bluuebunny/crossref_metadata_2025 huggingface dataset:
Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes
Authors: Zein N N , Germer J J , Wendt N K , Schimek C M , Thorvilson J N , Mitchell P S , Persing D H
Published: January 2020

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.
Clonal analysis and identification of epidemic strains of methicillin-resistant Staphylococcus aureus by antibiotyping and determination of protein A gene and coagulase gene polymorphisms
Authors: Hoefnagels-Schuermans A , Peetermans W E , Struelens M J , Van Lierde S , Van Eldere J
Published: January 2020

Forty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates with known genetic and epidemiological relatedness and different degrees of transmission were analyzed by antibiotyping, protein A gene polymorphism analysis, and coagulase gene polymorphism analysis. The three typing systems were evaluated for their performance and convenience to define clones and to discriminate between epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA). Antibiotyping and AluI restriction fragment length polymorphism analysis of the coagulase gene were able to define clones in the same way as DNA macrorestriction analysis (SmaI). However, both techniques presented disadvantages, making neither of them useful as a single typing method. Protein A gene polymorphism analysis appeared to be of no value for clonal analysis. None of the three typing methods was able to differentiate between EMRSA and SMRSA.
Phenotypic and genotypic changes in Vibrio cholerae O139 Bengal
Authors: Albert M J , Bhuiyan N A , Talukder K A , Faruque A S , Nahar S , Faruque S M , Ansaruzzaman M , Rahman M
Published: January 2020

To find reasons for the recent decline of Vibrio cholerae O139 Bengal cholera in Bangladesh, phenotypic and genotypic changes in O139 isolates obtained from patients with cholera from 1993 to 1996 were studied. The isolates were tested for the presence of ctx and tcpA genes, hemagglutinin/protease (HA/P), capsule, D-mannose-sensitive hemagglutinin (MSHA), L-fucose-sensitive hemagglutinin (FSHA), tube test (tube) and CAMP test (CAMP) hemolytic activities, resistance to 2,4-diamino-6,7-diisopropyl pteridine (O/129) and trimethoprim-sulfamethoxazole (TMP-SMX), and genotype by pulsed-field gel electrophoresis (PFGE). All isolates possessed ctx and tcpA genes, HA/P, and a capsule. Most isolates were negative for FSHA, but although the majority of the isolates were positive for MSHA, no discernible trend in the activity was found during the study period. All early isolates were CAMP hemolysin positive and resistant to the vibriostatic compound O/129 and TMP-SMX, the two properties that could be used for the presumptive diagnosis of O139 cholera. However, subsequently, isolates that were CAMP hemolysin negative and susceptible to TMP-SMX and O/129 were increasingly encountered, with all the 1996 isolates being so, which suggested that these properties can no longer be used for the presumptive diagnosis of O139 cholera. V. cholerae O139 isolates that were CAMP hemolysin positive and resistant to O/129 and TMP-SMX produced a disease of greater severity than that caused by the CAMP hemolysin-negative and susceptible isolates on the basis of the lengths of stay of the hospitalized patients. The study period witnessed the evolution of four different genotypes by PFGE. All of these data suggested that the V. cholerae O139 isolates have undergone changes in some properties. However, how these changes influenced their prevalence relative to that of V. cholerae O1 in human infection is not clear. Studies of the environmental factors will provide the key for an understanding of the relative abundance of these vibrios.
Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine
Authors: Goessens W H , Mouton J W , van der Meijden W I , Deelen S , van Rijsoort-Vos T H , Lemmens-den Toom N , Verbrugh H A , Verkooyen R P
Published: January 2020

We compared the Gen-Probe transcription-mediated amplification assay (AMP CT), the Abbott LCx assay, and the Roche COBAS AMPLICOR assay for the detection of Chlamydia trachomatis in a mixed population in urine samples. First-void urine, urethral specimens, and cervical specimens in females were obtained from 1,000 patients (544 males and 456 females) visiting the outpatient sexually transmitted disease clinic of our hospital. The prevalence of C. trachomatis infection was 7.7% as determined by tissue culture of urethral and cervical specimens. The sensitivities of LCx, COBAS AMPLICOR, and AMP CT compared to cell culture were 79, 86, and 78%, respectively. Sensitivity and specificity were recalculated by using a new "gold standard", i.e., a sample was considered to be true positive if two or more techniques yielded positive results. Specimens positive only by cell culture or positive in only one commercial amplification technique were retested by a previously described in-house PCR. After discordance analysis the sensitivities of LCx, COBAS AMPLICOR, and AMP CT were 84, 93, and 85%, respectively. Specificity exceeded 99% for all three assays. With each method the sensitivity was lower for urine samples from females compared to urine samples from males. By application of this new gold standard, existing differences between methods are highlighted; future evaluations of new techniques should be validated against two or more amplification assays.
Histoplasma capsulatum sinusitis
Authors: Butt A A , Carreon J
Published: January 2020

Sinusitis is commonly reported in patients with AIDS. In addition to the usual bacterial pathogens isolated from immunocompetent patients, sinusitis in patients with AIDS may be caused by a variety of unusual bacteria, viruses, fungi, parasites, and mycobacteria. Histoplasma capsulatum has not typically been associated with sinusitis in either group of patients. We report a case of sinusitis caused by H. capsulatum in a patient with AIDS.
A new medium for the presumptive identification of dermatophytes
Authors: Salkin I F , Padhye A A , Kemna M E
Published: January 2020

A new medium, Dermatophyte Identification Medium (DIM) (trade mark pending), was specifically developed to eliminate problems of false-positive results associated with commercially marketed media, such as dermatophyte test medium (DTM). Previous investigations had demonstrated that DTM only partially suppressed growth of nondermatophytes and that several of these nondermatophytic fungi that were morphologically similar to dermatophytes caused false-positive results. Presumptive identification of an unknown isolate as a dermatophyte required only the transfer of a portion of the suspected colony recovered from the specimen to DIM. Positive results, evidenced by a change in the color of the medium, were observed within 24 to 48 h. In studies of over 500 isolates of dermatophytes and common nondermatophyte molds, as well as close to 600 yeast isolates, false-positive results were always associated with bacterial contamination of the mold isolates while false negatives were only observed with occasional isolates of Trichophyton verrucosum. DIM culture was an inexpensive, rapid, and accurate method for the presumptive identification of dermatophytes in the clinical mycology laboratory.
Evaluation of the ESP Culture System II for recovery of mycobacteria from blood specimens collected in isolator tubes
Authors: Tholcken C A , Huang S , Woods G L
Published: January 2020

The reliability of the ESP Culture System II (ESP II; AccuMed International, Westlake, Ohio), a continuously monitoring, nonradiometric mycobacterial culture system, for recovery of mycobacteria from sediments of blood collected in an Isolator tube was evaluated by comparing its performance to inoculation of the sediment onto Middlebrook 7H11/7H11 selective biplates. Of 1,704 blood specimens, 73 (4.3%) were positive for mycobacteria (68 Mycobacterium avium complex and 5 M. tuberculosis). Fifty-three specimens were positive by both methods; 13 were positive by ESP II only, and 7 were positive by Middlebrook agar only (chi square = 1.8; P > 0.05). The mean times to positivity were 15.6 days for ESP II and 19.0 days for Middlebrook agar (P < 0.01). The time to detection was the same for 13 specimens; ESP II was positive first for 33, and agar plates were positive first for 7. ESP II allowed recovery of more mycobacteria (90.4% of all isolates versus 82.2% for Middlebrook agar) from sediments of blood specimens collected in Isolator tubes, and it provided significantly faster detection than did Middlebrook plates.
Proficiency testing of clinical microbiology laboratories using modified decontamination procedures for detection of nontuberculous mycobacteria in sputum samples from cystic fibrosis patients. The Nontuberculous Mycobacteria in Cystic Fibrosis Study Group
Authors: Whittier S , Olivier K , Gilligan P , Knowles M , Della-Latta P
Published: January 2020

An improved decontamination method has been demonstrated to reduce overgrowth of mycobacterial media by Pseudomonas aeruginosa and allow the successful recovery of nontuberculous mycobacteria (NTM) from cystic fibrosis (CF) patients. Twenty microbiology laboratories participating in a multicenter investigation designed to determine the significance of NTM in CF patients were required to demonstrate proficiency in the incorporation of this improved method; this was accomplished by successful decontamination and culture workup of a panel of simulated sputum samples seeded with P. aeruginosa and various NTM. All laboratories successfully recovered NTM from samples with acid-fast bacillus (AFB) smear scores of 3+/4+ (i.e., 2 to 18 or > 18 organisms/field). Low-inoculum samples (1+/2+ AFB smears [2 to 18 organisms in 100 or 10 fields]) were problematic in that processed specimens were often smear and/or culture negative.
Evaluation of three commercial enzyme-linked immunosorbent assays and two latex agglutination assays for diagnosis of primary Epstein-Barr virus infection
Authors: Svahn A , Magnusson M , Jägdahl L , Schloss L , Kahlmeter G , Linde A
Published: January 2020

Three commercially available enzyme-linked immunosorbent assays (ELISAs) from Gull, Biotest, and Behring (Enzygnost) and two latex agglutination tests for heterophile antibodies (Monolatex [Biotest] and Mono-Lex [Trinity Laboratories]) were evaluated for the diagnosis of primary Epstein-Barr virus (EBV) infection and EBV seropositivity. Two hundred fourteen consecutive samples from 197 patients with symptoms of primary EBV infection were analyzed by the five assays at a clinical microbiology laboratory. The samples were also analyzed independently by immunofluorescence methods at a reference laboratory. According to the reference methods, 37 patients (40 serum samples) had primary EBV infections, 120 patients (127 serum samples) had had past EBV infections, 33 patients (36 serum samples) were seronegative, and 7 patients (11 serum samples) exhibited atypical reactions. The respective sensitivities and specificities for the diagnosis of primary EBV infection were 95 and 100% for the Gull assays, 100 and 94% for the Biotest assays, and 100 and 89%, for the Enzygnost assays. The Monolatex and Mono-Lex methods showed similar sensitivities and specificities (78 to 85% and 100 to 99%, respectively) for the diagnosis of primary EBV infection. This study demonstrates the usefulness of commercially available assays for the rapid diagnosis of primary EBV infection, but also the importance of large-scale testing of routine samples before choosing an assay.
Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing
Authors: Goldenberger D , Künzli A , Vogt P , Zbinden R , Altwegg M
Published: January 2020

Broad-range PCR amplification of part of the 16S rRNA gene followed by single-strand sequencing was applied to samples of 18 resected heart valves from patients with infective endocarditis. The PCR results were compared with those of cultures of valves and with those of previous blood cultures. For two patients there was agreement with the cultures of the valves; for nine patients there was agreement with the previous blood cultures, which were positive, while the cultures of the valves were negative; a Streptococcus sp. and Tropheryma whippelii each were found in one patient with negative cultures (valve and blood); for two patients the cultures of the valves as well as the PCR results were negative but the blood cultures were positive; for one patient amplification was inhibited; and for two patients the PCR results were positive but the amplicons could not be sequenced. It is concluded that broad-range PCR is a promising tool for patients with culture-negative endocarditis and allows the detection of rare, noncultivable organisms.
Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses
Authors: Heaton P R , Johnstone P , McElhinney L M , Cowley R , O'Sullivan E , Whitby J E
Published: January 2020

A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.
Phenotypic and molecular characterization of three clinical isolates of Mycobacterium interjectum
Authors: Lumb R , Goodwin A , Ratcliff R , Stapledon R , Holland A , Bastian I
Published: January 2020

Introduction of molecular biology-based technology into an Australian mycobacterial reference laboratory has resulted in the identification of three isolates of Mycobacterium interjectum in the past 12 months. Conventional phenotypic methods failed to identify the species of these isolates, and high-performance liquid chromatography found that only one of the three isolates had a mycolic acid pattern similar to that of the type strain. In contrast, all three isolates were rapidly identified as M. interjectum by 16S rRNA gene sequence analysis. Two isolates were recovered from the lymph nodes of children with cervical lymphadenitis, confirming the pathogenicity of this organism. However, the third isolate was obtained from the sputum of an elderly male with chronic lung disease without evidence of clinical or radiological progression, suggesting that isolation of M. interjectum should not imply disease. With the increasing use of molecular biology-based technology in mycobacterial laboratories, M. interjectum may be recognized more frequently as a pathogen or commensal organism.
Culture of the causative organism of donovanosis (Calymmatobacterium granulomatis) in HEp-2 cells
Authors: Carter J , Hutton S , Sriprakash K S , Kemp D J , Lum G , Savage J , Bowden F J
Published: January 2020

We report successful culture of Calymmatobacterium granulomatis by standard cell culture methods. Swabs were obtained from lesions in three patients with a clinical diagnosis of donovanosis. For two patients, there was histological confirmation of the disease (i.e., the presence of Donovan bodies in Giemsa-stained smears). Specimens were inoculated onto cycloheximide-treated HEp-2 cell monolayers in RPMI 1640 medium (supplemented with fetal calf serum, NaHCO3, vancomycin hydrochloride, and benzylpenicillin). At 48 h, organisms resembling Donovan bodies were identified in monolayer cultures from all three specimens. The organisms appeared as pleomorphic bacilli with characteristic bipolar staining and "safety pin" appearance. Using a PCR designed to differentiate C. granulomatis from the Klebsiella species (which have a high degree of molecular homology), we were able to demonstrate that the cultured organisms produced a PCR product identical to that obtained from the original swab specimens. It is now possible to test in vitro susceptibility of C. granulomatis to antibiotics and to provide a ready source of DNA and antigenic material to enable the development of serological tests and, possibly in the future, a vaccine.
Virulence patterns of Escherichia coli K1 strains associated with neonatal meningitis
Authors: Bingen E , Bonacorsi S , Brahimi N , Denamur E , Elion J
Published: January 2020

The prevalence of the ibe10 gene, of the pap, afa, and sfa adhesin-encoding operons, and of a 14.9-kb rrn-containing HindIII fragment was studied for 67 Escherichia coli neonatal meningitis strains, 58 E. coli K1 commensal strains, and 47 E. coli blood isolates from neonates without meningitis. ibe10, sfa, and the 14.9-kb HindIII fragment were observed significantly more often in the meningitis strains than in blood or commensal strains.
Development of an antimicrobial susceptibility surveillance system for Neisseria gonorrhoeae in Malawi: comparison of methods
Authors: Daly C C , Hoffman I , Hobbs M , Maida M , Zimba D , Davis R , Mughogho G , Cohen M S
Published: January 2020

Susceptibility of Neisseria gonorrhoeae to gentamicin, the primary treatment for gonorrhea in Malawi since 1993, was determined by using agar dilution MICs, E-test MICs, disc diffusion, and clinical cure rate. Agar dilution MICs were slightly higher in 1996 than in 1993 isolates, with a concomitant drop in the clinical cure rate. E-test MICs were substantially lower than agar dilution determinations, with only 77.4% within 1 log2 concentration.
Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis
Authors: Haubek D , Dirienzo J M , Tinoco E M , Westergaard J , López N J , Chung C P , Poulsen K , Kilian M
Published: January 2020

Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans.
Highly sensitive single-step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients
Authors: Caballero O L , Menezes C L , Costa M C , Fernandes S C , Anacleto T M , de Oliveira R M , Viotti E A , Távora E R , Vilaça S S , Sabbaga E , de Paula F J , Távora P F , Villa L L , Simpson A J
Published: January 2020

A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.
Direct detection of Mycobacterium tuberculosis complex in clinical samples from patients in Norway by ligase chain reaction
Authors: Lindbråthen A , Gaustad P , Hovig B , Tønjum T
Published: January 2020

Our aim was to investigate the use of DNA amplification with the ligase chain reaction (LCR) for detection of the Mycobacterium tuberculosis complex directly in human clinical specimens. The LCR assay employed was the Abbott LCx MTB Assay, which uses the gene encoding protein antigen b as the target template. Four hundred eighty-two samples from 457 patients in one clinical microbiology laboratory in Norway were processed by routine culture analysis (BACTEC culture), direct microscopy (Ziehl-Neelsen staining) and LCR. Of the 118 specimens containing cultivable M. tuberculosis, 106 (90.6%) were detected by LCR. Among the 364 culture-negative specimens, 356 samples were negative also by LCR and 8 (1.6%) were positive by LCR. In five of the eight LCR-positive and culture-negative samples, another sample from the same patient was M. tuberculosis culture positive and/or the patient had symptoms of tuberculosis. In comparison with culture, the sensitivity of LCR was 96.7% for smear-positive samples and 72.0% for smear-negative samples, respectively. For all samples combined, the sensitivity, specificity, and positive and negative predictive values were 90.2, 99.2, 97.4, and 96.7%, respectively. Challenging the M. tuberculosis LCR test with DNAs and cultures from strains of Mycobacterium ulcerans and Mycobacterium marinum, which are the mycobacterial species most closely related to the M. tuberculosis complex, resulted in all-negative test results. The sensitivity, specificity, and positive and negative predictive values of BACTEC culture in comparison with the LCR test and clinical criteria were 95.9, 100, 100, and 98.6%, respectively. A certain prioritization of samples subjected to the LCR assay should be based on clinical indications and risks with regard to infection transmission and patient isolation policy. More automation and lower expenses are generally desired for nucleic acid amplification kits. However, this M. tuberculosis LCR assay represents a valuable tool in routine mycobacterial diagnostics.
Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcus spp. to vancomycin
Authors: Kohner P C , Patel R , Uhl J R , Garin K M , Hopkins M K , Wegener L T , Cockerill F R
Published: January 2020

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc., West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibility Staphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n = 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i.e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (> 1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < or = 8 microg/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > or = 64 microg/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. faecalis strain and one E. faecium strain carried only the vanC gene. The agar screen method may also require reformulation. The current agar screen plate contains 6 microg of vancomycin per ml, which may not detect all low-level resistance associated with vanC genotypes. Nevertheless, the clinical significance of this low-level vancomycin resistance remains unknown.
Quantitative urine cultures do not reliably detect renal candidiasis in rabbits
Authors: Navarro E E , Almario J S , Schaufele R L , Bacher J , Walsh T J
Published: January 2020

The significance of quantitative urine cultures in patients at risk for hematogenous disseminated candidiasis is controversial. While various concentrations of Candida spp. in urine have been suggested as critical cutoff points in the diagnosis of renal candidiasis, other investigators consider quantitative cultures less critical in diagnosing upper tract infections. To determine the significance of quantitative urine cultures in renal candidiasis, we studied serial quantitative urinary cultures of Candida albicans in a rabbit model of hematogenous infection. Of 197 urine samples from 34 infected animals, 144 were culture positive, with a sensitivity of 73.1% for urine cultures and a lower limit of detection of 10 CFU/ml. The yield of urine cultures varied according to severity and duration of infection. The mean renal and urinary concentrations of C. albicans from rabbits with subacute candidiasis differed significantly from those from rabbits with acute candidiasis (P = 0.013 and P < or = 0.001, respectively). During the first 4 days of subacute renal candidiasis, more than one-half of all urine cultures were negative for C. albicans. Only 12 (8.1%) of 148 urine cultures in animals with subacute renal candidiasis had concentrations of > 10(3) CFU/ml, 2.7% had concentrations of > 10(4) CFU/ml, and none were > or = 10(5) CFU/ml. By comparison, all urine cultures from the animals with lethal acute renal candidiasis had higher concentrations of C. albicans and were positive throughout the course of infection. Urinary concentrations of C. albicans were not predictive of the amount of Candida in the kidney (r < or = 0.49) and did not correlate with survival (r = 0.0232). However, the renal concentration of C. albicans (in CFU/gram) inversely correlated with the duration of survival (in days) of rabbits with renal candidiasis (r = 0.76; P < 0.001). These findings indicate that a negative urine culture in rabbits does not preclude the presence of renal candidiasis. The interpretation of a urine culture positive at any concentration, on the other hand, must involve an analysis of the risk factors for renal candidiasis, for any urinary concentration of C. albicans may reflect kidney infection.
Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996
Authors: Sharma C , Maiti S , Mukhopadhyay A K , Basu A , Basu I , Nair G B , Mukhopadhyaya R , Das B , Kar S , Ghosh R K , Ghosh A
Published: January 2020

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.
Identification and classification of Oxalobacter formigenes strains by using oligonucleotide probes and primers
Authors: Sidhu H , Allison M , Peck A B
Published: January 2020

Genomic DNAs of various strains of Oxalobacter formigenes were subjected to restriction endonuclease fragment length polymorphism- and PCR-based amplification analyses with DNA probes and primers complementary to sequences within either the oxc gene, encoding oxalyl coenzyme A (oxalyl-CoA) decarboxylase, or the frc gene, encoding formyl-CoA transferase. Oligonucleotide probes based on nonconserved sequences of oxc or frc were able to divide O. formigenes strains into at least two groups, consistent with the current separation of O. formigenes strains into groups I and II on the basis of 16S rRNA sequence similarities and lipid content. In contrast, an oligonucleotide probe based on the conserved 5' end of oxc appeared to bind all group I and the majority of group II strains. PCR amplification of the oxc gene showed even greater sensitivity in detecting O. formigenes and provided support for further division of the strains into subgroups. In addition, these oligonucleotides failed to hybridize to or amplify PCR products from whole fecal DNA isolated from fresh stool samples from an individual not colonized with O. formigenes, indicating unique specificity. Thus, these DNA analyses permit both detection as well as classification of O. formigenes strains.
A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens
Authors: Ignatius R , Lehmann M , Miksits K , Regnath T , Arvand M , Engelmann E , Futh U , Hahn H , Wagner J
Published: January 2020

The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.
Trichoderma longibrachiatum infection in a pediatric patient with aplastic anemia
Authors: Munoz F M , Demmler G J , Travis W R , Ogden A K , Rossmann S N , Rinaldi M G
Published: January 2020

Trichoderma longibrachiatum infection of the skin in an 11-year-old child with severe aplastic anemia and prolonged neutropenia is reported. The patient received systemic antifungal therapy and underwent bone marrow transplantation. To our knowledge, this is the first description of T. longibrachiatum infection in a pediatric patient. It also is the first case successfully treated with medical therapy. A review of the literature suggests that Trichoderma spp. are recognized as human pathogens with increasing frequency, particularly for immunocompromised patients, and should be considered in the differential diagnosis of fungal infections in the pediatric population.
Interlaboratory comparison of test results for detection of Lyme disease by 516 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Proficiency Testing Program
Authors: Bakken L L , Callister S M , Wand P J , Schell R F
Published: January 2020

In 1991, we reported that 55% of laboratories participating in the Wisconsin Proficiency Testing Program could not accurately identify serum samples from Lyme disease patients containing antibody against Borrelia burgdorferi. The purpose of this study was to determine whether the accuracy of Lyme disease test results reported by approximately 500 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Lyme Disease Survey had improved. From 1992 through 1994, 50 serum samples were sent to participants of the survey. Each laboratory received 28 serum samples from individuals with Lyme disease according to the case definition of the Centers for Disease Control and Prevention and 22 serum samples from healthy individuals. Unfortunately, the serodiagnosis of Lyme disease by participants had not improved. The specificity of the Lyme disease assays steadily decreased from approximately 95% to approximately 81% during the 3-year period of the survey. False-positive test results approached 55% with some of the serum samples from healthy donors. A serum sample containing antibody against Treponema pallidum was reported as positive by 70% of the participants. In addition, the sensitivity fluctuated between 93 and 75%, depending upon the conjugate used by the laboratories. These results suggest that stronger criteria must be applied for approving and continuing to approve commercially available kits for the serodiagnosis of Lyme disease.
A one-tube method of reverse transcription-PCR to efficiently amplify a 3-kilobase region from the RNA polymerase gene to the poly(A) tail of small round-structured viruses (Norwalk-like viruses)
Authors: Ando T , Monroe S S , Noel J S , Glass R I
Published: January 2020

Amplification of a 3-kb genome region from the RNA polymerase gene to the 3' poly(A) tail of small round-structured virus (SRSV) by reverse transcription-PCR (RT-PCR) has been difficult to achieve because of a stable secondary structure in a region between the RNA polymerase gene and the 5' end of the second open reading frame. We have developed a one-tube RT-PCR method to efficiently amplify this region. The method comprises three procedures: purification of poly(A)+ RNA from a starting RNA solution by oligo(dT)30 covalently linked to latex particles, buffer exchange, and continuous RT and PCR in a single tube containing all reaction components. The key elements of this method are (i) first-strand cDNA synthesis with the Superscript II version of RNase H- Moloney murine leukemia virus reverse transcriptase at 50 degrees C for 10 min by using the RNA-oligo(dT)30 hybrid on the latex particles as the template and primer, and (ii) PCR by Taq and Pwo DNA polymerases mixed together with a mixture of 12 phased oligo(dT)25 antisense primers. The detection threshold of the one-tube RT-PCR method was as little as 0.2 ng of the crude RNA used as the source of the template. Using this method, we obtained 3-kb products from 24 SRSV strains previously characterized into four genetic groups. These included 5 P1-A, 4 P1-B, 5 P2-A, and 10 P2-B strains. Because SRSVs have not yet been cultivated in vitro, this novel method should facilitate molecular characterization of SRSVs to provide a firm scientific foundation for improvements and refinements of SRSV diagnostics.
Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay
Authors: Schäfer P , Tenschert W , Gutensohn K , Laufs R
Published: January 2020

Quantitative cytomegalovirus antigenemia and DNAemia were determined in peripheral leukocytes of 25 patients stored for up to 72 h at room temperature (RT) and 4 degrees C before processing. Numbers of antigen-positive cells significantly decreased with time. The decline was greater at RT than at 4 degrees C. In contrast, no significant alterations in DNAemia occurred.
Diversity of the 32-kilodalton protein gene may form a basis for species determination of potentially pathogenic mycobacterial species
Authors: Soini H , Viljanen M K
Published: January 2020

In this study, partial gene sequences of the mycobacterial 32-kDa protein gene were determined by PCR-based sequencing. A total of 50 strains representing 18 potentially pathogenic mycobacterial species were studied. In 10 cases, all three strains of the species studied were identical, and intraspecies variability was found in 6 cases. Thus, the 32-kDa protein gene may be a good target for identification of mycobacteria by PCR-based sequencing.
Vulval swabs as alternative specimens for ligase chain reaction detection of genital chlamydial infection in women
Authors: Stary A , Najim B , Lee H H
Published: January 2020

A ligase chain reaction (LCR)-based assay was recently shown to be highly sensitive and specific for the detection of Chlamydia trachomatis not only in cervical specimens but also in first-void urine (FVU) specimens form women. The suitability of using vulval swabs as an alternative specimen that can be obtained by noninvasive means for the diagnosis of genital chlamydial infection by LCR was investigated. In a first study of 169 women, vulval, endocervical, and urethral swabs were tested by LCR, culture, and a combination of enzyme immunoassay (EIA) followed by confirmation by direct fluorescent-antibody assay (DFA), and the results were compared with those obtained by testing FVU specimens by LCR and EIA-DFA by using a specimen from an infected patient as a reference standard. Of the 169 women tested, 27 (16%) were shown to be infected. Whereas LCR showed high sensitivities with all specimen types (85.2% for vulval, urine, and endocervical specimens; 92.6% for urethral swabs), the sensitivities of culture and EIA-DFA were high only with endocervical swabs (74.1 and 70.4%, respectively), being 22.2 and 40.7%, respectively, with vulval swabs. In addition, urine testing by EIA-DFA also showed a poor sensitivity (48.1%). In order to further compare LCR performance with vulval specimens to that with FVU specimens, a second study was carried out with specimens from 312 women, of whom 26 were infected. Comparable sensitivity was obtained by LCR with vulval swabs (88.5%; 23 of 26) and FVU specimens (92.3%; 24 of 26). The results indicate that vulval swabs may serve as suitable alternative to specimens that can be obtained by noninvasive means for the detection of C. trachomatis by LCR.
Propagation of granulocytic Ehrlichia spp. from human and equine sources in HL-60 cells induced to differentiate into functional granulocytes
Authors: Heimer R , Van Andel A , Wormser G P , Wilson M L
Published: January 2020

Ehrlichia spp. from human and equine sources in the northeastern Unites States were detected by PCR, isolated, and propagated in the HL-60 promyelocytic leukemia cell line. Growth of Ehrlichia from both equine and human sources was enhanced by addition of retinoic acid, which causes granulocytic differentiation of the HL-60 cells. DNA sequencing of a portion of the 16S rDNA gene supported the hypothesis that the same pathogen was responsible for both equine and human granulocytic ehrlichiosis.
Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses
Authors: Li F , Drummer H E , Ficorilli N , Studdert M J , Crabb B S
Published: January 2020

Equine rhinovirus 1 (ERhV1) is a recognized cause of acute febrile respiratory disease in horse, although the virus is rarely isolated from such animals, despite seroprevalence rates as high as 50% in some horse populations. Recently, ERhV1 has been shown to be most closely related to foot-and-mouth disease virus, raising questions as to its disease associations in horses. We report that ERhV1 infection was the likely cause of two separate outbreaks of severe febrile respiratory disease which involved more than 20 horses. Attempts to isolate ErhV1 from nasopharyngeal swabs by conventional cell culture methods were unsuccessful, in that cytopathology was not observed. Viral antigen was detected by immunofluorescence assay in the cytoplasm of cells infected with 10 of 15 nasopharyngeal swab samples, indicating the presence and presumably replication of ERhV1. A rise in serum neutralizing antibody titer between acute- and convalescent-phase sera confirmed that ERhV1 was causatively associated with one of the outbreaks. ERhV1 RNA was detected in nasopharyngeal swabs collected from all horses during the acute phase of disease by reverse transcription-PCR. Nucleotide sequencing of amplified products showed that within each outbreak a single strain of ERhV1 was involved but that distinct viruses were involved in each outbreak. A retrospective study of samples from nine other outbreaks of respiratory disease in horses suggested ERhV1 etiology in at least two of these. We conclude that the relative importance of ERhV1 as a cause of acute febrile respiratory disease in horses has been underestimated due to failure in many instances to isolate virus by conventional cell culture methods.
Rapid diagnosis of Legionella pneumophila serogroup 1 infection with the Binax enzyme immunoassay urinary antigen test
Authors: Kazandjian D , Chiew R , Gilbert G L
Published: January 2020

The Binax legionella urinary antigen (LUA) enzyme immunoassay (Binax, Portland, Maine) was evaluated in 159 patients with suspected or proven legionellosis and 209 controls. A positive LUA test was found in 37% of patients with suspected legionellosis overall and in 83% of those with proven Legionella pneumophila serogroup 1 infection. The sensitivity of the LUA test was significantly greater than that of the direct fluorescent-antigen test (83 versus 42%; P < 0.0001) but not significantly different from that of culture (85%) or serology (91%); specificity was at least 99.5%.
Reproducibility problems with the AMPLICOR PCR Chlamydia trachomatis test
Authors: Peterson E M , Darrow V , Blanding J , Aarnaes S , de la Maza L M
Published: January 2020

In an attempt to use an expanded "gold standard" in an evaluation of an antigen detection test for Chlamydia trachomatis, the AMPLICOR (Roche Diagnostics Systems, Inc., Branchburg, N.J.) PCR Chlamydia trachomatis test and culture were used with 591 sets of cervical specimens. Of the 591 specimens assayed, 35 were retested due to either an equivocal result by the PCR (19 samples) or a discrepancy between the results of culture, PCR, and the antigen detection method. During the repeat testing of the samples with equivocal and discrepant results, all but one interpretation change was due to the PCR result. In addition, upon repeat testing the PCR assay value measured in optical density units varied widely for 13 of these specimens. These 13 specimens were then tested in triplicate by the manufacturer with primers to the chlamydia plasmid and in duplicate with primers to the major outer membrane protein. Only 3 of the 13 specimens gave the same interpretation with these five replicates. In summary, reproducibility problems with the AMPLICOR test should be considered before it is incorporated as part of routine testing or used as an expanded gold standard for chlamydia testing.
Widespread geographic distribution of oral Candida dubliniensis strains in human immunodeficiency virus-infected individuals
Authors: Sullivan D , Haynes K , Bille J , Boerlin P , Rodero L , Lloyd S , Henman M , Coleman D
Published: January 2020

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.
Genetic identification of Mycobacterium bovis BCG by restriction fragment length polymorphism analysis of the direct-repeat region
Authors: Howard S T , Laszlo A , Johnson W M
Published: January 2020

Restriction fragment length polymorphism (RFLP) analysis was performed on the direct repeat (DR) regions of 14 strains of Mycobacterium bovis BCG. With AluI-digested DNA, BCG Japanese, Russian, and Mexican had differing RFLP patterns but 11 strains, including Pasteur, Glaxo, and Tice, had an identical pattern not detected in over 60 strains of the M. tuberculosis complex. DR analysis can aid in confirming the identification of clinical BCG isolates.