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M D Cave

Publications and source records attributed to M D Cave.

At least 55 records · Page 3Linked to original sources
PubMed1997

DNA fingerprinting with two probes decreases clustering of Mycobacterium tuberculosis.

DNA fingerprinting of Mycobacterium tuberculosis is used to study the epidemiology of tuberculosis, but the specificity of the widely used IS6110 technique has not been validated. Isolates from Denver, Colorado from December 1988 through June 1994 were fingerprinted with the IS6110 technique. Available records were reviewed for patients whose isolates were within IS6110-defined clusters, and these isolates were fingerprinted with an independent technique (pTBN12). Of 189 isolates, 86 (46%) were in IS6110-defined clusters. Clustering was inversely related to the number of copies of IS6110, ranging from 12 of 12 (100%) to 37 of 48 (77%) and 37 of 129 (29%) for isolates having one, two to five, and more than five copies (p < 0.001). Of the 86 isolates clustered with the IS6110 technique, 35 (41%) had unique pTBN12 fingerprints. Discordant results with the two fingerprinting techniques were more common among isolates having five or fewer copies of IS6110. Epidemiologic links were identified among four of 35 (11%) patients whose isolates had discordant fingerprinting results, as compared with 40 of 51 (78%) of those whose isolates matched by both IS6110 and pTBN12. DNA fingerprinting with the IS6110 technique was not a specific marker of DNA clonality, particularly among isolates having fewer than five copies of IS6110. The use of a supplemental DNA fingerprinting technique decreased clustering and improved the correlation between the transmission links predicted by molecular techniques and epidemiologic investigation.

Cluster Analysis↗
PubMed1997

Chest radiographic findings in patients with tuberculosis with recent or remote infection.

To determine if chest radiographic findings differ in adult tuberculosis patients with recent and remote infection, we reviewed the chest radiographs of 103 patients with tuberculosis in Los Angeles and performed RFLP analyses of their Mycobacterium tuberculosis isolates. Patients whose isolates had identical or closely related RFLP patterns were considered a "cluster." Most patients in large clusters (more than seven patients) had tuberculosis from recent infection, whereas most unclustered patients had tuberculosis from remote infection. Mediastinal adenopathy or pleural effusions were classified as typical of recent infection, and upper lobe infiltrates, cavitation, or fibrosis were classified as characteristic of remote infection. Radiographic patterns were typical of remote infection in 62% of patients and were characteristic of recent infection in 23% of patients. The distribution of these radiographic patterns was similar in clustered and unclustered patients, both with or without human immunodeficiency virus (HIV) coinfection. However, mediastinal adenopathy and pleural effusions were significantly more common in HIV-infected patients. We conclude that: (1) chest radiographic findings in adults with tuberculosis of recent infection are similar to those in patients with remote infection; (2) the distinctive chest radiographic findings in HIV-infected patients with tuberculosis are not due to an increased frequency of recent infection.

AIDS-Related Opportunistic Infections↗
PubMed1996

Isolation and sequence of a Mycobacterium tuberculosis sigma factor.

A DNA segment from Mycobacterium tuberculosis containing a gene for a putative sigma factor was isolated and sequenced. The protein encoded by this gene is 92% similar to the Mycobacterium smegmatis sigma factor MysB, and has been designated Mtu SigB. A Mycobacterium leprae homologue of mysB and mtu sigB was identified in the database.

Amino Acid Sequence↗
PubMed1996

Strand displacement amplification and the polymerase chain reaction for monitoring response to treatment in patients with pulmonary tuberculosis.

Specific amplification of Mycobacterium tuberculosis DNA was investigated as an alternative to conventional microbiologic follow-up in 31 cases of smear- and culture-positive pulmonary tuberculosis. Strand displacement amplification (SDA) and the polymerase chain reaction (PCR) were applied to 438 sequential sputum specimens: 67 (15%) were positive by culture, 248 (57%) by SDA, and 231 (53%) by PCR (chi2=3.94, P=.05). Of 200 specimens collected >180 days after treatment started, none yielded positive cultures, while 50 (25%), representing 16 patients, were positive by both DNA assays. A weak correlation was demonstrated between DNA persistence in sputum and duration of culture positivity (r=0.45, P=.01), although no correlation was found with the radiographic extent of disease. The inability to distinguish live and dead organisms precludes DNA amplification from use in therapeutic monitoring. For this purpose, quantitative RNA assays are needed if such techniques are to supplant conventional microbiology.

Base Sequence↗
PubMed1996

Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA.

Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays.

Alkalies↗
PubMed1996

Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex.

The specificity of IS6110 for the Mycobacterium tuberculosis complex has recently been questioned. We observed no cross-reaction with 27 nontuberculous mycobacteria using strand displacement- and PCR-based amplification of the nucleotide 970 to 1026 and 762 to 865 regions of IS6110. These data support use of selected regions of IS6110 as M. tuberculosis complex-specific targets.

Bacteriological Techniques↗
PubMed1996

Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction.

Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110.

Animals↗
PubMed1996

Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis.

Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments.

Bacterial Typing Techniques↗
PubMed1996

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.

DNA Fingerprinting↗
PubMed1996

Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis.

Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.

Adenosine↗
PubMed1996

Polymerase chain reaction detection of Mycobacterium tuberculosis in formalin-fixed tissue.

Use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue would be of great diagnostic value. However, formaldehyde has been reported to decrease the efficiency of amplification by structurally altering the polynucleotide chain. We sought to determine the ability of the PCR assay to detect M. tuberculosis in formalin-fixed, paraffin-embedded tissue in a mouse experimentally infected with the H37Rv strain of M. tuberculosis. Lung tissue from the infected mouse was cultured to determine the number of organisms per gram of tissue. The remaining lung tissue was divided into eight portions, seven of which were fixed in 10% neutral buffered formalin for 24-h intervals over periods lasting from 1 to 7 d, and a control portion was placed in isotonic saline. The tissue samples were then paraffin-embedded, and sections were obtained from each tissue block for PCR analysis. We show that the PCR assay can detect as few as nine organisms in a 5-micron section of tissue, and that up to 7 d of fixation in 10% neutral buffered formalin has a negligible effect on the assay. The PCR assay can detect low numbers of M. tuberculosis organisms in formalin-fixed, paraffin-embedded tissue.

Animals↗
PubMed1995

Evolution of rifampin resistance in human immunodeficiency virus-associated tuberculosis.

Acquired rifampin resistance without preexisting isoniazid resistance is highly unusual in patients with tuberculosis. The purpose of this report is to describe and characterize that unusual pattern of acquired drug resistance in three patients with human immunodeficiency virus (HIV) infection. The patients originally had Mycobacterium tuberculosis strains that were susceptible to isoniazid and rifampin. During treatment in two patients and after completion of therapy in the remaining one, each patient developed active, rifampin-resistant, isoniazid-susceptible tuberculosis. One patient subsequently developed isoniazid resistance also. Studies on patients' M. tuberculosis isolates using IS6110 restriction fragment length polymorphism typing and rpoB gene sequencing indicated that rifampin resistance in each patient arose during therapy by an rpoB gene mutation in the original M. tuberculosis isolate. Detection of this unusual drug-resistance phenotype in three patients with HIV infection suggests that acquired rifampin resistance is somehow associated with co-infection due to HIV and tuberculosis.

AIDS-Related Opportunistic Infections↗
PubMed1994

Restriction fragment length polymorphism analysis detecting a community-based tuberculosis outbreak among persons infected with human immunodeficiency virus.

Analysis of restriction fragment length polymorphisms (RFLP) was used to investigate an increase in tuberculosis (TB) among noninstitutionalized human immunodeficiency virus (HIV)-infected persons in King County, Washington. Using the IS6110 insertion sequence, RFLP analysis was done on Mycobacterium tuberculosis isolates from 18 HIV-infected patients and 10 randomly selected patients without HIV risk factors. Six HIV-infected patients with the same M. tuberculosis strain had contact at one or more of three bars as their only common exposure. Two other HIV-infected persons, a patient and a health care worker who had close contact, had matching strains. Isolates from the 10 remaining HIV-infected patients and the 10 patients without HIV risk factors had different DNA patterns. Analysis of RFLP patterns revealed a community outbreak of TB among HIV-infected persons who had not been previously linked following conventional investigation by the health department. This technique deserves further evaluation as an epidemiologic tool in the investigation of TB.

AIDS-Related Opportunistic Infections↗
PubMed1994

Stability of DNA fingerprint pattern produced with IS6110 in strains of Mycobacterium tuberculosis.

To assess the stability of IS6110 restriction fragment length polymorphism patterns, DNA fingerprints of 6 Mycobacterium bovis isolates from 1 patient and of 41 Mycobacterium tuberculosis isolates from 18 patients were compared. The fingerprint pattern for a given patient remained identical or nearly identical despite recovery of the isolates during intervals which ranged from 8 months to 4.5 years. Changes in drug resistance profile did not alter a strain's fingerprint pattern.

DNA Fingerprinting↗
PubMed1993

Rapid, amplification-based fingerprinting of Mycobacterium tuberculosis.

Insertion element IS6110 occurs in multiple copies throughout the Mycobacterium tuberculosis genome, and the variability of its insertion sites is the basis for the IS6110 restriction fragment length polymorphism (RFLP) method for typing. We describe a novel gene amplification method to assess the variability of the location of IS6110. A unilateral-nested polymerase chain reaction and hybridization procedure was used to measure the variability in the distances between IS6110 elements and copies of a major polymorphic tandem repeat sequence of M. tuberculosis. The pattern of amplicons produced could be used to cluster epidemiologically related strains of M. tuberculosis into groups which correlated with the groups formed using IS6110-RFLP typing. Reliable patterns can be generated directly from sputum specimens as well as from M. tuberculosis cultures. We designated the novel method as IS6110-ampliprinting.

Bacterial Typing Techniques↗
PubMed1993

Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.

DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.

Base Sequence↗
PubMed1992

DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology.

Mycobacterium tuberculosis isolates were studied by comparing large restriction fragment (LRF) patterns produced by digestion of chromosomal DNA with infrequent-cutting endonucleases and pulsed-field gel electrophoresis. Four cultures of H37Rv and 36 clinical isolates of M. tuberculosis were compared by using DraI, AsnI, XbaI, and SpeI. DraI and AsnI allowed easy visual separation of 18 of 21 epidemiologically unrelated strains. XbaI and SpeI allowed discrimination of all 21 unrelated strains, including the 3 strains inseparable with DraI and AsnI, but comparison of LRF patterns was more tedious because of overlapping fragments. A total of 26 isolates belonging to 10 clusters of related isolates were compared by pulsed-field gel electrophoresis, with all related isolates giving identical LRF patterns. These included multiple isolates from the same patient or the same family. The same grouping of clustered isolates was obtained when BamHI DNA digests were hybridized with two probes from the insertion sequence IS6110. Long-term laboratory passage of H37Rv produced minimal detectable changes in LRF patterns. LRF patterns are useful tools for epidemiologic studies of tuberculosis without the need for radioactive or specific DNA probes.

DNA Fingerprinting↗