bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of numerous hepatitis C virus genotypes in Montreal, Canada
Authors: Bernier L , Willems B , Delage G , Murphy D G
Published: January 2020

Hepatitis C virus genotypes were determined for 358 viremic individuals in Montreal, Canada, by restriction endonuclease analysis of PCR products and phylogenetic analysis of core gene sequences. Types 1, 2, and 3 occurred in 62.8, 14.2, and 13.7%, respectively; types 4 and 5 were found in 3.9 and 4.5%, respectively; and genotypes 6a and 7c and a novel genotype each occurred in 0.3%. Types 4, 6, and 7 and the novel genotype were mostly from persons who had immigrated to Canada.
Evaluation of reformulated chemiluminescent DNA probe (AccuProbe) for culture identification of Mycobacterium kansasii
Authors: Tortoli E , Simonetti M T , Lavinia F
Published: January 2020

A panel of 104 isolates belonging to the species Mycobacterium kansasii and 78 mycobacterial isolates belonging to other species was tested in parallel with the present commercially available DNA probe (AccuProbe; Gen-Probe) and with a new probe just developed by the same manufacturer. While the old version of the probe confirmed the previously reported low sensitivity (only 59% of the M. kansasii isolates reacted), the new one was 100% sensitive. Only two non-M. kansasii strains, both M. gastri isolates, gave false-positive hybridization results.
Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus epidermidis
Authors: Martineau F , Picard F J , Roy P H , Ouellette M , Bergeron M G
Published: January 2020

Staphylococcus epidermidis is an aerobic gram-positive coccus that is now recognized among the coagulase-negative staphylococci as an etiological agent with an important range of pathogenicity in humans. Several diagnostic kits based on biochemical or immunological reactions can efficiently identify Staphylococcus aureus. However, these tests are often unreliable for the identification of coagulase-negative staphylococcal species including S. epidermidis. Since DNA-based assays for the species-specific identification of S. epidermidis remain unavailable, we have developed such tests in order to improve the accuracy and the rapidity of tests for the diagnosis of S. epidermidis infections. On the basis of the results of hybridization assays with clones randomly selected from an S. epidermidis genomic library, we identified a chromosomal DNA fragment which is specific and 100% ubiquitous for the identification of S. epidermidis. This 705-bp fragment was sequenced and used to design PCR amplification primers. PCR assays with the selected primers were also highly specific and ubiquitous for the identification from bacterial cultures of clinical isolates of S. epidermidis from a variety of anatomic sites. While three strains of S. capitis were misidentified as S. epidermidis with the API Staph-Ident system and 2.5% of the S. epidermidis identifications were inconclusive with the MicroScan Autoscan-4 system, the PCR assay was highly specific and allowed for the correct identification of all 79 S. epidermidis strains tested. The PCR assays developed are simple and can be performed in about 1 h. The DNA-based tests provide novel diagnostic tools for improving the diagnosis of S. epidermidis infections.
Enteroadherent Escherichia coli and diarrhea in children: a prospective case-control study
Authors: Forestier C , Meyer M , Favre-Bonte S , Rich C , Malpuech G , Le Bouguenec C , Sirot J , Joly B , De Champs C
Published: January 2020

The relative contribution of diarrheagenic Escherichia coli was examined during a 1-year prospective study of hospitalized children in Clermont-Ferrand, France, including 220 case patients (with diarrhea) and 211 matched controls. Fecal isolates were characterized by means of their pattern of adherence to HEp-2 cells and by colony hybridization with DNA probes specific for the six categories of diarrheagenic E. coli. No enteroinvasive or enterotoxigenic E. coli isolates were isolated. Twenty-eight (6.5%) eae-positive isolates and 39 (9%) enteroaggregative E. coli isolates characterized with the aggregative adherence probe and/or by their adherence pattern were detected; they were equally distributed among the patients and the controls. Diffusely adhering E. coli was the predominant pathotype: 30.7% were detected by their adherence pattern and 13.7% were detected with the daaC probe. They were isolated with similar frequencies from the patients and the controls, thereby showing no association with diarrhea. However, daaC-positive strains were significantly associated with a past record of urinary tract infections. These results suggest that the diffusely adhering E. coli organisms isolated in the present study are not true intestinal pathogens but may be regarded as resident colonic strains.
Characterization of multiple-antibiotic-resistant Salmonella typhimurium stains: molecular epidemiology of PER-1-producing isolates and evidence for nosocomial plasmid exchange by a clone
Authors: Vahaboglu H , Dodanli S , Eroglu C , Oztürk R , Soyletir G , Yildirim I , Avkan V
Published: January 2020

We characterized epidemiologic and genetic features of nosocomially originated multiple-antibiotic-resistant Salmonella typhimurium isolates from two hospitals. A total of 32 multiply resistant strains, isolated during a 28-month period, were studied. Four resistance phenotypes were distinguished on the basis of the results of disc diffusion tests. Group 1 was resistant to chloramphenicol, gentamicin, tobramycin, amikacin, and the newer cephalosporins because of the production of an extended-spectrum beta-lactamase (PER-1). Group 2 exhibited the same pattern plus resistance to sulfamethoxazole-trimethoprim (Sxt). Except for Sxt resistance, dominant phenotypes of both groups were transferred on an identical plasmid, pSTI1 (81 MDa). Group 3 was resistant to ampicillin, chloramphenicol, gentamicin, tobramycin, and Sxt. This pattern was also transferred on an 81-MDa plasmid (pSTI2) which differed from pSTI1 on the basis of EcoRI and HindIII restriction fragments. Group 4 was resistant to ampicillin, chloramphenicol, and tetracycline, and a 74-MDa nonconjugative plasmid was detected. Restriction fragment length polymorphism of RNA-encoding DNA and arbitrarily primed PCR tests revealed that bacteria from groups 1, 2, and 3 were clonally related. Epidemiologic data also supported the clonal-dissemination hypothesis. We concluded that S. typhimurium isolates acquire and exchange multiple-resistance plasmids in hospital microflora.
Isolation of a novel Helicobacter species, Helicobacter cholecystus sp. nov., from the gallbladders of Syrian hamsters with cholangiofibrosis and centrilobular pancreatitis
Authors: Franklin C L , Beckwith C S , Livingston R S , Riley L K , Gibson S V , Besch-Williford C L , Hook R R
Published: January 2020

A filamentous, gram-negative, motile bacterium with a single polar sheathed flagellum was isolated from gallbladders of hamsters with cholangiofibrosis and centrilobular pancreatitis. Bacteria grew under microaerophilic conditions at 37 and 42 degrees C, were oxidase, catalase, arginine aminopeptidase, and L-arginine arylamidase positive, reduced nitrate to nitrite, were resistant to cephalothin, and exhibited intermediate susceptibility to nalidixic acid. Sequence analysis of the 16S rRNA gene indicated that the bacterium was a novel member of the Helicobacter genus, most closely related to Helicobacter pametensis. We propose to name this bacterium Helicobacter cholecystus. In epidemiologic studies, isolation of H. cholecystus correlated strongly with the presence of cholangiofibrosis and centrilobular pancreatitis; however, further studies are needed to define the role of this bacterium in pathogenesis.
Application of lipovitellin-salt-mannitol agar for screening, isolation, and presumptive identification of Staphylococcus aureus in a teaching hospital
Authors: Merlino J , Gill R , Robertson G J
Published: January 2020

Lipovitellin-salt-mannitol (LSM) plate medium was examined for its ability to directly isolate, recover, and presumptively identify Staphylococcus aureus from 418 clinical specimens. The criteria for medium evaluation included colony morphology reactions, selectivity, and ease of isolation. For 298 specimens used for screening, LSM agar medium was compared with the other conventional media used, mannitol salt agar (MSA), 5% horse blood agar (HBA), and phenolphthalein phosphate agar (PPA), to detect and recover S. aureus and methicillin-resistant S. aureus. The results indicated that LSM agar is more effective than MSA, HBA, or PPA for the recovery and isolation of S. aureus and methicillin-resistant S. aureus. On a replicator multipoint inoculation system, we compared the reactions on LSM agar, MSA, and DNase agar of 227 different strains of staphylococci, which included 178 different strains of S. aureus and 49 different strains of coagulase-negative staphylococci isolated from clinical specimens. By using the lipovitellin precipitation activity and mannitol fermentation characteristics, LSM agar gave a 100% correlation in presumptively identifying S. aureus. LSM agar may be an alternative plate medium for large hospital extensive screening for the detection and isolation of S. aureus.
Identification of staphylococci with a self-educating system using fatty acid analysis and biochemical tests
Authors: Behme R J , Shuttleworth R , McNabb A , Colby W D
Published: January 2020

We characterized all of the 35 aerobic taxa of the genus Staphylococcus by using an objective, self-learning system combining both whole-cell fatty acid (FA) analysis and the results of 35 biochemical tests. Isolates were compared with the type strain for each taxon to generate an FA profile library and a biochemical table of test responses. Isolates were accepted into the system if they had a similarity index of > or = 0.6 for a taxon within the FA profile library and if they were identified as the same taxon by a computer program using a probability matrix constructed from the biochemical data. These stringent criteria led to acceptance of 1,117 strains assigned to legitimate taxa. Additional FA groups were assembled from selected strains that did not meet the inclusion criteria based on the type strains and were added to the system as separate entries. Currently, 1,512 isolates have bee accepted into the system. This approach has resulted in a comprehensive table of biochemical test results and a FA profile library, which together provide a practical system for valid identifications.
Improved sensitivity of direct microscopy for acid-fast bacilli: sedimentation as an alternative to centrifugation for concentration of tubercle bacilli
Authors: Miörner H , Ganlöv G , Yohannes Z , Adane Y
Published: January 2020

There is a great need for improved methods for the diagnosis of tuberculosis by techniques that are appropriate for control programs in low-income countries. Liquefaction of sputum with sodium hypochlorite followed by concentration of bacilli through overnight sedimentation significantly increases the sensitivity of direct microscopy, and this method could be an alternative for diagnostic centers not equipped with a centrifuge.
Veillonella parvula bacteremia without an underlying source
Authors: Fisher R G , Denison M R
Published: January 2020

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal human flora. It has rarely been identified as a pathogen in humans, and the most frequently reported infection caused by V. parvula is osteomyelitis. We report a case of bacteremia unrelated to a central venous catheter and without an underlying source of infection.
Molecular characterization of epidemic ciprofloxacin- and methicillin-resistant Staphylococcus aureus strains colonizing patients in an intensive care unit
Authors: Udo E E , al-Obaid I A , Jacob L E , Chugh T D
Published: January 2020

Eighteen methicillin-resistant Staphylococcus aureus (MRSA) samples isolated from patients and the environment in an intensive care unit (ICU) during a routine surveillance were tested for antimicrobial resistance and typed by pulsed-field gel electrophoresis. Three pulsed-field patterns were observed. Sixteen were ciprofloxacin resistant and had identical pulsed-field patterns. The results suggested that a ciprofloxacin-resistant MRSA clone had contaminated the environment and spread among patients. This study demonstrates the application of infection control surveillance combined with strain typing in detecting MRSA colonization in the ICU where it was not known to exist.
Comparison of two quantitative hepatitis C virus reverse transcriptase PCR assays
Authors: Roth W K , Lee J H , Rüster B , Zeuzem S
Published: January 2020

A quantitative hepatitis C virus reverse transcriptase PCR (HCV RT-PCR) assay established in our laboratory was compared with the Roche Amplicor HCV Monitor test kit for agreement of test results and intra-assay variability. Both assays rely on reverse transcription and amplification of extracted RNA from patients' sera together with an internal RNA standard derived from the 5'-noncoding region of HCV. A panel of clinical serum samples (n = 33) was quantitatively analyzed in parallel by both test systems. The methods demonstrated substantial agreement between 1 x 10(3) and 5 x 10(5) HCV RNA molecules per ml of serum. However, with sera containing more than 5 x 10(5) copies per ml, according to our in-house assay, the results diverged on average in a nonacceptable range of 2 orders of magnitude. Our in-house HCV RT-PCR assay measured up to 5 x 10(7) HCV-RNA molecules per ml in some serum samples. However, the Roche Amplicor HCV Monitor test kit did not detect more than 2 x 10(6) molecules in any of the serum samples tested. After dilution of serum samples prior to testing, an approximately 0.5 order of magnitude more HCV RNA molecules was detected by the Roche HCV test kit in sera containing high copy numbers (> 5 x 10(5) RNA copies according to the in-house assay). The in-house PCR and the Roche Amplicor HCV Monitor test kit revealed coefficients of variation of 6.2 and 7.5%, respectively.
Encephalocytozoon intestinalis-specific monoclonal antibodies for laboratory diagnosis of microsporidiosis
Authors: Beckers P J , Derks G J , Gool T , Rietveld F J , Sauerwein R W
Published: January 2020

Two monoclonal antibodies which can be used for the unambiguous identification by fluorescence microscopy of Encephalitozoon intestinalis spores in clinical specimens are described. Monoclonal antibody Si91 is specific for the extruded polar filament, and Si13 recognizes the surfaces of E. intestinalis spores. No cross-reaction with spores of Encephalitozoon hellem was observed. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. Combined in an indirect immunofluorescence assay, these antibodies are used to identify spores in feces. Although there was some cross-reaction with fecal bacteria and fungi, the typical morphology of the extruded polar filaments enabled proper identification of the E. intestinalis spores. Parasites could also be demonstrated to be present in urine, nasal swabs, lung brush biopsy specimens, and bronchoalveolar lavage fluid from a patient with disseminated infection with E. intestinalis. The use of these monoclonal antibodies facilitates the detection and species determination of E. intestinalis in clinical specimens.
Rejection criteria for endotracheal aspirates from pediatric patients
Authors: Zaidi A K , Reller L B
Published: January 2020

Endotracheal aspirates (ETAs) from mechanically ventilated pediatric patients frequently are cultured as part of an evaluation for suspected sepsis. There are now well-defined criteria for rejecting low-yield ETAs from adults, but it is uncertain whether the same criteria can be applied to ETAs from children. Therefore, we compared the Gram stain and culture results for 361 consecutive ETA specimens collected from pediatric patients over a 1-year period. Results for patients for whom a blood culture was performed within 48 h of the time that a culture of ETA was performed were also reviewed. Gram stains were examined under x100 magnification to quantitate the number of polymorphonuclear neutrophils and squamous epithelial cells (SECs) per low-power field and under x1,000 magnification for the presence of organisms. No organisms were seen by Gram staining in 225 (62%) of the ETAs. Culture of these specimens rarely yielded useful information: 52% were sterile, 32% grew rare to 1+ quantities of expected respiratory flora only, 12% grew rare to 1+ quantities of gram-negative rods mixed with expected respiratory flora, and only 10 (4%) yielded a pure or predominant growth of a potential respiratory pathogen. Unlike adult patients, we did not find the number of SECs to be a useful screening criterion. Only 17 (5%) of the ETAs had greater than 10 SECs per low-power field, and 5 (29%) of these yielded pure growth of a gram-negative rod. When blood culture results were positive, they correlated with ETA culture results in only 6 of 10 cases. On the basis of our findings, the absence of organisms on Gram staining is a useful criterion for rejecting ETAs from pediatric patients for culture and would have excluded 62% of the specimens from further processing.
Survey of type 6 group variants of hepatitis C virus in Southeast Asia by using a core-based genotyping assay
Authors: Mellor J , Walsh E A , Prescott L E , Jarvis L M , Davidson F , Yap P L , Simmonds P
Published: January 2020

Previous surveys of the prevalences of genotypes of hepatitis C virus (HCV) in different populations have often used genotyping assays based upon analysis of amplified sequences from the 5' noncoding region (5'NCR), such as restriction fragment length polymorphism (RFLP) or hybridization with type-specific probes (e.g., InnoLipa). Although highly conserved, this region contains several type-specific nucleotide polymorphisms that allow major genotypes 1 to 6 to be reliably identified. Recently, however, novel HCV variants found in Vietnam and Thailand that are distantly related to the type 6a genotype (type 6 group) by phylogenetic analysis of coding regions of the genome often have sequences in the 5'NCR that are similar or identical to those of type 1 and could therefore not be identified by an assay of sequences in this region. We developed a new genotyping assay based upon RFLP of sequences amplified from the more variable core region to investigate their distribution elsewhere in southeast (SE) Asia. Among 108 samples from blood donors in seven areas that were identified as type 1 by RFLP in the 5'NCR, type 6 group variants were found in Thailand (7 from 28 samples originally identified as type 1) and Burma (Myanmar) (1 of 3) but were not found in Hong Kong (n = 43), Macau (n = 8), Taiwan (n = 6), Singapore (n = 2), or Malaysia (n = 18). Although this small survey suggests a relatively limited distribution for type 6 group variants in SE Asia, larger studies will be required to explore their distribution in other geographical regions and the extent to which their presence would limit the practical usefulness of 5'NCR-based genotyping assays for clinical or epidemiological purposes.
Comparison of two methods for detecting varicella-zoster virus antibody with varicella-zoster virus cell-mediated immunity
Authors: Weinberg A , Hayward A R , Masters H B , Ogu I A , Levin M J
Published: January 2020

We evaluated an enzyme-linked immunoassay (EIA; BioWhittaker) and a latex agglutination (LA; Becton Dickinson) for varicella-zoster virus (VZV) antibody determination, using cell-mediated immunity (CMI) as a "gold standard." VZV EIA had a sensitivity, specificity, positive predictive value, and negative predictive value of 87, 91, 87, and 91%, respectively, compared with CMI. Correlation was excellent except when the varicella index was 0.9 to 1.2. We defined sera with varicella indices of 0.9 to 1.2 as indeterminate. LA had a sensitivity, specificity, positive predictive value, and negative predictive value of 96, 91, 97, and 90%, respectively, compared with EIA. LA reactivity only at a 1:2 dilution did not correlate with CMI, but sera reactive at dilutions of > or = 1:8 indirectly did. We defined indeterminate sera as those reactive at 1:2 and nonreactive at 1:8. EIA and LA were equivalent for determining VZV immune status, and both methods required modified criteria of interpretation to increase their specificity.
Infectious disease physicians rate microbiology services and practices
Authors: Baron E J , Francis D , Peddecord K M
Published: January 2020

Recent years have seen increasing emphasis on cost containment and quality improvement in clinical laboratory activities. Modifying those activities to enhance clinical relevance is one strategy that should be satisfying to both laboratory scientists and administrators. This guest commentary describes one approach to quality improvement--the use of user surveys to identify areas for improvement. As an initial attempt to define such areas in clinical diagnostic microbiology, infectious disease specialists, targeted for their particular interest and expertise in microbiology laboratory results, were polled and their responses were analyzed. Some of these data have been presented previously (E. J. Baron, D. P. Francis, and K. M. Peddecord, abstr. C-170, p. 520, in Abstracts of the 94th General Meeting of the American Society for Microbiology, 1994; K. M. Peddecord, E. J. Baron, D. P. Francis, and A. S. Benenson, abstr. C-172, p. 520, in Abstracts of the 94th General Meeting of the American Society for Microbiology, 1994; K. M. Peddecord, E. J. Baron, D. P. Francis, and J. A. Drew, Am. J. Clin. Pathol. 105:58-64, 1996). The discussion includes our recommendations for the use of these survey responses, and their limitations, as stimuli to initiate reexamination of certain microbiology laboratory practices in the interest of developing more cost-effective and clinically relevant protocols.
Home-use nebulizers: a potential primary source of Burkholderia cepacia and other colistin-resistant, gram-negative bacteria in patients with cystic fibrosis
Authors: Hutchinson G R , Parker S , Pryor J A , Duncan-Skingle F , Hoffman P N , Hodson M E , Kaufmann M E , Pitt T L
Published: January 2020

Inhalation of aerosols contaminated with gram-negative bacteria generated from home-use nebulizers used by cystic fibrosis (CF) patients may be a primary route for bacterial colonization of the lung. Burkholderia cepacia was isolated from 3 of [corrected] 35 home-use nebulizers, and Stenotrophomonas maltophilia was isolated from 4 of 35 home-use nebulizers. Sputum cultures for two patients whose nebulizers were contaminated with B. cepacia did not yield the organism. However, DNA macrorestriction analysis by pulsed-field gel electrophoresis confirmed that one of two strains of B. cepacia recovered from the nebulizer of a third patient was also present in the sputum of that patient. Although Pseudomonas aeruginosa was isolated from 34 patients, none of the nebulizers were positive for the organism. Sixty-nine percent of nebulizers were contaminated, and up to 16 different environmental colistin-resistant, gram-negative species were identified. The heaviest contamination was found beneath the chamber atomizer. A questionnaire survey showed that the majority of patients (28 of 34) were receiving nebulized colistin and/or gentamicin. Patients who followed recommended instructions for good nebulizer hygienic practice and paid particular attention to drying had minimal or no contamination of their nebulizers.
Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture
Authors: Munderloh U G , Madigan J E , Dumler J S , Goodman J L , Hayes S F , Barlough J E , Nelson C M , Kurtti T J
Published: January 2020

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.
Comparative evaluation of two serologic typing methods for hepatitis C virus
Authors: Cerino A , Cividini A , Asti M , Lanza A , Silini E , Mondelli M U
Published: January 2020

Serologic methods of typing for hepatitis C virus offer advantages over PCR-based typing methods in terms of speed and simplicity of sample preparation and in the use of standard laboratory equipment. We examined the sensitivities and specificities of two hepatitis C virus serotyping assays which use sets of type-specific antigenic peptides derived from the core or the nonstructural 4 (NS4) regions and compared the results with those of molecular typing with type-specific primers from the core region. Although there was a good concordance between serotyping by either assay and genotyping, we found that the sensitivities of both serologic assays were less than optimal compared with that of molecular typing, with only about 50% of samples being unequivocally typed. Moreover, amino acid sequence similarities within the regions of the genome used for serotyping preclude differentiation into subtypes, which may have important clinical and therapeutic implications.
Development of immuno-PCR for diagnosis of bovine herpesvirus 1 infection
Authors: Mweene A S , Ito T , Okazaki K , Ono E , Shimizu Y , Kida H
Published: January 2020

A highly sensitive immuno-PCR method specific for the detection of antigens of and antibodies to bovine herpesvirus 1 was established. This assay attained a high sensitivity of up to 10(7.0) times higher than that of enzyme-linked immunosorbent assay (ELISA) or PCR for antigen detection and 10(5.0) times higher than that of ELISA for antibody detection.
Emergence of fluconazole-resistant strains of Blastoschizomyces capitatus causing nosocomial infections in cancer patients
Authors: D'Antonio D , Mazzoni A , Iacone A , Violante B , Capuani M A , Schioppa F , Romano F
Published: January 2020

Blastoschizomyces capitatus strains resistant to fluconazole were isolated in three cancer patients. All of the strains had identical genomic DNA restriction profiles. Our findings give evidence for the emergence of fluconazole-resistant B. capitatus and suggest a nosocomial acquisition emanating from a common source within the hospital environment.
Molecular study of nosocomial nocardiosis outbreak involving heart transplant recipients
Authors: Exmelin L , Malbruny B , Vergnaud M , Prosvost F , Boiron P , Morel C
Published: January 2020

Randomly amplified polymorphic DNA analysis and rRNA gene restriction patterns (ribotyping) were compared as methods of investigating a nosocomial outbreak of nocardiosis involving three heart transplant recipients. No clear distinctions between three clinically related isolates and four unrelated strains were obtained by ribotyping. On the contrary, randomly amplified polymorphic DNA analysis with two selected primers, primers 2650 and DKU49, showed one pattern for the three related isolates and four patterns for the unrelated strains.
Simultaneous rapid culture for four respiratory viruses in the same cell monolayer using a differential multicolored fluorescent confirmatory stain
Authors: Brumback B G , Wade C D
Published: January 2020

A simultaneous rapid culture for influenza virus types A and B, parainfluenza virus, and respiratory syncytial virus was developed in a 96-well plate format with a culture-confirmatory stain using multiple fluorescent tags. Performance characteristics were comparable to those of standard and/or single rapid-culture methods as shown by parallel testing of 590 fresh clinical specimens and retrospective testing of 190 previously positive frozen specimens. The quadruple culture required less specimen volume than separate cultures, was significantly quicker than standard tube culture, was less labor intensive than separate cultures, and was less expensive than the other methods.
Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae
Authors: Sciortino C V , Johnson J A , Hamad A
Published: January 2020

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.
Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology
Authors: Detmer J , Lagier R , Flynn J , Zayati C , Kolberg J , Collins M , Urdea M , Sánchez-Pescador R
Published: January 2020

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.
Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe
Authors: Tevere V J , Hewitt P L , Dare A , Hocknell P , Keen A , Spadoro J P , Young K K
Published: January 2020

Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections.
Variability in 3' end of 16S rRNA sequence of Mycobacterium ulcerans is related to geographic origin of isolates
Authors: Portaels F , Fonteyene P A , de Beenhouwer H , de Rijk P , Guédénon A , Hayman J , Meyers M W
Published: January 2020

Mycobacterium ulcerans causes extensive ulcers (Buruli ulcers) in the skin of humans. Analysis of the 3'-terminal region of the 16S rRNA gene sequence of 17 strains of M. ulcerans from Africa, the Americas, and Australia revealed three subgroups corresponding to the continent of origin, and some variable phenotypic characteristics. This sequence is useful for the rapid detection of M. ulcerans and discriminates M. marinum and M. shinshuense from M. ulcerans.
Simplified testing for antibodies to human immunodeficiency virus
Authors: Pagcharoenpol P , Burgess-Cassler A , Schramm W
Published: January 2020

Test strips for the detection of antibodies to human immunodeficiency virus type 1 were investigated using specimens from risk groups in Thailand (141 reactive; 445 nonreactive) in a local Thai laboratory. The diagnostic sensitivity and specificity were both 100%. Using a set of seroconversion panels, the sensitivity of the test strips was within the range of sensitivities obtained with enzyme immunoassays. The test was developed for performance at decentralized settings under nonlaboratory conditions.
Clinical evaluation of the Roche AMPLICOR PCR Mycobacterium tuberculosis test for detection of M. tuberculosis in respiratory specimens
Authors: Bergmann J S , Woods G L
Published: January 2020

The reliability of the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB) for the diagnosis of pulmonary tuberculosis was evaluated by testing 956 respiratory specimens from 502 patients and comparing results with results by culture and medical history. Of those 135 specimens that were culture positive for mycobacteria, 61 specimens from 31 patients grew M. tuberculosis. Fifty-two specimens were smear positive for acid-fast bacteria (AFB); M. tuberculosis was isolated from 41 of these specimens. On initial testing, the sensitivity and specificity of the AMPLICOR MTB assay, compared with culture, were 78.7 and 99.3%, respectively. After resolution of discrepancies (by review of medical history), the sensitivity, specificity, and positive and negative predictive values of the AMPLICOR MTB assay were 79.4, 99.6, 92.6, and 98.6%, respectively. Two specimens from two patients with no clinical evidence of tuberculosis were AMPLICOR MTB positive and culture positive for Mycobacterium avium complex. For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 97.6, 100, 100, and 90.9%, respectively. For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 40.0, 99.5, 69.2, and 98.7%, respectively. Our results support the use of AMPLICOR MTB for rapid diagnosis of tuberculosis in patients whose respiratory specimens are AFB smear positive. Further studies are needed to determine the most clinically relevant and cost-effective use of this assay with AFB smear-negative specimens.
Most Corynebacterium xerosis strains identified in the routine clinical laboratory correspond to Corynebacterium amycolatum
Authors: Funke G , Lawson P A , Bernard K A , Collins M D
Published: January 2020

A comprehensive study was performed on 25 bacterial clinical isolates originally identified as Corynebacterium xerosis. Three reference strains of C. xerosis were also included in the study. On the basis of a variety of phenotypic characteristics tested, all strains could be divided into two separate clusters: reference strains ATCC 373 (the type strain of C. xerosis) and ATCC 7711 showed yellow-pigmented, dry, rough colonies, fermented 5-keto-gluconate, exhibited strong leucine arylamidase and alpha-glucosidase activities, produced lactate as the major end product of glucose metabolism, were susceptible to most of the 19 antimicrobial agents tested, and showed an inhibition zone around disks containing the vibriocidal compound O/129. In contrast, the remaining 26 strains including reference strain NCTC 7243 as well as all clinical isolates formed white-grayish, dry, slightly rough colonies, did not ferment 5-keto-gluconate, exhibited only weak leucine arylamidase and no alpha-glucosidase activity, produced large amounts of propionic acid as the end product of glucose metabolism, and were resistant to most antimicrobial agents tested, including O/129. Chemotaxonomic (cellular fatty acids, mycolic acids, and G+C content) and molecular genetic (16S rRNA gene sequence) investigations revealed that the strains of the second cluster unambiguously belonged to the species C. amycolatum. Our data suggest that most strains reported in the literature as C. xerosis are probably misidentified and correspond to C. amycolatum.
Evaluation of three commercial enzyme immunoassays compared with the 13C urea breath test for detection of Helicobacter pylori infection
Authors: Marchildon P A , Ciota L M , Zamaniyan F Z , Peacock J S , Graham D Y
Published: January 2020

The diagnostic significance of the serological detection of antibodies to Helicobacter pylori has been established by numerous investigators. Reports of the clinical reliabilities of commercial enzyme immunoassay (EIA) kits available for this purpose vary as a result of the different H. pylori antigen sources and reference methods used. The 13C urea breath test (UBT) has been shown to be an extremely accurate and reliable method of detecting H. pylori infection. We used the 13C urea breath test as the confirmatory method for H. pylori status to evaluate three commercially available EIA kits designed to detect immunoglobulin G antibodies to H. pylori. These kits were the HM-CAP EIA kit (Enteric Products, Inc.), the PYLORI STAT EIA kit (BioWhittaker, Inc.), and the G.A.P. kit (Bio-Rad Laboratories/Biomerica, Inc.). The evaluations were performed in a double-blind manner with samples from 473 clinically characterized patients. This group included patients with symptomatic gastrointestinal disorders as well as nonsymptomatic volunteers. The sensitivities of the kits were as follows: HM-CAP, 98.4%; PYLORI STAT, 99.2%; and G.A.P., 100%. The specificities were as follows: HM-CAP, 96.4%; PYLORI STAT, 90.1%; and G.A.P., 26.0%. Although the HM-CAP and PYLORI STAT kits performed comparably, the G.A.P. test yielded significantly more false-positive results and an unacceptably high number of indeterminate results.
Outbreak of histoplasmosis among employees in a paper factory--Michigan, 1993
Authors: Stobierski M G , Hospedales C J , Hall W N , Robinson-Dunn B , Hoch D , Sheill D A
Published: January 2020

In December 1993, four reported cases of histoplasmosis among employees in a Michigan pulp paper factory prompted an investigation. A cohort of employees was surveyed to identify additional cases. A case of acute histoplasmosis was defined as an influenza-like illness in a plant employee with the onset of illness during October or November 1993 and laboratory evidence of recent infection with Histoplasma capsulatum. Among the 96 employees surveyed, 18 persons met the case definition; all of these had illness onset during the last week of October in 1993. The attack rate among maintenance employees (16 of 53 [30%]) was much greater than that among nonmaintenance employees (2 of 43 [5%]) (relative risk = 6.5; 95% confidence interval = 1.6 to 26.7; P = 0.003). On October 22, a dry, windy day, one maintenance worker swept bird guano, approximately 10 cm deep, from an adjacent roof < 20 m from the maintenance building. The disturbance of the bird guano was the likely event which caused this outbreak. H. capsulatum remains an important pathogen among immunocompetent hosts. We recommended that the plant authorities (i) discourage birds from roosting at the facility and (ii) use safe procedures for the cleanup and disposal of soil contaminated with bird droppings.
Metronidazole and clarithromycin resistance in Helicobacter pylori determined by measuring MICs of antimicrobial agents in color indicator egg yolk agar in a miniwell format. The Gastrointestinal Physiology Working Group of Universidad Peruana Cayetano Heredia and the Johns Hopkins University
Authors: Vasquez A , Valdez Y , Gilman R H , McDonald J J , Westblom T U , Berg D , Mayta H , Gutierrez V
Published: January 2020

Resistance of Helicobacter pylori to metronidazole often causes failure of commonly used combination drug treatment regimens. We determined the MICs of metronidazole and clarithromycin against 18 H. pylori strains from Peru using tetrazolium egg yolk (TEY) agar. The MIC results obtained by agar dilution with petri dishes were compared with the results found through a miniwell format. The results of the two protocols for measuring drug susceptibility differed by no more than 1 dilution in all cases. On TEY agar, bright-red H. pylori colonies were easy to identify against a yellow background. Sixty-one percent (11 of 18) of the strains were resistant to metronidazole (MIC, > or = 4 micrograms/ml) and 50% (9 of 18) were resistant to clarithromycin (MIC, > or = 0.125 micrograms/ml), whereas none (0 of 5) of the strains tested were resistant to tetracycline (MIC, > or = 1 micrograms/ml). Thus, the prevalence of metronidazole and clarithromycin resistance in Peru is higher than that in developed regions of the world. The miniwell plate with TEY agar allows easy H. pylori colony identification, requires about one-third less of the costly medium necessary for petri dish assaying, conserves space, and yields MICs equivalent to those with agar dilution in petri dishes.
Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting
Authors: Sorrell T C , Chen S C , Ruma P , Meyer W , Pfeiffer T J , Ellis D H , Brownlee A G
Published: January 2020

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.
Necrotizing cerebritis in an allogeneic bone marrow transplant recipient due to Cladophialophora bantiana
Authors: Emmens R K , Richardson D , Thomas W , Hunter S , Hennigar R A , Wingard J R , Nolte F S
Published: January 2020

We describe a necrotizing cerebritis in an allogeneic bone marrow transplant recipient caused by the neurotropic, dematiaceous fungus Cladophialophora bantiana. The patient presented 7 months after bone marrow transplantation with fever and sudden onset of left-sided weakness, followed shortly by cranial nerve III and VI palsies. The patient had a lesion (3.0 by 2.0 by 2.0 cm) of the right midbrain with extension to the pons, the left brain stem, and the right superior and the middle cerebellar peduncles. The diagnosis was made by microscopic examination and culture of a brain biopsy.
Evaluation of CMV Brite kit for detection of cytomegalovirus pp65 antigenemia in peripheral blood leukocytes by immunofluorescence
Authors: Landry M L , Ferguson D , Stevens-Ayers T , de Jonge M W , Boeckh M
Published: January 2020

The CMV Brite antigenemia kit was compared with culture and an established cytomegalovirus pp65 antigenemia assay (CMV AG). Of 300 clinical specimens tested, 92 were positive by CMV Brite, 83 were positive by CMV AG, and 34 were positive by culture. Discrepancies could be attributed to anticytomegalovirus therapy or low-level antigenemia.