bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemiology of Hepatitis B, C, E, and G Virus Infections and Molecular Analysis of Hepatitis G Virus Isolates in Bolivia
Authors: Konomi Nami , Miyoshi Chiaki , La Fuente Zerain Carlos , Li Tian-Cheng , Arakawa Yasuyuki , Abe Kenji
Published: December 2019

Prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis G virus (HGV), and hepatitis E virus (HEV) was investigated among 574 healthy blood donors in Bolivia. HCV RNA and HGV RNA in the serum were identified by a nested reverse transcription-PCR using primers derived from the 5′ untranslated region (5′ UTR). We also tested for hepatitis B surface antigen (HBsAg) and for the antibody to HEV. The results revealed that HGV RNA was present in 84 of 574 (14.6%) tested blood donors, whereas HBsAg was detected in only 2 (0.3%) donors, and no individuals positive for HCV RNA were found. Anti-HEV immunoglobulin G (IgG) was detected in 93 (16.2%) individuals and anti-HEV IgM was found in 10 (1.7%) individuals among the same population. Phylogenetic analysis of 44 HGV isolates in the 5′ UTR showed that 27 (61%) isolates were genotype 3 (Asian type) and the remaining 17 (39%) isolates were genotype 2 (United States and European type). Moreover, we obtained a full-length nucleotide sequence of the HGV genome (designated HGV-BL230) recovered from a Bolivian blood donor. The BL230 was composed of 9,227 nucleotides and had a single open reading frame, encoding 2,842 amino acid residues. Interestingly, the BL230 belonged to genotype 2 of HGV at the level of a full-length sequence, although this was classified as genotype 3 by a phylogenetic analysis based on the 5′ UTR sequence. The BL230 differed from previously reported HGV/hepatitis GB virus type C isolates by 12 to 13% of the nucleotide sequence and 4% of the amino acid sequence. Our data indicate a high prevalence of HGV in native Bolivians, and the major genotype of HGV was type 3.
Detection of Multiple Human Papillomavirus Types in Condylomata Acuminata Lesions from Otherwise Healthy and Immunosuppressed Patients
Authors: Brown Darron R. , Schroeder Jill M. , Bryan Janine T. , Stoler Mark H. , Fife Kenneth H.
Published: December 2019

Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.
Improved Detection of
Streptococcus pneumoniae
in Middle-Ear Fluid Cultures by Use of a Gentamicin-Containing Medium
Authors: Peled Nechama , Yagupsky Pablo
Published: December 2019

The performance of Columbia agar medium with added sheep blood and gentamicin (CAG) for isolation of
Streptococcus pneumoniae
from middle-ear fluid cultures was compared to that of routine blood agar medium (BA). Of 238 pneumococcal isolates recovered, CAG plates detected 233 (97.9%) but BA plates detected only 208 (87.4%) (
P
< 0.001).
Phenotypic and Phylogenetic Characterization of a New
Corynebacterium
Species from Dogs: Description of
Corynebacterium auriscanis
sp. nov
Authors: Collins Matthew D. , Hoyles Lesley , Lawson Paul A. , Falsen Enevold , Robson Robert L. , Foster Geoffrey
Published: December 2019

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus
Corynebacterium
sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus
Corynebacterium
; this subline is close to but distinct from
C. falsenii
,
C. jeikeium
, and
C. urealyticum
. The unknown bacterium from dogs was distinguished from all currently validated
Corynebacterium
species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species,
Corynebacterium auriscanis
. The type strain of
C. auriscanis
is CCUG 39938
T
.
Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of
Actinobacillus actinomycetemcomitans
,
Bacteroides forsythus
, and
Porphyromonas gingivalis
Authors: Tran Simon Dangtuan , Rudney Joel D.
Published: December 2019

Among putative periodontal pathogens,
Actinobacillus actinomycetemcomitans
,
Bacteroides forsythus
, and
Porphyromonas gingivalis
are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects
A. actinomycetemcomitans
and
P. gingivalis
. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six
A. actinomycetemcomitans
, one
B. forsythus
, and four
P. gingivalis
strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10
A. actinomycetemcomitans
, 10
P. gingivalis
, and 100
B. forsythus
cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
strains tested, and (iii) no amplicons were detected for the eight other bacterial species.
A. actinomycetemcomitans
,
B. forsythus
, and
P. gingivalis
were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.
Evaluation of Reverse Transcription-PCR and a Bacteriophage-Based Assay for Rapid Phenotypic Detection of Rifampin Resistance in Clinical Isolates of
Mycobacterium tuberculosis
Authors: Eltringham I. J. , Drobniewski F. A. , Mangan J. A. , Butcher P. D. , Wilson S. M.
Published: December 2019

New rapid phenotypic assays for the detection of rifampin resistance in
Mycobacterium tuberculosis
have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of
M. tuberculosis
. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of
M. tuberculosis
to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible
dnaK
mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 μg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries.
Monitoring Treatment of Patients with Pulmonary Tuberculosis: Can PCR Be Applied?
Authors: Thomsen Vibeke Østergaard , Kok-Jensen Axel , Buser Mauro , Philippi-Schulz Sabine , Burkardt H.-J.
Published: December 2019

To assess whether PCR is applicable for monitoring the efficacy of antituberculous treatment, respiratory specimens obtained during treatment and follow-up from sputum smear-positive tuberculosis (TB) patients were examined. First, results of smear, culture, and PCR for
Mycobacterium tuberculosis
complex (MTB) and an internal inhibition control (MCC) were correlated retrospectively on 1,601 respiratory specimens from patients with no previous cultures of MTB. MTB optical density (OD) values increased to a maximum level of 3.5 to 4.0, with both increasing numbers of acid-fast bacilli and CFU. MTB/MCC OD ratios also increased with both smear and culture grading and correlated significantly better with both than the MTB OD value. Second, changes in MTB OD values and MTB/MCC OD ratios were compared with microscopy and culture for MTB in monthly sputa obtained during treatment and follow-up in 22 smear-positive pulmonary TB patients. Declines in MTB/MCC OD ratios during antituberculous treatment and follow-up were observed. Patients with moderate disease reached the baseline after 6 to 8 months of standard antituberculous treatment regimen, whereas patients with extensive disease were predicted to reach the baseline 1 year or more after the initiation of treatment. Although PCR detects both dead and live bacteria, we believe that PCR can be used to assess the efficacy of antituberculous treatment since increases or slow reductions in MTB/MCC OD ratios would indicate nonoptimal treatment, noncompliance, reduced bioavailability of drugs, or resistant strains of MTB and thereby would identify patients at risk for treatment failure or reactivation.
Excretion of Hepatitis A Virus (HAV) in Adults: Comparison of Immunologic and Molecular Detection Methods and Relationship between HAV Positivity and Infectivity in Tamarins
Authors: Polish Louis B. , Robertson Betty H. , Khanna Bhawna , Krawczynski Krzysztof , Spelbring John , Olson Fred , Shapiro Craig N.
Published: December 2019

Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools.
Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper
Authors: Frisk A. L. , König M. , Moritz A. , Baumgärtner W.
Published: December 2019

Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.
Comparison of Selective Broth Medium Plus Neomycin-Nalidixic Acid Agar and Selective Broth Medium Plus Columbia Colistin-Nalidixic Acid Agar for Detection of Group B Streptococcal Colonization in Women
Authors: Dunne W. Michael
Published: December 2019

The combination of neomycin-nalidixic acid (NNA) agar and a selective broth medium (SBM) has recently been shown to improve the sensitivity of screening cultures for group B streptococcal (GBS) carriage in women. Because of the relatively high cost of NNA agar, a study was initiated to determine whether Columbia colistin-nalidixic acid (CNA) agar would be an equally sensitive, more economical alternative. A total of 580 cervical-vaginal and/or rectal specimens submitted for detection of GBS were included in the study. Each was plated onto NNA and CNA agar and then inoculated into SBM. GBS were recovered from 95 of 580 (16.4%) specimens, including 61 isolates from CNA, 74 from NNA, 73 from the CNA-SMB combination, and 86 from the NNA-SMB tandem. Of those, 22 isolates were recovered on NNA but not CNA, 9 were cultured on CNA but not NNA, 52 were isolated on both media, and 12 were recovered from subcultures of SBM only. The overall sensitivity of CNA alone (64.2%) was statistically significantly less than that of NNA agar (77.9%), as was the sensitivity of combination of CNA plus SBM (76.8%) compared to that of NNA plus SBM (90.5%). Based on these findings, CNA should not be considered an acceptable alternative to NNA for the detection of GBS colonization in women despite potential cost savings.
Effects of OspA Vaccination on Lyme Disease Serologic Testing
Authors: Aguero-Rosenfeld Maria E. , Roberge Janet , Carbonaro Carol A. , Nowakowski John , Nadelman Robert B. , Wormser Gary P.
Published: December 2019

This study presents the effects of OspA vaccination on two-step testing for
Borrelia burgdorferi
antibodies. Although vaccinees developed enzyme-linked immunosorbent assay reactivity, immunoblots did not fulfill Centers for Disease Control and Prevention criteria for positivity. Furthermore, OspA reactivity did not interfere with interpretation of immunoblots with sera from patients who developed early Lyme disease despite vaccination.
Characterization of Extended-Spectrum β-Lactamase-Producing
Salmonella typhimurium
by Phenotypic and Genotypic Typing Methods
Authors: Ait Mhand Rajaa , Brahimi Naima , Moustaoui Najat , El Mdaghri Naima , Amarouch Hamid , Grimont Francine , Bingen Edouard , Benbachir Mohamed
Published: December 2019

During 1994, 10 isolates of extended-spectrum β-lactamase-producing
Salmonella typhimurium
were recovered from children transferred to our hospital from two different centers. Two additional isolates were recovered from two nurses from one of these centers. The aim of this study was to determine if there is any relationship between these isolates. The characterization was done by phenotypic and genotypic methods: biotyping, phage typing, antibiotic susceptibility pattern determination, plasmid analysis, ribotyping (by the four endonucleases
Eco
RI,
Sma
I,
Bgl
II, and
Pvu
II), pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with
Xba
I, and randomly amplified polymorphic DNA (RAPD) pattern determination (with the three primers 217 d2, B1, and A3). The same biotype, the same serotype, and an identical antibiotype were found. All isolates were resistant to oxyimino-β-lactams, gentamicin, tobramycin, and sulfamethoxazole-trimethoprim. All isolates showed an indistinguishable pattern by ribotyping and very similar patterns by PFGE and RAPD. The overall results indicated the spread of a closely related strain of
S. typhimurium
in children and nurses.
Spread of a
Salmonella typhimurium
Clone Resistant to Expanded-Spectrum Cephalosporins in Three European Countries
Authors: Tassios P. T. , Gazouli M. , Tzelepi E. , Milch H. , Kozlova N. , Sidorenko S. , Legakis N. J. , Tzouvelekis L. S.
Published: December 2019

Twelve
Salmonella typhimurium
strains resistant to broad-spectrum cephalosporins were isolated from cases of gastroenteritis during 1996 to 1998 in Russia, Hungary, and Greece. Resistance was due to the production of CTX-M-type extended-spectrum β-lactamases encoded by similar 12-kb plasmids. By pulsed-field gel electrophoresis, all strains shared the same chromosomal type. These data suggest that an
S. typhimurium
clone resistant to broad-spectrum cephalosporins is present in at least three European countries.
Molecular Epidemiological Investigation Using a Randomly Amplified Polymorphic DNA Assay of
Burkholderia cepacia
Isolates from Nosocomial Outbreaks
Authors: Okazaki Mitsuhiro , Watanabe Takashi , Morita Koji , Higurashi Yoshimi , Araki Koji , Shukuya Naoko , Baba Shigeyuki , Watanabe Noboru , Egami Teruo , Furuya Nobushige , Kanamori Masato , Shimazaki Shuji , Uchimura Hidemasa
Published: December 2019

We experienced two
Burkholderia cepacia
outbreaks over a 1-year period. During this period, 28
B. cepacia
isolates were obtained from clinical specimens, and 2 were obtained from environmental specimens (i.e., from a nebulizer solution and a nebulizer tube). These 30 isolates were subjected to the PCR-based randomly amplified polymorphic DNA (RAPD) assay as well as to pulsed-field gel electrophoresis (PFGE). In the first outbreak, in which eight patients hospitalized in the Trauma and Critical Care Center were involved, the RAPD assay revealed that all 20 isolates obtained from clinical specimens and the 2 isolates from environmental specimens had identical DNA profiles. These RAPD data enabled us to pinpoint a possible source and to take countermeasures to prevent further spread of the epidemic-causing strain. In the second outbreak, two consecutive
B. cepacia
infection/colonization cases were seen in the surgery ward. The RAPD profiles of four isolates obtained were again identical, but they were distinct from those seen in the first outbreak, clearly indicating that the second outbreak was not related to the first. Thus, our experience demonstrated that the RAPD assay is a useful and reliable tool for epidemiological studies of
B. cepacia
isolates from nosocomial outbreaks. Since the RAPD assay could provide discriminatory potential and reproducibility comparable to those of the widely used PFGE assay with less complexity and in a shorter time, the introduction of the RAPD assay into hospital microbiology laboratories as a routine technique may help prevent nosocomial outbreaks.
Determination of Bovine Rotavirus G and P Serotypes in Italy by PCR
Authors: Falcone E. , Tarantino M. , Di Trani L. , Cordioli P. , Lavazza A. , Tollis M.
Published: December 2019

Determination of the G and P serotypes of group A bovine rotaviruses from 149 samples of feces or intestinal contents collected from calves showing clinical signs of neonatal diarrhea was performed by a nested reverse transcription-PCR typing assay. The G6 serotype was the most prevalent, accounting for viruses in 55.7% of the samples; viruses of the G10 and G8 serotypes were found in 34.9 and 4.7% of the samples, respectively. The virus in one sample (0.7%) was not classified due to concomitant infection with G6 and G8 strains, whereas viruses in six samples (4.0%) could not be characterized with any of the three G serotype-specific primers selected for the present study. When examined for their P-serotype specificities, viruses in 55 and 42.3% of the samples were characterized as P[11] and P[5], respectively, no P[1] serotype was identified, and viruses in 2.7% of the samples could not be classified due to multiple reactivity with both P[5]- and P[11]-specific primers. Various combinations of G and P serotypes were observed, the most frequent being G6,P[5] (38.3%), G10,P[11] (31.5%), and G6,P[11] (15.4%). The results of the present study, while contributing to a better understanding of the epidemiology of bovine rotaviruses in Italy, address the relevance of serotype specificity with regard to the constancy of the quality of bovine rotavirus vaccines under different field conditions.
Molecular Epidemiology of Vancomycin-Resistant
Enterococcus faecium
in a Large Urban Hospital over a 5-Year Period
Authors: Bischoff Werner E. , Reynolds Tammy M. , Hall Gaye O. , Wenzel Richard P. , Edmond Michael B.
Published: December 2019

To investigate the dissemination of vancomycin-resistant
Enterococcus faecium
(VREF) in a 728-bed tertiary-care hospital, all clinical VREF isolates recovered from June 1992 to June 1997 were typed by pulsed-field gel electrophoresis, and the transfer histories of the patients were documented. A total of 413 VREF isolates from urine (52%), wounds (16%), blood (11%), catheter tips (6%), and other sites (15%) were studied. VREF specimens mostly came from patients on wards (66%) but 34% came from patients in an intensive care unit. The number of VREF isolates progressively increased over time, with higher rates of isolation during the winter months and lower rates in the late summer months. Four distinct banding patterns were detected by pulsed-field gel electrophoresis among 316 samples (76%). Strain A (122 samples; 30%) appeared in June 1992 as the first VREF strain and was found until December 1994 throughout the entire hospital. Type B (92 samples; 22%) was initially detected in January 1994 and disappeared in November 1996. Strain C (10 samples; 2%) was limited to late 1996 and early 1997. Strain D (92 samples; 22%) showed two major peaks during March 1996 to August 1996 and January 1997 to February 1997. Unrelated strains (97 samples; 24%) appeared 1 year after the appearance of the first VREF isolate, and the numbers increased slightly over the years. Nosocomial acquisition (i.e., no known detection prior to admission and first isolation from cultures performed with samples retrieved ≥2 days after hospitalization) was found for 316 (91%) of 347 patients. Despite the implementation of Centers for Disease Control and Prevention guidelines, the proportion of related strains and high number of nosocomial cases of infection indicate a high transmission rate inside the hospital. The results imply an urgent need for stringent enforcement of more effective infection control measures.
Rapid Diagnosis of Tuberculous Meningitis by a Dot Immunobinding Assay To Detect Mycobacterial Antigen in Cerebrospinal Fluid Specimens
Authors: Sumi M. G. , Mathai A. , Sarada C. , Radhakrishnan V. V.
Published: December 2019

In the present prospective study, a dot immunobinding assay (Dot-Iba) was standardized to measure the circulating mycobacterial antigen in cerebrospinal fluid (CSF) specimens for the laboratory diagnosis of tuberculous meningitis (TBM). Immunoglobulin G antibody specific for
Mycobacterium tuberculosis
in a CSF specimen from a patient with culture-proven TBM was isolated and was coupled with activated cyanogen bromide-Sepharose 4B. By immunosorbent affinity chromatography, a 14-kDa antigen was isolated from the culture filtrate of
M. tuberculosis
. Antibody to the 14-kDa mycobacterial antigen was raised in rabbits. The Dot-Iba in this study gave no false-positive results with CSF specimens from patients with nontuberculous neurological diseases. The assay gave positive results for all five patients with culture-proven TBM. The Dot-Iba described in the present report is simple, rapid, sensitive, specific, and, more importantly, suitable for routine application in laboratories in developing countries.
Microdilution Susceptibility Testing of Amphotericin B, Itraconazole, and Voriconazole against Clinical Isolates of
Aspergillus
and
Fusarium
Species
Authors: Arikan Sevtap , Lozano-Chiu Mario , Paetznick Victor , Nangia Sunaina , Rex John H.
Published: December 2019

We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical
Aspergillus
(
n
= 82) and
Fusarium
(
n
= 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for
Aspergillus
isolates, with minor variations among species. MICs for
Fusarium
isolates were higher than those for
Aspergillus
isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant
Aspergillus fumigatus
isolates and
Fusarium solani
. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for
F. solani
were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B.
Differentiation of Two Biovars of
Ureaplasma urealyticum
Based on the 16S-23S rRNA Intergenic Spacer Region
Authors: Harasawa Ryô , Kanamoto Yasuo
Published: December 2019

The 16S-23S rRNA intergenic spacer regions of 14 strains representing the 14 serovars of
Ureaplasma urealyticum
were amplified by PCR and sequenced for genetic differentiation between the two biovars Parvo and T960. Although the spacer region of the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly, the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found to be identical. The nucleotide sequence of the T960 biovar contains multiple restriction sites for restriction endonuclease
Ssp
I, which allows differentiation of the T960 biovar from the Parvo biovar.
Rare Incidence of Interspousal Transmission of
Helicobacter pylori
in Asymptomatic Individuals in Japan
Authors: Suzuki Junko , Muraoka Hiroe , Kobayasi Intetsu , Fujita Toshiro , Mine Tetsuya
Published: December 2019

PCR-restriction fragment length polymorphism electrophoretic patterns of amplified
ureB
and
ureC
of
Helicobacter pylori
were compared between spouses after digestion with restriction endonucleases. Twenty of 21 couples, both members of which were positive for
H. pylori
, showed
ureB
and
ureC
patterns that differed between spouses. We concluded that in Japan, interspousal transmission of
H. pylori
occurs rarely.
Direct Detection of Sabin Poliovirus Vaccine Strains in Stool Specimens of First-Dose Vaccinees by a Sensitive Reverse Transcription-PCR Method
Authors: Buonagurio Deborah A. , Coleman John W. , Patibandla Sai A. , Prabhakar Bellur S. , Tatem Joanne M.
Published: December 2019

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in ∼70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.
Evaluation of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus
Authors: Arai Hiroyasu , Petchclai Bencha , Khupulsup Kalayanee , Kurimura Takashi , Takeda Katsumichi
Published: December 2019

A new immunochromatographic rapid test, Determine HIV-1/2, for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 in human whole blood, serum, and plasma was evaluated. Determine HIV-1/2 is a sandwich immunoassay and uses a nitrocellulose strip with a capture site for the patient’s results and a procedural control site to confirm the validity of the assay. The results can be read visually, and a positive result is indicated by the formation of a red line within 15 min after sample application. The test showed 100% sensitivity for HIV-1 with 102 whole-blood, 152 serum, and 144 plasma samples obtained from Ramathibodi Hospital, Bangkok, Thailand. The sensitivity of the test for HIV-2 was 100% with 100 serum or plasma samples obtained from Ivory Coast. The sensitivity of the test with 4 anti-HIV-1 seroconversion panels from Boston Biomedica Inc. was equivalent to or better than those of another agglutination assay with serum or plasma and the enzyme immunoassay licensed by the U.S. Food and Drug Administration. The specificity was 100% with 367 sets of whole-blood, serum, and plasma samples from Ramathibodi Hospital. This method had an analytical sensitivity for the detection of HIV-1 equivalent to or better than that of another agglutination assay with serum or plasma. This test had an analytical sensitivity for the detection of HIV-1 better than that of another immunochromatographic test with whole blood. This evaluation demonstrated the excellent performance of this immunochromatographic test with EDTA-anticoagulated whole-blood, serum, and plasma samples. We conclude that this test is suitable for use in emerging countries and is an excellent alternative to HIV antibody testing at remote sites, as well as in traditional laboratories.
Evaluation of a Microplate Latex Agglutination Method (Verotox-F Assay) for Detecting and Characterizing Verotoxins (Shiga Toxins) in
Escherichia coli
Authors: Karmali Mohamed A. , Petric Martin , Bielaszewska Martina
Published: December 2019

The performance of a commercial microplate latex agglutination assay, the Verotox-F assay, was compared with that of the Vero cell assay for the detection and characterization of
Escherichia coli
verocytotoxins (VTs). Culture filtrates of 68 VT-positive
E. coli
strains (65 human isolates [33 of serotype O157:H7/H−, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were investigated. The toxin phenotypes and genotypes of the 68 VT-positive isolates were VT1 only (18 strains), VT2 and/or VT2c (33 strains), and VT1 plus VT2 (17 strains). The Verotox-F assay involved incubation of serial dilutions of culture filtrates with equal volumes of latex particles sensitized with anti-VT1 antibody or anti-VT2 antibody in 96-well microtiter plates with appropriate controls and examination for latex agglutination after 20 to 24 h. Compared to the results of the Vero cell assay, the Verotox-F assay was 100% sensitive and 100% specific for the detection of VTs in culture filtrates and correctly identified the toxin types of all 68 VT producers. By checkerboard titration with purified toxins, the sensitivity of the Verotox-F assay was found to be 14 pg (0.7 ng/ml) for VT1, 12 pg (0.6 ng/ml) for VT2, and 350 pg (17.5 ng/ml) for VT2c; this sensitivity is comparable to that of the bioassay. The anti-VT2 latex reagent detected both VT2 and VT2c and did not cross-react with VT1. The anti-VT1 reagent showed a low-level cross-reaction with VT2c only at levels (≥4.5 μg/ml) that were about 1,000-fold higher than those found in culture filtrates. We conclude that the Verotox-F assay is highly sensitive and specific for the detection and characterization of VTs in culture filtrates of human
E. coli
isolates. The test is rapid, reliable, and easy to perform; its results are easy to interpret; and it should allow testing for VT to become more widely performed.
Test Characteristics of Acridine Orange, Gram, and May-Grünwald-Giemsa Stains for Enumeration of Intracellular Organisms in Bronchoalveolar Lavage Fluid
Authors: De Brauwer Els , Jacobs Jan , Nieman Fred , Bruggeman Cathrien , Drent Marjolein
Published: December 2019

For enumeration of intracellular organisms (ICO) in bronchoalveolar lavage fluid samples, the May-Grünwald-Giemsa (MGG) stain displayed higher interobserver agreement than the acridine orange and Gram stains. The MGG stain offered a reliable enumeration of ICO when 200 cells were counted by one observer.
PCR–Enzyme-Linked Immunosorbent Assay for Detection and Identification of
Campylobacter
Species: Application to Isolates and Stool Samples
Authors: Metherell L. A. , Logan J. M. J. , Stanley J.
Published: December 2019

We report a PCR–enzyme-linked immunosorbent assay which identifies
Campylobacter
species by capture hybridization of a single-stranded 16S rRNA gene amplicon with species-specific probes in a microtiter plate format. Specificities were confirmed for both reference and field strains, but the type strain of
Campylobacter coli
was atypical. The assay was rapid, informative, and usable with stool-extracted DNA.
Baculovirus Expression of Chimeric Hepatitis B Virus Core Particles with Hepatitis E Virus Epitopes and Their Use in a Hepatitis E Immunoassay
Authors: Touze Antoine , Enogat Nathalie , Buisson Yves , Coursaget Pierre
Published: December 2019

Two hepatitis B core proteins bearing the immunodominant region of the hepatitis E virus (HEV) capsid protein, one at the C terminus of hepatitis B virus core antigen (HBcAg) and the other within the HBcAg immunodominant loop, were constructed. Both chimeric proteins exhibited HEV reactivity, but only the first construct retained HBcAg reactivity. The second construct was used to develop an anti-HEV test which is equivalent to a commercial test for the detection of anti-HEV immunoglobulin G (IgG) but is more sensitive for the detection of anti-HEV IgM.
Utility of PCR Assay in Diagnosis of En-Plaque Tuberculoma of the Brain
Authors: Singh K. K. , Nair M. D. , Radhakrishnan K. , Tyagi J. S.
Published: December 2019

This is the first report of a case in which diagnosis of en-plaque tuberculoma on the basis of magnetic resonance imaging (MRI) findings was confirmed by a
Mycobacterium tuberculosis
complex-specific PCR assay of cerebrospinal fluid. The accuracy of the diagnosis was supported by good response to antitubercular drugs, which was shown by repeat MRI studies performed after treatment.
Retrospective Analysis of the Beijing Family of
Mycobacterium tuberculosis
in Preserved Lung Tissues
Authors: Qian Lishi , Van Embden Jan D. A. , Van Der Zanden Adri G. M. , Weltevreden Evert F. , Duanmu Hongjin , Douglas James T.
Published: December 2019

Direct repeat spoligotyping of 85 paraffin-embedded lung biopsies was used to investigated the occurrence around Beijing of the Beijing family of
Mycobacterium tuberculosis
. Samples ranged in time from 1956 to 1990. Hybridization patterns were found with 49 (58%) samples, and 45 (92%) produced typical Beijing family patterns extending over the 34-year period.
Detection and Quantitation of Human Papillomavirus by Using the Fluorescent 5′ Exonuclease Assay
Authors: Josefsson Agnetha , Livak Ken , Gyllensten Ulf
Published: December 2019

A method for the detection and quantitation of oncogenic human papillomavirus (HPV) was developed by using the fluorescent 5′ exonuclease assay. The method is based on the amplification of a 180-bp fragment from the 3′ part of the E1 open reading frame in a single PCR with type-specific probes for HPV types 16, 18, 31, 33, and 35. The probes can be used separately or in combinations of up to three probes per assay. Quantitation over a range of 10
1
to 10
6
initial HPV copies was possible by using real-time detection of the accumulation of fluorescence with cycle number. Reconstitution experiments, performed to mimic mixed infections, showed that individual HPV types can be detected down to a ratio of about 1% in a mixture. The performance of the assay depends on DNA quality, the presence of PCR inhibitors, and the number of different probes used simultaneously. This homogeneous assay provides a fast and sensitive way of screening for oncogenic HPV types in biopsy specimens as well as cervical smear samples. The closed-tube nature of the assay and the inclusion of uracil
N
′-glycosylase reduces cross contamination of PCR products to a minimum. A similar assay for β-actin was used in parallel for quantitation of genomic DNA. After normalizing the samples for genomic DNA content, the mean number of HPV copies per cell could be calculated.
Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay
Authors: Andeweg Arno C. , Bestebroer Theo M. , Huybreghs Martijn , Kimman Tjeerd G. , de Jong Jan C.
Published: December 2019

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.
Trends in Antifungal Use and Epidemiology of Nosocomial Yeast Infections in a University Hospital
Authors: Berrouane Yasmina F. , Herwaldt Loreen A. , Pfaller Michael A.
Published: December 2019

This report describes both the trends in antifungal use and the epidemiology of nosocomial yeast infections at the University of Iowa Hospitals and Clinics between fiscal year (FY) 1987–1988 and FY 1993–1994. Data were gathered retrospectively from patients’ medical records and from computerized databases maintained by the Pharmacy, the Program of Hospital Epidemiology, and the Medical Records Department. After fluconazole was introduced, use of ketoconazole decreased dramatically but adjusted use of amphotericin B decreased only moderately. However, the proportion of patients receiving antifungal therapy who were treated with amphotericin B declined markedly. In FY 1993–1994, 26 patients of the gastrointestinal surgery service received fluconazole. Among these patients, fluconazole use was prophylactic in 16 (61%), empiric in 3 (12%), and directed to a documented fungal infection in 7 (27%). Rates of nosocomial yeast infection in the adult bone marrow transplant unit increased from 6.77/1,000 patient days in FY 1987–1988 to 10.18 in FY 1989–1990 and then decreased to 0 in FY 1992–1993. Rates of yeast infections increased threefold in the medical and surgical intensive care units, reaching rates in FY 1993–1994 of 6.95 and 5.25/1,000 patient days, respectively. The rate of bloodstream infections increased from 0.044/1,000 patient days to 0.098, and the incidence of catheter-related urinary tract infections increased from 0.23/1,000 patient days to 0.68. Although the proportion of infections caused by yeast species other than
Candida albicans
did not increase consistently,
C. glabrata
became an important nosocomial pathogen.
Comparison of Agar Dilution, Disk Diffusion, MicroScan, and Vitek Antimicrobial Susceptibility Testing Methods to Broth Microdilution for Detection of Fluoroquinolone-Resistant Isolates of the Family
Enterobacteriaceae
Authors: Steward Christine D. , Stocker Sheila A. , Swenson Jana M. , O’Hara Caroline M. , Edwards Jonathan R. , Gaynes Robert P. , McGowan John E. , Tenover Fred C.
Published: December 2019

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family
Enterobacteriaceae
collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.
Multicenter Comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with the National Committee for Clinical Laboratory Standards M27-A Reference Method for Testing Clinical Isolates of Common and Emerging
Candida
spp.,
Cryptococcus
spp., and Other Yeasts and Yeast-Like Organisms
Authors: Espinel-Ingroff A. , Pfaller M. , Messer S. A. , Knapp C. C. , Killian S. , Norris H. A. , Ghannoum M. A.
Published: December 2019

National Committee for Clinical Laboratory Standards (NCCLS) standard guidelines are available for the antifungal susceptibility testing of common
Candida
spp. and
Cryptococcus neoformans
, but NCCLS methods may not be the most efficient and convenient procedures for use in the clinical laboratory. MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined by the commercially prepared Sensititre YeastOne Colorimetric Antifungal Panel and by the NCCLS M27-A broth microdilution method for 1,176 clinical isolates of yeasts and yeast-like organisms, including
Blastoschizomyces capitatus
,
Cryptococcus
spp., 14 common and emerging species of
Candida
,
Hansenula anomala
,
Rhodotorula
spp.,
Saccharomyces cerevisiae
,
Sporobolomyces salmonicolor
, and
Trichosporon beigelii
. Colorimetric MICs of amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first purple or blue well. Three comparisons of MIC pairs by the two methods were evaluated to obtain percentages of agreement: 24- and 48-h MICs and 24-h colorimetric versus 48-h reference MICs. The best performance of the YeastOne panel was with 24-h MICs (92 to 100%) with the azoles and flucytosine for all the species tested, with the exception of
C. albicans
(87 to 90%). For amphotericin B, the best agreement between the methods was with 48-h MIC pairs (92 to 99%) for most of the species tested. The exception was for isolates of
C. neoformans
(76%). These data suggest the potential value of the YeastOne panel for use in the clinical laboratory.