Pneumocystis carinii has been shown to cause extra-alveolar infections in humans, but the lack of a reproducible animal model has hindered the elucidation of mechanisms of P. carinii dissemination. In the present study, PCR and the immunosuppressed rat model were used to gain further insight into the dissemination of P. carinii organisms in extrapulmonary (EP) tissues. Primer sequences specific to major surface glycoprotein (MSG) and dihydrofolate reductase (DHFR) were used to detect P. carinii in lungs and EP tissues. Sprague-Dawley rats were grouped into two classes: one group included rats that had primary episodes of pneumocystosis and the other group included rats that had undergone treatment for P. carinii infection and that had second episodes of pneumocystosis. PCR analysis with MSG primers with tissues obtained from both groups of rats showed the presence of P. carinii DNA in adrenal tissue, bone marrow, blood, and heart, kidney, liver, lymph node, spleen, and thyroid tissues. Reverse transcriptase-PCR (RT-PCR) analysis was carried out with DHFR primers with lung, spleen, heart, kidney, and liver tissues from both groups of rats. Only those tissues that showed a positive PCR result and hybridization signal for the MSG gene were used for the RT-PCR experiments. RT-PCR analysis showed that the P. carinii DHFR gene is actively transcribed in these tissues, thereby indicating the presence of viable P. carinii organisms in EP tissues. Our observations suggest that P. carinii dissemination is influenced by factors other than P. carinii chemotherapy and that heavy organism load and destruction of lung tissue may contribute to the dissemination of P. carinii. The study provides an animal model that can be used for further investigations of the causes of EP pneumocystosis.
Bartonella henselae infection in cats: evaluation during primary infection, treatment, and rechallenge infection
Authors: Greene C E , McDermott M , Jameson P H , Atkins C L , Marks A M
Published: January 2020

Bartonella henselae infection was established in eight cats of various ages by experimental inoculation. All cats remained persistently bacteremic until they were treated 4 to 7 weeks after primary inoculation. Antibody titers increased and peaked between 4 and 12 weeks for all cats. Treatment with doxycycline for 1 week was effective in suppressing bacteremia in all cats but was effective in clearing infection from only four cats. Amoxicillin, given subsequently, was effective in clearing the infection from three of the remaining cats. One kitten that remained bacteremic was treated unsuccessfully with enrofloxacin, and its bacteremia was finally cleared when it was treated with a clavulanate-amoxicillin combination. After the bacteremia was cleared, with a corresponding reduction in serum antibody titers, all eight cats were rechallenged with B. henselae. None of the cats became bacteremic after secondary challenge, and all had higher and more rapid increases in serum antibody titers than after primary inoculation. The cats became resistant to reinfection following recovery from infection, indicating that immunoprophylaxis in cats might be beneficial in helping to reduce their public health risk.
Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains
Authors: Patel S , Wall S , Saunders N A
Published: January 2020

A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes.
Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates
Authors: Pfaller M A , Messer S A , Bolmström A , Odds F C , Rex J H
Published: January 2020

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.
Rapid diagnosis of poliovirus infection by PCR amplification
Authors: Chezzi C
Published: January 2020

A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections.
Simple method for detecting fluconazole-resistant yeasts with chromogenic agar
Authors: Patterson T F , Revankar S G , Kirkpatrick W R , Dib O , Fothergill A W , Redding S W , Sutton D A , Rinaldi M G
Published: January 2020

A method for detecting fluconazole-resistant yeasts was developed that uses chromogenic agar containing fluconazole. Yeasts were plated on media with fluconazole at 0, 8, and 16 micrograms/ml. On media without fluconazole, normal growth of susceptible yeasts (defined as those having a fluconazole MIC of < 8 micrograms/ml) was detected, while fluconazole-containing media suppressed susceptible strains and normal colonies of resistant yeasts (fluconazole MICs of > or = 8 micrograms/ml) were detected. This method was used to screen for resistance in oropharyngeal candidiasis. Isolates having fluconazole MICs of > or = 8 micrograms/ml and < 8 micrograms/ml were correctly predicted in 43 of 45 cultures and 115 of 116 cultures, respectively. This screening method appears to be rapid and sensitive for detection of fluconazole-resistant yeasts.
Automated DNA fingerprinting analysis of Mycobacterium tuberculosis using fluorescent detection of PCR products
Authors: Butler W R , Haas W H , Crawford J T
Published: January 2020

DNA fingerprints of Mycobacterium tuberculosis are produced by restriction fragment length polymorphism analysis of the insertion element IS6110. We modified a PCR-based subtyping method, mixed-linker PCR with fluorescent-labeled IS6110-specific oligonucleotides, to demonstrate rapid, automated, and unattended electrophoretic analysis. Variation in band sizing (normally occurring with fragment mobility), an artifact of lane-to-lane and gel-to-gel differences, was controlled with an internal lane standard, resulting in accurate and precise DNA sizing. By using this method, fingerprint analysis can be performed using actual fragment length rather than estimated position analysis.
A single strain of Candida albicans associated with separate episodes of fungemia and meningitis
Authors: Porter S D , Noble M A , Rennie R
Published: January 2020

Four isolates of Candida albicans recovered from the blood and cerebral spinal fluid of a 66-year-old man during episodes of systemic infection separated by 3 months and antifungal therapy were analyzed by a variety of molecular typing methods. All four isolates were shown to represent the same strain, indicating a relapse of infection rather than reinfection.
PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients
Authors: Costa J M , Durand R , Deniau M , Rivollet D , Izri M , Houin R , Vidaud M , Bretagne S
Published: January 2020

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.
Detection of extended-spectrum beta-lactamase (ESBL)-producing strains by the Etest ESBL screen
Authors: Cormican M G , Marshall S A , Jones R N
Published: January 2020

Resistance to contemporary broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase (ESBL) enzymes, is an increasing problem worldwide. The Etest (AB Biodisk, Solna, Sweden) ESBL screen uses stable gradient technology to evaluate the MIC of ceftazidime alone compared with the MIC of ceftazidime with clavulanic acid (2 micrograms/ml) to facilitate the recognition of strains expressing inhibitable enzymes. In the present study, ESBL-producing strains (17 Escherichia coli transconjugants) were studied to define "sensitive" interpretive criteria for the Etest ESBL screen. These criteria (reduction of the ceftazidime MIC by > 2 log2 dilution steps in the presence of clavulanic acid) defined a group of 92 probable ESBL-positive organisms among the 225 tested strains of Klebsiella species and E. coli having suspicious antibiogram phenotypes. With a subset of 82 clinical strains, the Etest ESBL screen was more sensitive (100%) than the disk approximation test (87%) and was more convenient. The MICs of ciprofloxacin, gentamicin, and tobramycin at which 50% of isolates are inhibited were 16- to 128-fold higher (coresistance) for the ESBL screen-positive group of strains than for the ESBL screen-negative group of strains. Some strains for which cephalosporin MICs were elevated and which were Etest ESBL screen negative were also cefoxitin resistant, i.e., consistent with a chromosomally mediated AmpC resistance phenotype. The Etest ESBL screen test with the ceftazidime substrate appears to be a useful method for detecting or validating the presence of enteric bacilli potentially producing this type of beta-lactamase.
Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures
Authors: Duffey P S , Guthertz L S , Evans G C
Published: January 2020

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.
Characterization of two unusual clinically significant Francisella strains
Authors: Clarridge J E , Raich T J , Sjösted A , Sandström G , Darouiche R O , Shawar R M , Georghiou P R , Osting C , Vo L
Published: January 2020

We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F. tularensis biovar novicida, and F. philomiragia. Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar. Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase. They were additionally shown to belong to the species F. tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene. Also, RNA probe hybridization confirmed their belonging to the species F. tularensis. However, the new strains, which are not identical, are distinguished from other F. tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests. Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative. Commercial F. tularensis antiserum agglutinated stock F. tularensis strains but not Fx1, Fx2, F. tularensis biovar novicida, or F. philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate. Fx1 and Fx2 produced beta-lactamase. Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used.
Prospective clinical evaluation of Amplicor Mycobacterium tuberculosis PCR test as a screening method in a low-prevalence population
Authors: Cartuyvels R , De Ridder C , Jonckheere S , Verbist L , Van Eldere J
Published: January 2020

Of 656 respiratory samples analyzed for Mycobacterium tuberculosis by microscopy, culture, and the Amplicor PCR method, 25 were positive by culture, 12 were positive by microscopy, and 17 were positive by the Amplicor PCR method; 16 samples were Amplicor PCR positive and culture negative. No patient except one with culture-negative, Amplicor PCR-positive samples had clinical indications of tuberculosis. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 68 and 97.4%, respectively. For culture-positive, smear-negative samples, the sensitivity of the Amplicor PCR was 46%.
Species belonging to the "Streptococcus milleri" group: antimicrobial susceptibility and comparative prevalence in significant clinical specimens
Authors: Bantar C , Fernandez Canigia L , Relloso S , Lanza A , Bianchini H , Smayevsky J
Published: January 2020

The association between the three species belonging to the "Streptococcus milleri" group and different sites of isolation was examined for 73 successive strains recovered from clinically significant, purulent infections. Susceptibility testing was performed on 64 of these strains. The present study supports the association of particular species with different clinical sources. Susceptibility data suggest that emerging penicillin resistance among Streptococcus anginosus and Streptococcus intermedius isolates may represent a potential clinical problem in the therapeutic management of infections caused by these species.
Incubation time for feline immunodeficiency virus cultures
Authors: Giannecchini S , Matteucci D , Mazzetti P , Bendinelli M
Published: January 2020

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.
Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests
Authors: Jahn B , Stüben A , Bhakdi S
Published: January 2020

Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used.
Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia
Authors: Mochizuki M , Horiuchi M , Hiragi H , San Gabriel M C , Yasuda N , Uno T
Published: January 2020

Twenty-seven feline parvovirus (FPV) isolates were recovered from cats clinically diagnosed with feline panleukopenia (FPL) for assessing antigenic and genomic properties of FPL viruses (FPLV) recently prevalent among cats in Japan. All isolates, with the exception of one novel isolate, FPV-314, possessed homologous properties, and their subgroups in FPVs were identified as FPLV. The FPV-314 isolate, which was from a 1.5-year-old cat which manifested clinical signs of FPL and died on the 13th day after the first medical examination, was finally identified as canine parvovirus (CPV) because it lacked a specific antigenic epitope commonly detected in FPLV and mink enteritis virus and because the nucleotide sequence of the capsid protein gene was almost identical to those of CPV-2a and -2b antigenic type strains recently prevalent among dogs in Japan. The present result together with our previous findings (M. Mochizuki, R. Harasawa, and H. Nakatani. Vet. Microbiol. 38:1-10, 1993) indicates the possibility that CPV and FPLV undergo mutual interspecies transmission between dogs and cats, and it is postulated that they may cause disease in some adventitious hosts.
Rapid diagnosis of adenoviral conjunctivitis by PCR and restriction fragment length polymorphism analysis
Authors: Saitoh-Inagawa W , Oshima A , Aoki K , Itoh N , Isobe K , Uchio E , Ohno S , Nakajima H , Hata K , Ishiko H
Published: January 2020

To detect and identify adenovirus (Ad), we used a combination of PCR and restriction fragment length polymorphism (RFLP) analysis. Nested PCR with two primer sets that hybridize to the conserved region for hexon proteins of 14 prototypes of Ad, Ad serotype 1 (Ad1) to Ad8, -11, -14, -19, -37, -40, and -41, amplified a 956-bp DNA fragment. The amplified fragments from the 14 prototypes were completely differentiated with a combination of three restriction endonucleases, EcoT14I, HaeIII, and HintI. We applied this new method for 127 samples of conjunctival scrapings from patients with conjunctivitis and compared the results with those obtained with the combination of culture isolation and a neutralization test (NT). PCR gave a positive result in 69 of 127 cases (54.3%), while only 61 of the 127 samples (48.0%) tested positive by culture isolation. Compared with isolation, the PCR method had a sensitivity of 100% (61 of 61). Positive PCR samples were further classified as Ad37 (59.5%), -3(31.9%), -11 (4.3%), -8 (2.9%), and -4 (1.4%) by PCR-RFLP analysis. Of eight samples that were PCR positive and culture isolation negative, six were Ad37 and two were Ad8 by PCR-RFLP analysis. These differentiations of isolation-positive samples were identical to the results obtained by the NT. It took only 3 days to detect and identify Ad by PCR-RFLP analysis, whereas it took at least 3 weeks by culture isolation and NT. Our newly developed method of detecting and typing human Ad by PCR-RFLP analysis is more sensitive, accurate, and rapid than the conventional method of culture isolation and an NT.
Thin agar film for enhanced fungal growth and microscopic viewing in a new sealable fungal culture case
Authors: Hill D R
Published: January 2020

This project was undertaken to find ways to enhance fungus colony maturation, to make viewing of fungal cultures easier, and to reduce disruption of the fungal structures to be observed for identification. Accordingly, a technique using a thin (0.2-mm) agar film that avoids problems inherent in traditional methods of fungal culture and identification was developed. In addition, to accommodate the 0.2-mm layer of agar film and a contiguous thicker 4-mm section of agar, a sealable fungal culture case that fits within microscope stage calipers and under the objective lenses was invented. The growth and identification of 28 organisms were evaluated in the sealable fungal culture cases and on double-pour agar plates by using potato dextrose agar in both. Compared with results obtained with the double-pour agar plates (rated as "good"), fungal growth and identification with the sealable fungal culture case were superior (rated as "excellent") (P < 0.05, chi-square test). The thin agar film limits excessive mycelial growth, while it often promotes complete sporulation or other forms of maturation of the fungal colony. More importantly, the thin agar film allows direct microscopic viewing of the developing fungal colonies. The portion of the sealable fungal culture case with the 4-mm layer of agar can be used for evaluation of colony pigment and texture. In conclusion, this new sealable fungal culture case allows direct viewing and earlier fungal species identification with greater intrinsic safety.
Diagnosis of extrapulmonary tuberculosis by Gen-Probe amplified Mycobacterium tuberculosis direct test
Authors: Ehlers S , Ignatius R , Regnath T , Hahn H
Published: January 2020

A total of 294 specimens collected from nonrespiratory sites of 268 patients were tested for direct detection of Mycobacterium tuberculosis complex by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (AMTD). The specimens included ascitic, pleural, pericardial, and synovial fluids, abscess aspirates, and tissue and lymph node biopsy samples, as well as gastric aspirates and cerebrospinal fluid samples. All samples were processed by the N-acetyl-L-cysteine-sodium hydroxide decontamination procedure prior to testing. Twenty samples showed acid-fast bacilli on auramine staining, and 48 samples were positive by AMTD, 9 of which were negative for M. tuberculosis complex by culture. After reviewing the patients clinical charts to resolve discrepancies, the test result of one cerebrospinal fluid sample was considered to be false positive by AMTD. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 83.9, 99.6, 97.9, and 96.3%, respectively. No significant differences were found when AMTD results obtained with specimens of nonrespiratory origin were compared with assay results obtained with samples of respiratory origin (P > 0.05). In conclusion, our results demonstrate that AMTD performs equally well with all types of specimens.
Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay
Authors: Nelson S , Matlow A , McDowell C , Roscoe M , Karmali M , Penn L , Dyster L
Published: January 2020

In order to improve detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) in our laboratory, a PCR-based assay was optimized, and a study was designed (i) to compare results obtained by PCR to those obtained by culture and (ii) to evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) by comparing it to agarose gel electrophoresis (PCR-gel) for detection of the PCR product. DNA for the PCR was extracted with a guanidine thiocyanate buffer and used in a PCR mixture containing primers directed against a reiterated gene sequence in B. pertussis (Q. He, J. Mertsola, H. Soini, M. Skurnik, O. Ruuskanen, and M. K. Viljanen, J. Clin, Microbiol. 31:642-645, 1993). Of 96 NPAs submitted from a targeted study group, 23 were positive by culture, 27 were positive by PCR-gel, and 31 were positive by PCR-plate. All culture-positive specimens were also positive by PCR. Of nine patients with culture-negative-PCR-positive results, six had discharge diagnoses of pertussis. Thus, PCR with plate-based product detection is a sensitive method for the laboratory detection of B. pertussis in NPAs. Additional advantages of the plate assay include rapidity, objectivity in reading results, specificity, and the capability of being adapted to a high-volume, automated system.
Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea
Authors: Yamamoto T , Nakazawa M
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.
Protein characterization of Australian spotted fever group rickettsiae and monoclonal antibody typing of Rickettsia honei
Authors: Stenos J , Ross B , Feng H M , Crocquet-Valdes P , Walker D
Published: January 2020

Rickettsial proteins rOmp A and rOmp B exist in both Rickettsia australis and Rickettsia honei but differ in molecular weight and antigenicity; in addition, they produce distinct immunogenic responses and appear to be to conformationally dependent antigens. Species-specific monoclonal antibodies for other spotted fever group rickettsial species did not react with R. honei. A PCR product of the repeat region of the rOmp A gene from R. honei was amplified and calculated to contain 11 repeat units.
Genomic fingerprinting of Haemophilus somnus by a combination of PCR methods
Authors: Appuhamy S , Parton R , Coote J G , Gibbs H A
Published: January 2020

Twenty-three isolates of Haemophilus somnus were typed by repetitive extragenic palindromic (REP) element-based PCR, enterobacterial repetitive intergenic consensus (ERIC)-based PCR, and PCR ribotyping. A total of 11 types were distinguished by REP-PCR, 13 types were distinguished by ERIC-PCR, and 5 types were distinguished by PCR ribotyping. PCR ribotyping produced a relatively simple pattern and a small number of distinct types, whereas REP- and ERIC-PCR both produced more complex banding patterns but increased the discrimination between strains. Clearly distinguishable profiles were obtained for respiratory and genital isolates of H. somnus by all three typing methods. The results suggest that a combination of all three primer sets provides a high-resolution fingerprinting method for epidemiological studies of H. somnus and for its differentiation from related species.