bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide for Rapid Detection of Rifampin-ResistantMycobacterium tuberculosis
Authors: Mshana Robert N. , Tadesse Genet , Abate Getahun , Miörner Håkan 
Published: December 2019

ABSTRACTWe describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-culturedMycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitiveM. bovis(BCG) andM. tuberculosisexposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistantM. tuberculosisthat is affordable even by low-income countries where tuberculosis is a major public health problem.
Detection of Kanamycin-Resistant
Mycobacterium tuberculosis
by Identifying Mutations in the 16S rRNA Gene
Authors: Suzuki Yasuhiko , Katsukawa Chihiro , Tamaru Aki , Abe Chiyoji , Makino Masanao , Mizuguchi Yasuo , Taniguchi Hatsumi 
Published: December 2019

ABSTRACT

In
Mycobacterium smegmatis
and a limited number of
Mycobacterium tuberculosis
strains, the involvement of alterations of the 16S rRNA gene (
rrs
) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the
rrs
gene and the kanamycin-resistant phenotype in clinically isolated
M. tuberculosis
strains were correlated, 43 kanamycin-resistant strains (MICs, ≧200 μg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the
rrs
gene and the intervening sequence between the
rrs
gene and 23S rRNA (
rrl
) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases
Tai
I and
Tsp
45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease,
Bst
UI or
Dde
I.
Typing of
Helicobacter pylori vacA
Gene and Detection of
cagA
Gene by PCR and Reverse Hybridization
Authors: van Doorn L. J. , Figueiredo C. , Rossau R. , Jannes G. , van Asbroeck M. , Sousa J. C. , Carneiro F. , Quint W. G. V. 
Published: December 2019

ABSTRACT

The present report describes an analysis of two virulence genes of
Helicobacter pylori
. Parts of the
cagA
gene, as well as parts from the signal (s) and middle (m) regions of the mosaic
vacA
gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the
vacA
s region (s1a, s1b, and s2 alleles), the
vacA
m region (m1 and m2 alleles), and the
cagA
gene. A total of 103
H. pylori
-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (
n
= 55) and The Netherlands (
n
= 48) were tested by the PCR-LiPA.
cagA
was detected in 84 and 73% of the Portuguese and Dutch patients, respectively.
vacA
typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type s1b, whereas most Dutch patients (61%) contained type s1a (
P
< 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (
P
= 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of
vacA
type s1 (s1a or s1b;
P
= 0.008) and
cagA
(
P
= 0.003) genes.
Molecular Epidemiological Study of
Aspergillus fumigatus
in a Bone Marrow Transplantation Unit by PCR Amplification of Ribosomal Intergenic Spacer Sequences
Authors: Radford Sarah A. , Johnson Elizabeth M. , Leeming John P. , Millar Michael R. , Cornish Jacqueline M. , Foot Annabel B. M. , Warnock David W. 
Published: December 2019

ABSTRACT

We have developed a PCR-based method for the subspecific discrimination of
Aspergillus fumigatus
types by using two primers designed to amplify the intergenic spacer regions between ribosomal DNA transcription units. The method permitted the reproducible discrimination of 11 distinct DNA types among a total of 119 isolates of
A. fumigatus
collected from patients and from the environment of a bone marrow transplantation (BMT) unit over a three-year period. Ten DNA types of
A. fumigatus
were isolated from patients in the BMT unit; eight of these types were also found in the hospital environment, and six of these were present in the unit itself. Thirteen BMT patients developed infection with one of three DNA types some months after these had first been found in the environment of the unit. In other instances, the same DNA types of
A. fumigatus
were isolated from BMT patients that were later recovered from the environment of the unit. Several DNA types of
A. fumigatus
were found in the hospital environment over an 18-month period. Molecular typing of multiple isolates of
A. fumigatus
, obtained from postmortem tissue samples, showed that one patient was infected with a single DNA type, but two others had up to three different DNA types. Our findings suggest that
A. fumigatus
infection in BMT recipients may be nosocomial in origin and underline the need for careful environmental monitoring of units in which high-risk patients are housed.
Persistence of a Multidrug-Resistant
Pseudomonas aeruginosa
Clone in an Intensive Care Burn Unit
Authors: Hsueh Po-Ren , Teng Lee-Jene , Yang Pan-Chyr , Chen Yu-Chi , Ho Shen-Wu , Luh Kwen-Tay 
Published: December 2019

ABSTRACT

Long-term colonization of various body sites with a multidrug-resistant
Pseudomonas aeruginosa
clone (resistant to piperacillin, cefoperazone, ceftazidime, aztreonam, imipenem, cefepime, cefpirome, ofloxacin, ciprofloxacin, minocycline, and aminoglycosides) with subsequent severe infections in burn patients has not been reported previously. Thirty-nine isolates of multidrug-resistant
P. aeruginosa
(resistant to ceftazidime and at least three of the agents listed above) recovered from various clinical samples from three patients in an intensive care burn unit from April 1997 to May 1997 and seven preserved isolates recovered from six patients in other medical wards at National Taiwan University Hospital from April 1996 to May 1997 were studied for their epidemiological relatedness. The epidemic could be attributed to a multidrug-resistant
P. aeruginosa
clone belonging to serogroup O:F (serogroup O:4) by means of antimicrobial susceptibility testing, O serogrouping, and analysis of the randomly amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates. The epidemic strain persisted in the three patients for weeks to months; in the meantime, these patients had received multiple antimicrobial agents for the management of intervening episodes of invasive infections (bacteremia, ventilator-associated pneumonia, and/or catheter-related sepsis) caused by this strain, as well as concomitant infections due to other organisms. The strain had been isolated only once previously, from a burn patient who was on the unit in December 1996. The present report, describing a small outbreak due to
P. aeruginosa
, documents the fact that a single clone of multidrug-resistant
P. aeruginosa
can cause long-term persistence in different body sites of burn patients and that the colonization can subsequently result in various severe infections.
Reverse Transcription-PCR Detection of
Mycobacterium leprae
in Clinical Specimens
Authors: Kurabachew Mekonnen , Wondimu Assefa , Ryon Judith J. 
Published: December 2019

ABSTRACT

A reverse transcription (RT)-PCR assay targeting the 16S rRNA of
Mycobacterium leprae
was developed to detect the organism in clinical specimens. A 171-bp fragment was amplified when
M. leprae
RNA was used as a template but not when a panel of RNAs from 28 potentially cross-reacting mycobacterial species, seven genera related to
Mycobacterium
, and three organisms normally found among skin or nose flora were tested. As few as 10 organisms isolated from infected tissue could be detected, confirming the sensitivity of the assay. When the test was applied to clinical specimens,
M. leprae
was detected in 82% of skin biopsy specimens obtained from untreated leprosy patients, while skin biopsy specimens from healthy volunteers and patients with other dermatological disorders were negative. The sensitivity of the RT-PCR was higher than that of slit skin smear staining for acid-fast bacilli or acid-fast staining of fixed biopsy specimens since 53% of acid-fast bacillus-negative biopsy specimens were RT-PCR positive. Because 16S rRNA is rapidly degraded upon cell death, the assay may detect only viable organisms and may prove to be useful in assessing the efficacy of chemotherapy.
Identification of Acinetobacters on Blood Agar in Presence of
d
-Glucose by Unique Browning Effect
Authors: Siau Hong , Yuen Kwok-Yung , Ho Pak-Leung , Luk Wei-Kwang , Wong Samson S. Y. , Woo Patrick C. Y. , Lee Rodney A. , Hui Wai-Ting 
Published: December 2019

ABSTRACT

A positive phenotypic characteristic of glucose-oxidizing acinetobacters was demonstrated with blood agar containing
d
-glucose. Glucose-oxidizing
Acinetobacter baumannii
,
Acinetobacter
genospecies 3,
Acinetobacter lwoffii
, and
Acinetobacter
genospecies 13 sensu Tjernberg and Ursing caused a unique brown discoloration of media supplemented with 5% blood (of horse, sheep, or human origin) and an aldose sugar (0.22 M
d
-glucose,
d
-galactose,
d
-mannose,
d
-xylose, or lactose). The browning effect was not observed when a ketose sugar (
d
-fructose or sucrose) was substituted for the aldose sugar or under high osmolarity in the presence of mannitol, glycerol, or sodium chloride. Other gram-negative nonfermenters (non-glucose-oxidizing acinetobacters,
Pseudomonas aeruginosa
, other
Pseudomonas
spp.,
Stenotrophomonas maltophilia
,
Flavobacterium
spp., and
Moraxella
spp.) did not cause similar discoloration. This novel browning effect may serve as an alternative trait for identifying glucose-oxidizing acinetobacters.
Nonrandom Association of IS6110andMycobacterium tuberculosis: Implications for Molecular Epidemiological Studies
Authors: McHugh T. D. , Gillespie S. H. 
Published: December 2019

ABSTRACTIS6110restriction fragment length polymorphism typing is now established as the primary typing method forMycobacterium tuberculosis. It has been assumed that the position of bands is random. Thus, the discrimination of the technique increases in proportion to the copy number. Two collections ofM. tuberculosiswere investigated to test this hypothesis. We identified 33 positions in isolates from a Tanzanian collection and 25 positions in isolates from a London, United Kingdom, collection where bands were significantly more likely to be present than would be expected by chance. These data suggest that band position is not random, and this possibility may have an impact on the interpretation of molecular epidemiological studies ofM. tuberculosis.
Simplification of a Locus-Specific DNA Typing Method (Vir Typing) for
Streptococcus pyogenes
Authors: Hartas J. , Hibble M. , Sriprakash K. S. 
Published: December 2019

ABSTRACT

We describe a simplification of a highly discriminatory molecular typing method, called Vir typing, for
Streptococcus pyogenes
(D. Gardiner, J. Hartas, B. Currie, J. D. Mathews, D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl. 4:288–293, 1995). The procedure can be completed within a day, is reproducible, and can be applied directly to colonies growing on primary culture plates, allowing rapid establishment of strain identity in an outbreak.
Association of
Providencia alcalifaciens
with Diarrhea in Children
Authors: Albert M. John , Faruque A. S. G. , Mahalanabis D. 
Published: December 2019

ABSTRACT

It has been demonstrated in previous studies that
Providencia alcalifaciens
can produce diarrhea by an invasive mechanism. In the present study,
P. alcalifaciens
was isolated from the stool specimens of 17 of 814 diarrheal children younger than 5 years of age (2.1%) and from those of 4 of 814 matched controls (0.49%) (
P
= 0.004), indicating that the organism is significantly associated with diarrhea. However, 71% of
P. alcalifaciens
-positive diarrheal children had simultaneous infections with other recognized enteric pathogens.
Isolation of Influenza C Virus during an Outbreak of Influenza A and B Viruses
Authors: Greenbaum Evgenia , Morag Avraham , Zakay-Rones Zichria 
Published: December 2019

ABSTRACT

During the winter of 1996 to 1997 two cases of influenza C were confirmed, one by isolation and the second by serology (fourfold increase in hemagglutination inhibition antibodies). The cases of influenza C occurred during an outbreak of influenza A (H
3
N
2
) and B viruses. The positive isolation was from one of three throat washings sent to the laboratory, and the other case was from a group of 51 students participating in a study of influenza virus vaccination. It seems, therefore, that influenza C virus should also be considered when examining patients with respiratory infections during the influenza season.
Number of Days Required for Recovery of Mycobacteria from Blood and Other Samples
Authors: Esteban Jaime , Molleja Ana , Fernández-Roblas Ricardo , Soriano Francisco 
Published: December 2019

ABSTRACT

From 1991 to 1996, 541 blood samples were tested for the presence of mycobacteria; 56 were positive (30 patients, 26 human immunodeficiency virus positive). The species found were
Mycobacterium avium
(41 samples from 18 patients),
Mycobacterium tuberculosis
(12 samples from 9 patients), and three other species (1 sample each). The average time to detection was 25.23 days (22.65 for
M. avium
and 35.33 for
M. tuberculosis
). For 10 patients, the blood isolate was the only mycobacterium detected (4
M. tuberculosis
).
Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates
Authors: Ravyn M. Dana , Goodman Jesse L. , Kodner Carrie B. , Westad Deborah K. , Coleman Lisa A. , Engstrom Suzanne M. , Nelson Curt M. , Johnson Russell C. 
Published: December 2019

ABSTRACTHuman granulocytic ehrlichiosis (HGE) is an emerging infection caused by anEhrlichiaspecies closely related toEhrlichia equiandEhrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused byEhrlichia chaffeensis,Rickettsia rickettsii, andCoxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples fromR. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests forBorrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n= 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure toEhrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.
Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital
Chlamydia trachomatis
Infection
Authors: Puolakkainen Mirja , Hiltunen-Back Eija , Reunala Timo , Suhonen Satu , Lähteenmäki Pekka , Lehtinen Matti , Paavonen Jorma 
Published: December 2019

ABSTRACT

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx
Chlamydia trachomatis
assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland.
Chlamydia trachomatis
was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of
C. trachomatis
infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital
C. trachomatis
infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic
C. trachomatis
infections.
Hospital Specificity, Region Specificity, and Fluconazole Resistance of
Candida albicans
Bloodstream Isolates
Authors: Pfaller M. A. , Lockhart S. R. , Pujol C. , Swails-Wenger J. A. , Messer S. A. , Edmond M. B. , Jones R. N. , Wenzel R. P. , Soll D. R. 
Published: December 2019

ABSTRACT

In a survey of bloodstream infection (BSI) isolates across the continental United States, 162
Candida albicans
isolates were fingerprinted with the species-specific probe Ca3 and the patterns were analyzed for relatedness with a computer-assisted system. The results demonstrate that particular BSI strains are more highly concentrated in particular geographic locales and that established BSI strains are endemic in some, but not all, hospitals in the study and undergo microevolution in hospital settings. The results, however, indicate no close genetic relationship among fluconazole-resistant BSI isolates in the collection, either from the same geographic locale or the same hospital. This study represents the first of three fingerprinting studies designed to analyze the origin, genetic relatedness, and drug resistance of
Candida
isolates responsible for BSI.
Identification of New Influenza B Virus Variants by Multiplex Reverse Transcription-PCR and the Heteroduplex Mobility Assay
Authors: Zou Shimian , Stansfield Carol , Bridge Jodi 
Published: December 2019

ABSTRACT
A quick genetic approach for the screening of influenza virus variants was developed in this laboratory (S. Zou, J. Clin. Microbiol. 35:2623–2627, 1997). It uses multiplex reverse transcription and multiplex PCR to amplify and differentiate the variable region of the hemagglutinin genes of different types and subtypes of influenza viruses. Variants within the same type or subtype are then identified by the heteroduplex mobility shift assay of the amplicons. The method was used to screen influenza virus isolates received from provincial laboratories during the 1996–1997 season and was able to identify new influenza B virus variants. Sequencing of the amplicons derived from the hemagglutinin gene of the identified variants and comparison with the vaccine strain B/Harbin/7/94 showed substitution rates of 2.26 to 2.55% at the nucleotide level and 4.26 to 4.68% at the amino acid level. The result further demonstrated that the approach provides a quick, sensitive, and reliable screening for influenza virus variants. It also suggested the necessity of close monitoring of influenza B virus isolates in the 1997–1998 season and critical evaluation of the reference strain for the type B influenza virus.