bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of BBL CHROMagar orientation medium for detection and presumptive identification of urinary tract pathogens
Authors: Hengstler K A , Hammann R , Fahr A M
Published: January 2020

The microbiological performance of BBL CHROMagar Orientation medium and CPS ID2 agar was compared to that of Columbia agar with 5% sheep blood and MacConkey agar without crystal violet for the enumeration and presumptive identification of bacteria responsible for urinary tract infections. Of a total of 658 clinical urine specimens, 118 specimens yielded no growth, 402 specimens yielded growth with cell counts of > or = 10(5) CFU/ml, and 138 specimens yielded growth with cell counts of < 10(5) CFU/ml. Of the specimens with cell counts of > or = 10(5) CFU/ml, 163 were pure cultures and 239 were mixed cultures. A total of 266 Escherichia coli organisms were isolated on both chromogenic media, 260 were isolated on blood agar, and 248 were isolated on MacConkey agar. One strain (0.4%) failed to develop the expected pink color on CHROMagar Orientation medium, and 23 strains (8.7%) failed to develop the expected pink color on CPS ID2 agar. Enterococci (CHROMagar Orientation medium, n = 266; CPS ID2 agar, n = 265) produced small blue-green colonies on both chromogenic media. Fifty of the mixed cultures contained enterococci that were detected only on the chromogenic media. The Klebsiella-Enterobacter-Serratia (KES) and the Proteus-Morganella-Providencia (PMP) groups could be identified on both chromogenic media. Of 66 isolates of the KES group, 63 grew with the expected color on CHROMagar Orientation medium and 58 of 64 isolates grew with the expected color on CPS ID2 agar. Other microorganisms required further identification. The use of chromogenic medium formulations offers a time-saving method for the reliable detection, enumeration, and presumptive identification of urinary tract pathogens. One of the greatest advantages of these media is the easy recognition of mixed cultures.
Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper
Authors: Cassol S , Gill M J , Pilon R , Cormier M , Voigt R F , Willoughby B , Forbes J
Published: January 2020

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.
Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods
Authors: Seifert H , Dijkshoorn L , Gerner-Smidt P , Pelzer N , Tjernberg I , Vaneechoutte M
Published: January 2020

At least 19 genomic species are recognized as constituting the genus Acinetobacter. However, little is known about the natural reservoirs of the various members of the genus. An epidemiological study was therefore performed to investigate the colonization with Acinetobacter spp. of the skin and mucous membranes of 40 patients hospitalized in a cardiology ward and 40 healthy controls. Single samples were obtained once from each of nine different body sites, i.e., forehead, ear, nose, throat, axilla, hand, groin, perineum, and toe web. Identification of Acinetobacter isolates was achieved by using phenotypic properties and was compared to identification by amplified ribosomal DNA restriction analysis. Selected isolates were further investigated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ribotyping, and DNA-DNA hybridization. Plasmid profile analysis was used for epidemiological typing. Thirty patients (75%) and 17 controls (42.5%) were found to be colonized with Acinetobacter spp., and the colonization rates of patients increased during their hospital stay. The most frequently isolated species were Acinetobacter lwoffii (47%), A. johnsonii (21%), A. radioresistens (12%), and DNA group 3 (11%). In contrast, A. baumannii and DNA group 13TU, the most important nosocomial Acinetobacter spp., were found only rarely on human skin (0.5 and 1%, respectively) and their natural habitat remains to be defined. A good correlation between phenotypic and genotypic methods for identification of Acinetobacter spp. was observed, and only two isolates could not be assigned to any of the known DNA groups.
Comparison of a photometric method with standardized methods of antifungal susceptibility testing of yeasts
Authors: Clancy C J , Nguyen M H
Published: January 2020

We determined the fluconazole MICs for 101 clinical isolates of Candida and Cryptococcus neoformans using the macro- and microdilution methods recommended by the National Committee for Clinical Laboratory Standards. We compared the MICs obtained by these methods with those obtained by a photometric assay that quantified the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by viable fungi. The MIC determined by this method was defined as the highest fluconazole concentration associated with the first precipitous drop in optical density. For Candida, both the MTT and the microdilution methods demonstrated excellent agreement with the standard macrodilution method. The MTT method, however, generated MICs at 24 h that were comparable to those generated by the standard macrodilution method, whereas the microdilution method required 48 h. For C. neoformans, the levels of agreement between the MICs determined by the MTT and microdilution methods after 48 h and those determined by the standard 72-h macrodilution method were 94% (29 of 31) and 94% (29 of 31), respectively. The MTT method therefore provided results comparable to those of currently recommended methods and had the advantages of a more rapid turnaround time and potential adaptability to use as an automated system. Furthermore, the MICs determined by the MTT method were determined photometrically, thereby eliminating reader bias.
Seroprevalence of Bartonella henselae infection and correlation with disease status in cats in Switzerland
Authors: Glaus T , Hofmann-Lehmann R , Greene C , Glaus B , Wolfensberger C , Lutz H
Published: January 2020

The prevalence of infection with Bartonella henselae was investigated in cats from different areas of Switzerland. Serum samples of 728 cats were examined for antibodies to B. henselae by immunofluorescent antibody testing, and the results were analyzed with a view to a possible correlation between a positive titer and signalment, clinical signs, infection with feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV), or feline spumavirus (FeSFV), and the living environments of the cats. The seroprevalence in all cats was 8.3%. No significantly different prevalence was found in sick versus healthy cats (9.2 versus 7.2%); however, in sick cats seropositive for B. henselae, there was an increased frequency of stomatitis and a variety of diseases of the kidneys and the urinary tract. There was an increased prevalence of B. henselae in cats positive for FCoV (P = 0.0185) or FeSFV (P = 0.0235) and no statistically significant increased prevalence in cats infected with FeLV or FIV. There was no correlation between a positive titer and sex or breed. The same prevalence of B. henselae antibodies was found in cats with and without access to the outdoors and in cats from single- and multicat households. The seroprevalence was increased in cats living south of the Alps (12.1%); however, this difference was not significant (P = 0.0616).
Growth and morphological transformations of Helicobacter pylori in broth media
Authors: Andersen A P , Elliott D A , Lawson M , Barland P , Hatcher V B , Puszkin E G
Published: January 2020

Helicobacter pylori, a cause of peptic ulcer disease and certain types of gastric cancers, has usually been cultured on diverse agar-based media, resulting in a requirement for 2 to 4 days of growth at 37 degrees C. We have developed a novel broth medium consisting of a base medium supplemented with 2% newborn calf serum, Mg2+, Cu2+, Fe2+, Zn2+, Mn2+, and 1 mg of lysed human erythrocytes per ml. This medium supports rapid growth of H. pylori, with a doubling time of about 50 min. Optimal growth was obtained in a pH range higher than that supporting most other gram-negative bacteria (at pH 8.5). H. pylori cultured in this supplemented broth retains the spiral morphology seen in both histological sections and cultures from agar-based media and also retains a high urease activity. After 18 h in this broth, H. pylori transforms to a coccal form with a complete loss of urease activity. Previously these cocci have been reported to be senescent, since they could not be subcultured on agar medium. Our experiments suggest that some of the cocci can revert back to the spiral morphology with full recovery of urease activity when subcultured in fresh microaerobic broth medium.
Rapid diagnosis of human brucellosis by peripheral-blood PCR assay
Authors: Queipo-Ortuño M I , Morata P , Ocón P , Manchado P , Colmenero J D
Published: January 2020

A single-step PCR assay with genus-specific primers for the amplification of a 223-bp region of the sequence encoding a 31-kDa immunogenetic Brucella abortus protein (BCSP31) was used for the rapid diagnosis of human brucellosis. We examined peripheral blood from 47 patients, with a total of 50 cases of brucellosis, and a group of 60 control subjects, composed of patients with febrile syndromes of several etiologies other than brucellosis, asymptomatic subjects seropositive for Brucella antibodies, and healthy subjects. Diagnosis of brucellosis was established in 35 cases (70%) by isolation of Brucella in blood culture and in the other 15 cases (30%) by clinical and serological means. The sensitivity of our PCR assay was 100%, since it correctly identified all 50 cases of brucellosis, regardless of the duration of the disease, the positivity of the blood culture, or the presence of focal forms. The specificity of the test was 98.3%, and the only false-positive result was for a patient who had had brucellosis 2 months before and possibly had a self-limited relapse. In those patients who relapsed, the results of our PCR assay were positive for both the initial infection and the relapse, becoming negative once the relapse treatment was completed and remaining negative in the follow-up tests at 2, 4, and 6 months. In conclusion, these results suggest that the PCR assay is rapid and easy to perform and highly sensitive and specific, and it may therefore be considered a useful tool for diagnosis of human brucellosis.
Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 mycobacterial species
Authors: Devallois A , Goh K S , Rastogi N
Published: January 2020

PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. C. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175-178, 1993) was applied to 108 mycobacterial isolates representing 34 species to evaluate its potential as a rapid reference method. A total of 49 distinct patterns were obtained; 25 species were characterized by a single PRA pattern, while 9 species gave more than one specific pattern. An algorithm describing these 34 species (which includes five additional species and new subgroups of Mycobacterium kansasii, M. abscessus, and M. peregrinum) is proposed. A relatively simple and inexpensive method, PRA may be particularly helpful in routine clinical microbiology laboratories.
Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization
Authors: Goh S H , Santucci Z , Kloos W E , Faltyn M , George C G , Driedger D , Hemmingsen S M
Published: January 2020

A previous study (S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996) demonstrated that a 600-bp region of the chaperonin 60 (Cpn60) genes from various bacterial isolates could be amplified by PCR with a pair of degenerate primers and that the products could be used as species-specific probes for Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. lugdunensis, S. saprophyticus, and S. schleiferi. To further validate the utility of bacterial Cpn60 genes as universal targets for bacterial identification (ID), reverse checkerboard chemiluminescent hybridization experiments were performed with DNA probes from 34 different Staphylococcus species and subspecies. With the exception of probes from the Cpn60 genes of S. intermedius and S. delphini, which cross hybridized, all were species specific. Two subspecies of both S. capitis and S. cohnii were differentiated from one another, while DNAs from the two S. schleiferi subspecies cross hybridized. When 40 known Staphylococcus isolates were tested in a blind experiment by the Cpn60 gene method, 36 strains, representing six species and one subspecies (S. sciuri, S. caseolyticus, S. hominis, S. warneri, S. hyicus, S. haemolyticus, and S. capitis subsp. ureolyticus), were correctly identified. DNA from the four remaining isolates, known to be S. hyicus bovine strains, failed to hybridize to DNA from the S. hyicus target strain or any other Staphylococcus species. However, DNAs from these S. hyicus isolates did cross hybridize with each other. New DNA sequence data and evidence from previous studies suggest some genetic divergence between the two groups of S. hyicus isolates. Our results demonstrate that this Cpn60 gene-based ID method has the potential to be a basic method for bacterial ID. Studies are in progress to further validate the utility of this Cpn60 gene system for ID of Staphylococcus and other genera, including those of slow-growing microorganisms.
Disk diffusion test interpretive criteria and quality control recommendations for testing linezolid (U-100766) and eperezolid (U-100592) with commercially prepared reagents
Authors: Biedenbach D J , Jones R N
Published: January 2020

Two new oxazolidinones were tested to determine interpretive susceptibility testing criteria for MIC and disk diffusion methods. Commercial lots of linezolid (formerly U-100766) and eperezolid (formerly U-100592) disks containing 30 microg of drug were tested against 728 isolates of bacteria with defined mechanisms of resistance. Results from linezolid were highlighted because of its choice for clinical development. By using preliminary pharmacokinetic data, a tentative susceptibility breakpoint of < or = 4 microg/ml was selected. Corresponding breakpoint zone diameters for linezolid were > or = 21 mm (< or = 4 microg/ml) for susceptibility and < or = 17 mm (> or = 16 microg/ml) for resistance. Regression statistics demonstrated a high correlation coefficient (r > or = 0.98), and absolute categorical agreement between methods was obtained, when staphylococci and enterococci were tested with the cited criteria. When Streptococcus spp. (including S. pneumoniae) were tested, only the susceptibility breakpoint was suggested. Quality control (QC) guidelines for linezolid disk diffusion tests were established by a multilaboratory trial as follows: 27 to 31 mm for Staphylococcus aureus ATCC 25923 and 28 to 34 mm for S. pneumoniae ATCC 49619. More than 95% of all QC results were within these proposed ranges. Although not advanced to clinical trials, eperezolid demonstrated potency comparable to that of linezolid and had identical interpretive testing criteria. These preliminary interpretive criteria and QC limits (accepted by the National Committee for Clinical Laboratory Standards) should be applied to linezolid tests during the clinical-trial phases of oxazolidinone drug development in order to ensure test accuracy and reproducibility.
Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis
Authors: Cegielski J P , Devlin B H , Morris A J , Kitinya J N , Pulipaka U P , Lema L E , Lwakatare J , Reller L B
Published: January 2020

Nucleic acid amplification techniques for the diagnosis of tuberculosis (TB) are rapidly being developed. Scant work, however, has focused on pericardial TB. Using cryopreserved specimens from a prior study of pericarditis, we compared PCR to culture and histopathology for the diagnosis of tuberculous pericarditis in 36 specimens of pericardial fluid and 19 specimens of pericardial tissue from 20 patients. Fluid and tissue were cultured on Lowenstein-Jensen and Middlebrook solid media and in BACTEC radiometric broth. Tissue specimens were stained with hematoxylin-eosin, Ziehl-Neelsen, auramine O, and Kinyoun stains and were examined for granuloma formation and acid-fast bacilli. PCR was performed with both fluid and tissue with IS6110-based primers specific for the Mycobacterium tuberculosis complex by published methods. Sixteen of the 20 patients had tuberculous pericarditis and 4 patients had other diagnoses. TB was correctly diagnosed by culture in 15 (93%) patients, by PCR in 13 (81%) patients, and by histology in 13 of 15 (87%) patients. PCR gave one false-positive result for a patient with Staphylococcus aureus pericarditis. Considering the individual specimens as the unit of analysis, M. tuberculosis was identified by culture in 30 of 43 specimens (70%) from patients with tuberculous pericarditis and by PCR in 14 of 28 specimens (50%) from patients with tuberculous pericarditis (P > 0.15). The sensitivity of PCR was higher with tissue specimens (12 of 15; 80%) than with fluid specimens (2 of 13; 15%; P = 0.002). In conclusion, the overall accuracy of PCR approached the results of conventional methods, although PCR was much faster. Therefore, PCR merits further development in this regard. The sensitivity of PCR with pericardial fluid was poor, and false-positive results with PCR remain a concern.
Detection of antibodies to Candida albicans germ tubes for diagnosis and therapeutic monitoring of invasive candidiasis in patients with hematologic malignancies
Authors: García-Ruiz J C , del Carmen Arilla M , Regúlez P , Quindós G , Alvarez A , Pontón J
Published: January 2020

We prospectively investigated the ability of detection of antibodies to Candida albicans germ tubes (CAGT) to diagnose invasive candidiasis in 95 consecutive admissions of 73 patients with hematologic disorders undergoing intensive chemotherapy. The episodes were divided into three groups according to clinical and microbiological diagnosis. Group 1 comprised eight admissions of eight patients with invasive candidiasis. Group 2 comprised 42 admissions of 34 patients without evidence of invasive candidiasis. Group 3 comprised the remaining 45 admissions of 37 patients with febrile episodes which were not diagnosed by microbiological culture. Antibodies to CAGT were detected in 87.5% of group 1 patients. Detection of antibodies to CAGT in patients with Candida fungemia was delayed somewhat relative to the time the blood culture was positive, but antibodies to CAGT were detected earlier than a diagnosis was made in patients with deep-tissue candidiasis. Sera from 2 admissions in group 2 and 12 admissions in group 3 revealed antibodies to CAGT. At a titer of > or = 1:20, detection of antibodies to CAGT had a sensitivity of 87.5%, specificity of 95.2%, positive predictive value of 77.8%, and negative predictive value of 97.6%. Antibodies to CAGT were usually detected before beginning of empiric antifungal therapy. Titers of antibodies to CAGT were maintained in most patients who died but declined and eventually disappeared in the patients who survived. Since antibodies to CAGT were detected in all patients with tissue-proven invasive candidiasis but negative by blood culture, detection of antibodies to CAGT complemented blood cultures for diagnosis and therapeutic monitoring of patients with hematologic malignancies and invasive candidiasis.
Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis
Authors: Hald B , Madsen M
Published: January 2020

Living in a household with a dog or cat has previously been identified as a significant risk factor for acquiring campylobacteriosis, in particular, with reference to Campylobacter upsaliensis infection. In a cross-sectional study carried out in Denmark between August and December 1996, 72 healthy puppies and 42 healthy kittens, aged between 11 and 17 weeks, were sampled for fecal campylobacter shedding by culture of rectal swab specimens on blood-free agar base with cefoperazone at 32 mg/liter and amphotericin at 10 mg/liter and on blood-free agar base with cefoperazone at 8 mg/liter, teicoplanin at 4 mg/liter, and amphotericin at 10 mg/liter. Additionally, with respect to the C. upsaliensis transmission potential of poultry, a chicken cloacal swab sample from each of 100 different broiler flocks was included in the study for comparison. We found 21 (29%) of the puppies positive for Campylobacter spp., with a species distribution of 76% C. jejuni, 5% C. coli, and 19% C. upsaliensis. Of the kittens examined, two (5%) excreted campylobacters; both strains were C. upsaliensis. None of the chicken samples examined were found to be positive for C. upsaliensis. We concluded that young puppies and kittens are potential transmitters of human-pathogenic Campylobacter spp., including C. upsaliensis, while poultry seems negligible in C. upsaliensis epidemiology.
Characterization of Aeromonas spp. isolated from humans with diarrhea, from healthy controls, and from surface water in Bangladesh
Authors: Kühn I , Albert M J , Ansaruzzaman M , Bhuiyan N A , Alabi S A , Islam M S , Neogi P K , Huys G , Janssen P , Kersters K , Möllby R
Published: January 2020

Aeromonas isolates from patients with diarrhea in Bangladesh (n = 69), from healthy controls (n = 11), and from surface water (n = 40) were analyzed with respect to their hybridization groups (HGs) by the aid of fatty acid methyl ester (FAME) characterization and DNA fingerprinting by AFLP, biochemical phenotypes (Phe-nePlate [PhP] types), and the production of hemolysin and cytotoxin. The aim of the investigation was to find out whether certain strains carrying virulence factors predominated among patient isolates. According to FAME and/or AFLP analysis, most human isolates were allocated to DNA HGs 4 (Aeromonas caviae) and 1 (A. hydrophila). Most environmental strains were allocated to HG8 (A. veronii biogroup sobria) and HG4 (A. caviae), and only one was of HG1. According to PhP typing, the diversity among patient isolates was lower than that among other strains, and two dominating PhP types (types BD-1 and BD-2) were identified in 29 and 30% of the patient isolates, respectively. PhP type BD-1 was also common among the environmental isolates, whereas PhP type BD-2 was only identified in two of the other isolates. Twenty-five of 26 isolates belonging to HG1 were of the same PhP type (BD-2), whereas isolates of other common HGs were more diverse according to their PhP types. Hemolytic and cytotoxin-producing strains occurred more frequently among the environmental isolates than among patient isolates. However, the hemolytic and cytotoxic activities among human isolates was strongly correlated to the HG1/BD-2 type, which, in addition, showed high cytotoxin titers (median values, 1/512 compared to 1/128 for cytotoxin-positive isolates belonging to other types). Thus, the HG1/BD-2 type may represent a pathogenic A. hydrophila type that is able to produce diarrhea in humans.
Invasive Nattrassia mangiferae infections: case report, literature review, and therapeutic and taxonomic appraisal
Authors: Sigler L , Summerbell R C , Poole L , Wieden M , Sutton D A , Rinaldi M G , Aguirre M , Estes G W , Galgiani J N
Published: January 2020

We report on a case of subcutaneous infection of the arm caused by the coelomycetous fungus Nattrassia mangiferae (formerly Hendersonula toruloidea) in a steroid-dependent diabetic man with chronic obstructive lung disease. The man was a resident of Arizona, where the fungus is known to be endemic on Eucalyptus camaldulensis and on citrus trees. Diagnosis of fungal infection was made by observation of narrow hyphal filaments by histopathology of biopsy specimens and isolation of a fast-growing black mold which demonstrated hyphae and arthroconidia of varying widths typical of the Scytalidium synanamorph (S. dimidiatum). The formation of pycnidia, which at maturity expressed conidia with a central median dark band, allowed for the confirmation of the isolate as N. mangiferae. Remission of the lesions occurred following intravenous therapy with amphotericin B, followed by topical clotrimazole treatment. We use this patient's case report as an opportunity to review the literature on cases of deep infection caused by Scytalidium species, to evaluate the antifungal susceptibilities of a spectrum of Scytalidium isolates, and to review the taxonomy of Scytalidium species isolated from human infections.
Seroepidemiology of Helicobacter pylori infection in a cohort of children monitored from 6 months to 11 years of age
Authors: Granström M , Tindberg Y , Blennow M
Published: January 2020

A cohort of Swedish children was monitored from 6 months to 11 years of age. Immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori were measured in 1,857 serum samples, drawn at the ages of 6, 8, 10, 18 months and 2, 4, and 11 years. Of the 294 children, 40 (13.6%) were found to have been infected at some time. However, at 11 years of age, only 6 of 201 (3%) children were seropositive. The highest seroprevalence of positive results, 10%, was found at 2 years of age, and the highest incidence of 13.3% could be calculated for the period between 18 months and 2 years of age. There were no confirmed additional cases for children between 4 and 11 years of age. Infection with H. pylori thus occurs at an early age in a developed country (as well as in developing countries), and spontaneous clearance seems to be common.
Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing
Authors: Kim B J , Kim S Y , Park B H , Lyu M A , Park I K , Bai G H , Kim S J , Cha C Y , Kook Y H
Published: January 2020

Rifampin susceptibility of 32 rifampin-resistant and 26 rifampin-susceptible Mycobacterium tuberculosis strains was analyzed by PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing within the 157-bp region of the rpoB gene (Ala500 to Val550). Two false-positive PCR-SSCP results were observed among the susceptible strains due to the silent mutation Gln513 (CAA-->CAG) and the deletion mutation Thr508 and Ser509. Another silent mutation [Leu511 (CTG-->CTA)], combined with the mutation Ser531-->Leu, was observed in a resistant strain. These results suggest that to rule out false-positive PCR-SSCP results, sequencing of the target DNA is required.
Fulminating bacteremia and pneumonia due to Bacillus cereus
Authors: Miller J M , Hair J G , Hebert M , Hebert L , Roberts F J , Weyant R S
Published: January 2020

We present two cases of rapidly progressing, fatal pneumonia caused by Bacillus cereus. These cases are interesting in that B. cereus, even from blood or sputum specimens, may often be considered a contaminant and receive inadequate attention. Also of interest was the fact that the two patients resided in the same area of the state, were welders by trade, and became ill within a few days of each other, yet there was no epidemiologic link between them.
Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis
Authors: Hjelle B , Jenison S , Torrez-Martinez N , Herring B , Quan S , Polito A , Pichuantes S , Yamada T , Morris C , Elgh F , Lee H W , Artsob H , Dinello R
Published: January 2020

To develop a rapid antibody test for Sin Nombre hantavirus (SNV) infection for diagnosis of hantavirus pulmonary syndrome (HPS) in field settings where advanced instrumentation is not available, a strip immunoblot assay bearing four immobilized antigens for SNV and a recombinant nucleocapsid protein antigen of Seoul hantavirus (SEOV) was prepared. The SNV antigens included a full-length recombinant-expressed nucleocapsid (N) protein (rN), a recombinant-expressed G1 protein (residues 35 to 117), and synthetic peptides derived from N (residues 17 to 59) and G1 (residues 55 to 88). On the basis of the observed reactivities of hantavirus-infected patient and control sera, we determined that a positive assay requires reactivity with SNV or SEOV rN antigen and at least one other antigen. Isolated reactivity to either viral rN antigen is indeterminate, and any pattern of reactivity that does not include reactivity to an rN antigen is considered indeterminate but is unlikely to represent hantavirus infection. Fifty-eight of 59 samples from patients with acute SNV-associated HPS were positive according to these criteria, and one was initially indeterminate. Four of four samples from patients with HPS due to other hantaviruses were positive, as were most samples from patients with SEOV and Puumala virus infections. Of 192 control serum samples, 2 (1%) were positive and 2 were indeterminate. Acute SNV infection was distinguishable from remote SNV infection or infection with hantaviruses other than SNV by the presence of G1 peptide antigen reactivities in the former. The strip immunoblot assay shows promise for the detection of SNV antibodies early in the course of HPS.
Antigenic and molecular analyses of different Chlamydia pneumoniae strains
Authors: Jantos C A , Heck S , Roggendorf R , Sen-Gupta M , Hegemann J H
Published: January 2020

Chlamydia pneumoniae is an important human respiratory pathogen. Classification of C. pneumoniae isolates into distinguishable serovars or genotypes has not yet been reported. To determine whether antigenic or molecular variants among C. pneumoniae isolates exist, six strains were studied via immunoblot analysis and DNA sequence determination of the entire major outer membrane protein (MOMP) gene omp1. The strains included four prototype strains and two clinical isolates from our laboratory. Immunoblot analysis of sera from patients infected with C. pneumoniae revealed antigenic differences between the C. pneumoniae strains. Strong reactivity of one serum sample with a 65-kDa protein in two C. pneumoniae strains which was not observed with the other strains was the most prominent finding. All sera reacted with the 40-kDa MOMP. Comparison of the omp1 DNA sequences revealed that the omp1 genes of all strains were identical and were 100% identical to the sequence of the omp1 gene of C. pneumoniae AR-39. The results of this study demonstrate that unlike C. trachomatis, the omp1 gene is conserved in C. pneumoniae. Furthermore, it was shown that C. pneumoniae strains are antigenically different. This finding indicates that more than one serovar of C. pneumoniae exist.
Evaluation of the Gen-Probe Chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women
Authors: Pasternack R , Vuorinen P , Miettinen A
Published: January 2020

We evaluated the Gen-Probe Chlamydia trachomatis transcription-mediated amplification (TMA) assay with urine specimens for the detection of C. trachomatis infections in women. The novel test, based on the isothermal amplification of chlamydial RNA, was compared with the Roche Amplicor PCR with urine and cell culture with endocervical specimens. First-catch urine and endocervical swab specimens were collected from a total of 561 patients, of whom 70 (12.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of TMA with urine were 91.4 and 99.6%, respectively, and those of Amplicor PCR were 97.1 and 99.8%, respectively. By repeated analysis of the specimens with discrepant results, the sensitivity of TMA could be increased to 99%, indicating that some methodological improvements in the assay are still to be expected. The sensitivity of PCR could be increased to 100% by the elimination of DNA polymerase inhibitors in a repeated analysis. The sensitivity and specificity of cell culture with cervical specimens were 85.7 and 100%, respectively. The results indicate that TMA with urine specimens from women is a sensitive and specific assay for the detection of C. trachomatis, providing a new noninvasive technique for the screening of chlamydial infections in women.
Characterization of mycobacterial isolates phylogenetically related to, but different from Mycobacterium simiae
Authors: Tortoli E , Piersimoni C , Kirschner P , Bartoloni A , Burrini C , Lacchini C , Mantella A , Muzzi G , Tosi C P , Penati V , Scarparo C , Simonetti M T , Böttger E C
Published: January 2020

The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade resistance to the majority of antimycobacterial drugs. Three of the isolates were clinically significant according to stringent criteria. Sophisticated techniques, like genetic sequencing or HPLC, by now seem indispensable for differentiating unusual and new mycobacteria from well-established ones.
Restriction enzyme analysis of the virulence plasmids of VapA-positive Rhodococcus equi strains isolated from humans and horses
Authors: Nicholson V M , Prescott J F
Published: January 2020

Restriction enzyme digestion patterns of the large virulence plasmids of 8 human and 37 foal isolates of virulence-associated protein (VapA)-positive Rhodococcus equi strains from different sources were compared. Foal isolates came from five continents. Digestion with EcoRI divided these plasmids into three closely related types, and digestion with BamHI divided them into three major types which corresponded to the EcoRI types. The only EcoRI and BamHI type 3 plasmid was from a single foal isolate obtained from Japan. There are thus two major but related virulence plasmids in isolates from foals. Geographic differences were noted, since foal isolates with the EcoRI type 1 plasmid digestion pattern tended to come mostly from the United States, Canada, European countries, India or Zimbabwe and foal isolates with EcoRI type 2 pattern tended to come mostly from Latin American countries. Only 8 of 38 different human isolates, mostly from AIDS patients, were VapA positive, in contrast to 37 of 42 foal isolates. VapA-positive isolates from humans possessed virulence plasmids of either EcoRI type 1 or EcoRI type 2. These results confirm that only a small proportion of human patients with R. equi infections acquire foal virulent R. equi.
Genetic analysis of a meningococcal population based on polymorphism of the pilA-pilB locus: a molecular approach for meningococcal epidemiology
Authors: Guibourdenche M , Giorgini D , Guèye A , Larribe M , Riou J Y , Taha M K
Published: January 2020

The genetic relationships between 88 meningococcal strains were analyzed by using the polymorphism of the pilA gene and the multilocus enzyme electrophoresis. While a good agreement was observed, correlation with antigenic formula (serogroup, serotype, and serosubtype) was incomplete. The inadequacy of serological classification alone in outbreak surveillance may be overcome by DNA-based approaches.
An Ehrlichia strain from a llama (Lama glama) and Llama-associated ticks (Ixodes pacificus)
Authors: Barlough J E , Madigan J E , Turoff D R , Clover J R , Shelly S M , Dumler J S
Published: January 2020

An ehrlichia was identified in the blood of a diseased llama (lama glama). Sequencing of its 16S rRNA gene showed the ehrlichia to be closely related to members of the Ehrlichia phagocytophila genogroup. The agent was also found in a pool of ticks (Ixodes pacificus) collected at the llama site.
Fatal sepsis caused by Corynebacterium amycolatum in a premature infant
Authors: Berner R , Pelz K , Wilhelm C , Funke A , Leititis J U , Brandis M
Published: January 2020

Corynebacterium amycolatum has not been reported as a cause of human infections up to now, but usually the bacterium is misidentified in clinical specimens as Corynebacterium xerosis. We report the first case of neonatal sepsis due to Corynebacterium amycolatum with a fatal outcome in a premature infant.
Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis, and Eperythrozoon suis
Authors: Rikihisa Y , Kawahara M , Wen B , Kociba G , Fuerst P , Kawamori F , Suto C , Shibata S , Futohashi M
Published: January 2020

Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed clinical signs and splenomegaly, and three isolates were maintained by passage in mice. Tetracyclines were effective in preventing infection of mice with these agents, but streptomycin and penicillin were ineffective. The agents did not grow in bacterial growth media or chicken embryos. In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes. Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter. On the basis of these results, the isolates were identified as Haemobartonella muris. There was no antigenic cross-reactivity with Rickettsia or Ehrlichia spp. or other related organisms. Western immunoblot analysis of three strains of H. muris with mouse antisera to H. muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa. By heteroduplex analysis of the three PCR-amplified segments of the 16S rRNA genes, the three strains of H. muris were found to be identical. The 16S rRNA genes of one of the H. muris strains, four strains of H. felis, and two strains of Eperythrozoon suis were sequenced and compared. The sequences of two strains of H. felis from cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida, but the similarity of sequences between the California and the Ohio-Florida strains was only 85%. The sequence of an H. muris strain was unique and was more closely related to that of the Ohio-Florida strain of H. felis (89%) than to that of the California strain of H. felis (84%). The sequence of E. suis from a pig in Illinois was identical to that from another pig from Taiwan. The similarity of the 16S rRNA gene sequence of E. suis with those of three Haemobartonella strains was 84 to 92%, with that of E. suis being most similar to that of the H. felis strain from California. In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonella spp. and E. suis formed a distinct clade more closely related to Mycoplasma spp. (79 to 83% similarity) than to Anaplasma marginale (72 to 75% similarity). Our results suggest that the Haemobartonella spp. and E. suis may be reclassified in the same genus in the family Mycoplasmataceae.
PCR detection of Pneumocystis carinii in bronchoalveolar lavage specimens: analysis of sensitivity and specificity
Authors: Ribes J A , Limper A H , Espy M J , Smith T F
Published: January 2020

Although PCR detection of Pneumocystis carinii DNA has been described, little is known about the sensitivity or specificity of the assay in routine laboratory practice. We had the unique opportunity to use a bronchoalveolar lavage (BAL) specimen bank with samples for which the direct examination results for P. carinii were known. DNA purified from 129 selected specimens was amplified by using the primers described previously (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moton, and J. M. Hopkin, Mol. Biochem. Parasitol. 43:69-76, 1990). Of the 129 specimens, 37 were positive for P. carinii by direct examination. All 37 specimens were positive for P. carinii by PCR, yielding a 100% sensitivity and 100% negative predictive value for the assay. An additional 23 specimens were repeatedly positive for P. carinii by PCR but were not positive by direct examination. Review of the patient charts for these specimens with discordant results demonstrated that five of the patients were actually positive for P. carinii, as determined by either biopsy or examination of repeat or prior BAL specimens. A response to empiric therapy for P. carinii pneumonia was seen in an additional two patients. Of the remaining specimens, 8 produced no significant isolates other than P. carinii, while 12 contained culture-confirmed significant respiratory pathogens in addition to P. carinii (two fungal, nine bacterial, and one viral pathogen). Cytomegalovirus, which was of unknown significance, was isolated from 16 additional specimens. Overall, the specificity of the PCR assay was 79.3% compared to the results of direct examination. We hypothesized that the apparently poor specificity of the PCR assay was due to the increased sensitivity of the assay compared to that of direct examination. The sensitivity of the PCR assay was therefore assessed with BAL specimens containing P. carinii cysts. Serial dilutions of this preparation were evaluated by direct examination and PCR. PCR was found to be 100-fold more sensitive than direct examination, which detected one to two cysts per amplification. No false-positive results were detected in controls containing no DNA or by using target DNA from various fungal, viral, or bacterial respiratory pathogens. We conclude that PCR detection of P. carinii in BAL specimens is very sensitive and should be considered for patients whose specimens do not yield a diagnosis. The increased sensitivity of the PCR assay may help to identify those patients with low-titer infections who might benefit from directed antibiotic therapy for P. carinii and would otherwise be missed by direct examination alone.
Determination of anti-streptolysin O antibody titer by a new passive agglutination method using sensitized toraysphere particles
Authors: Kodama T , Ichiyama S , Morishita Y , Fukatsu T , Shimokata K , Nakashima N
Published: January 2020

We developed a new method for determining the anti-streptolysin O (ASO) titer on the basis of a passive agglutination reaction. This new method uses Toraysphere particles sensitized with streptolysin O. This method was used to measure the ASO titer in 130 serum samples (from 31 patients with post-streptococcal glomerulonephritis, 26 patients with group A streptococcal pharyngitis, 5 patients with scarlet fever, 29 patients with non-group A streptococcal pharyngitis, 20 healthy children, and 19 healthy adults). The results were compared with those obtained simultaneously by two conventional methods, the Rantz-Randall and the NA-Latex-ASL methods. The results obtained by the Toraysphere method showed an excellent correlation with those obtained by each conventional method (correlation coefficients, 0.932 for the Rantz-Randall method and 0.913 for the NA-Latex-ASL method). The performance of each method was evaluated with respect to the clinical and microbiological diagnoses for each patient. The sensitivity, specificity, positive predictive value, and negative predictive value for the Toraysphere method were 76, 84, 81, and 79%, respectively. The corresponding values for the Rantz-Randall method were 48, 78, 67, and 62%, respectively, while those for the NA-Latex-ASL method were 60, 82, 76, and 69%, respectively. Because the Toraysphere method is simple and reproducible, has a short reaction time, and has a clear-cut endpoint for the analysis, we recommend it for routine use in determining the ASO titer in the clinical microbiology laboratory.