Biomedical subjects

G H Mazurek

Publications and source records attributed to G H Mazurek.

18 recordsLinked to original sources

PubMed2001

Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States.

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.

Amidohydrolases↗

PubMed2000

Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis.

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were > or =100 microg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 microg/ml, 3 had the same mutation (Thr47-->Ala) and 18 had the wild-type sequence. For the three Thr47-->Ala mutants PZA MICs were 12.5 microg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 microg of PZA per ml and the third was resistant to 800 microg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to > or =100 microg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47-->Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to > or =100 microg of PZA per ml.

Amidohydrolases↗

PubMed1999

Safe determination of susceptibility of Mycobacterium tuberculosis to antimycobacterial agents by flow cytometry.

We showed previously that susceptibility testing for Mycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.

Antitubercular Agents↗

PubMed1998

Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours.

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.

Agar↗

PubMed1997

Genetic similarity among Mycobacterium avium isolates from blood, stool, and sputum of persons with AIDS.

Large-restriction-fragment pattern comparison of Mycobacterium avium from 85 blood, stool, and respiratory specimens from 25 human immunodeficiency virus-infected San Francisco patients revealed 4 strains that infected multiple people (3 groups of 2 patients and 1 group of 3 patients). Most patients harbored a single M. avium strain, but 2 strains were recovered from 8 patients. The significance of recovering 2 strains is not clear, since the second strain was seldom recovered more than once. The strain recovered from blood was recovered from stool of 4 patients and respiratory secretions of 6 patients >4 weeks before detection of bacteremia, indicating that the intestinal and respiratory tracts are entry portals from which M. avium can disseminate. M. avium from 21 cities outside of California served as controls. Thus, a single M. avium strain can cause disseminated infection in multiple patients. This may represent infection from a common environmental source or person-to-person spread.

AIDS-Related Opportunistic Infections↗

PubMed1996

Tuberculosis outbreak in a Texas prison, 1994.

In 1994 a Texas prison containing a population of mentally retarded inmates experienced a large tuberculosis outbreak. Fifteen cases of tuberculosis were identified (8 confirmed by positive cultures for Mycobacterium tuberculosis) and more than 100 inmates became infected. The culture-confirmed patients were infected with an identical strain of tuberculosis as demonstrated by polymerase chain reaction (PCR) based DNA fingerprinting technique. The prison followed standard tuberculosis infection control policies, but these controls were inadequate to prevent tuberculosis transmission in this special population. Two hundred and thirty inmates (119 inmates showing evidence of new tuberculosis infection or active disease and 111 healthy controls) were enrolled in the investigation. Inmate cell assignments, job duties, and educational classes were identified and medical chart reviews were conducted on all inmates. Tuberculosis transmission was associated with residing on the D Wing of the prison (OR = 25.84, P < 0.01), attending school in Classroom A (OR = 8.34, P = 0.01) and working on the prison utility work crew (OR = 2.52, P < 0.01). The index case in the outbreak had been prescribed 6 months of isoniazid (INH) chemoprophylaxis in 1988.

Case-Control Studies↗

PubMed1996

DNA fingerprinting by infrequent-restriction-site amplification.

Identification of bacterial strains by DNA fingerprinting facilitates epidemiologic studies and improves disease control. For some species of organisms, no typing method is available; for others, typing methods are tedious. We developed a method of amplifying DNA sequences flanking infrequent restriction sites by PCR and used the method to produce strain-specific electrophoretic patterns from crude bacterial lysates. This method of fingerprinting is rapid, sensitive, and widely applicable. Identical enzymes, adaptors, primers, and PCR conditions were used to characterize 32 Mycobacterium avium-M. intracellulare isolates, 4 Pseudomonas aeruginosa isolates, and 4 Staphylococcus aureus isolates.

DNA Fingerprinting↗

PubMed1996

Detection of Mycobacterium tuberculosis in cerebrospinal fluid following immunomagnetic enrichment.

The detection of Mycobacterium tuberculosis by culture of cerebrospinal fluid (CSF) is unacceptably slow. Low numbers of organisms and the presence of reaction inhibitors may prevent detection of M. tuberculosis by PCR. We used immunomagnetic enrichment to accelerate and enhance the detection of mycobacteria in CSF after demonstrating the utility of the method with pure suspensions. Growth was detected earlier in Bactec cultures of magnetically recovered mycobacteria than in untreated CSF (7 versus 15 days). We detected M. tuberculosis DNA by PCR in the immunomagnetically enriched sample but not in untreated CSF. PCR fingerprintings of the immunomagnetically recovered M. tuberculosis and of the isolate subsequently recovered by culture were identical.

Adult↗

PubMed1995

Genetic differences between BCG substrains.

SETTING: University-affiliated Mycobacteriology Reference Laboratory. OBJECTIVE: To determine the genetic differences of 25 BCG isolates representing 16 referenced substrains. DESIGN: Non-randomized, observational study based on the visual comparison of the large restriction fragment (LRF) patterns created by digesting each BCG isolate's DNA with an infrequent cutting restriction endonuclease (DraI, AsnI, XbaI or SpeI) and separating the resultant DNA fragments with pulsed field gel electrophoresis. RESULTS: The 25 BCG isolates gave 13 different DraI LRF patterns, 11 different XbaI LRF patterns, 11 different AsnI LRF patterns, and 15 different SpeI LRF patterns. Examples of the same BCG substrains from different sources produced the same LRF patterns for only 2 of 6 substrains studied. These findings suggest a significant degree of genetic diversity in this group of isolates despite a common origin. Four clinical BCG isolates gave LRF patterns identical to BCG Tice, BCG Connaught or BCG Glaxo. The BCG LRF patterns more closely resembled patterns of Mycobacterium bovis than M. tuberculosis. CONCLUSIONS: LRF patterns can accurately identify specific BCG substrains and will be useful in epidemiologic studies, monitoring vaccine production and studies of BCG vaccine efficacy.

DNA Restriction Enzymes↗

PubMed1995

Tuberculosis outbreak among healthcare workers in a community hospital.

Twenty-nine healthcare workers (HCW) were exposed to an active case of unrecognized drug-susceptible pulmonary tuberculosis in a community hospital for as long as 2 h in the emergency room and 10 h in a medical intensive care unit. Twelve of the 29 exposed HCW could not be evaluated for tuberculosis infection because 10 of them had a previously positive tuberculin skin test and two were lost to follow-up. Of the remaining 17 tuberculin skin test negative HCW, 13 (76%) either converted their skin test to positive (10 HCW) or developed active disease (three HCW) after exposure to the index case. The Mycobacterium tuberculosis isolates from the three HCW had identical DNA restriction fragment length polymorphism (RFLP) patterns when studied by pulsed field gel electrophoresis. This case of drug-susceptible tuberculosis was associated with unusually high rates of tuberculosis infection and disease in HCW. Prevention of similar occurrences in HCW may be difficult because of the short exposure time required for transmission of tuberculosis and the absence of consensus on optimal respiratory protective measures.

Adult↗

PubMed1995

DNA fingerprinting of pathogenic bacteria by fluorophore-enhanced repetitive sequence-based polymerase chain reaction.

Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.

Base Sequence↗

PubMed1994

Initial clarithromycin monotherapy for Mycobacterium avium-intracellulare complex lung disease.

Sputum conversion rates in Mycobacterium avium-intracellulare (MAI) complex lung disease have ranged from only 50 to 80% despite the use of three to five antituberculosis agents. We initiated a prospective, open, noncomparative trial of initial clarithromycin monotherapy at 500 mg twice a day for 4 months in HIV-negative patients with MAI lung disease. The primary study end point was microbiologic improvement. Of 30 patients enrolled, 20 completed therapy. This latter group was predominantly male (60%), smokers (70%), older than 45 yr of age (90%), infected with Mycobacterium intracellulare (70%) and with bilateral disease (85%). Of 19 patients with pretreatment minimum inhibitory concentrations (MIC) for clarithromycin < 16 micrograms/ml, 58% became sputum-negative, and 21% showed significant reductions in sputum positivity. Heavily positive sputum cultures (> 200 colonies) were reduced from 30 to 47 samples pretherapy (64%) to three of 54 (6%) post-therapy (p < 0.0001); 18 of 19 patients (95%) showed an improvement in sputum cultures, chest radiographs, or both. Only two patients (7%) discontinued the drug because of adverse events. Only three (16%) of 19 isolates developed clarithromycin resistance (MIC > 32 micrograms/ml). Clarithromycin-susceptible and -resistant MAI isolates from the same patient had identical DNA large-restriction fragment patterns. Clarithromycin is the first single agent to be shown efficacious in the treatment of MAI lung disease.

Adult↗

PubMed1993

DNA large restriction fragment patterns of sporadic and epidemic nosocomial strains of Mycobacterium chelonae and Mycobacterium abscessus.

Large restriction fragment (LRF) pattern analysis of genomic DNA using pulsed-field gel electrophoresis was performed on three reference strains, 32 sporadic isolates, and 92 nosocomial isolates from 12 epidemics of Mycobacterium chelonae and Mycobacterium abscessus. Only 17 of 30 (57%) unrelated strains of M. abscessus, compared with 10 of 11 (91%) of M. chelonae strains, gave satisfactory DNA extractions, with the remainder resulting in highly fragmented DNA. DraI, AsnI, XbaI, and SpeI gave satisfactory LRF patterns. Sporadic isolates of the two species had highly variable LRF patterns, except for one reference strain and one sporadic isolate of M. chelonae that differed by only two to five bands. Evaluation of repeat isolates from five patients monitored for 8 months to 13 years (mean, 5.8 years) revealed LRF patterns to be stable, with changes of not more than two bands. LRF analysis of the seven nosocomial outbreaks with evaluable DNA revealed identical patterns in most or all of the patient isolates and in three outbreaks revealed identity with environmental isolates. These outbreaks included endoscope contamination, postinjection abscesses, and surgical wound infections. LRF analysis of genomic DNA is a useful technique for epidemiologic studies of M. abscessus and M. chelonae, although improved technology is needed for the approximately 50% of strains of M. abscessus with unsatisfactory DNA extractions.

Cross Infection↗

PubMed1993

Large DNA restriction fragment polymorphism in the Mycobacterium avium-M. intracellulare complex: a potential epidemiologic tool.

Mycobacterium avium-M. intracellulare complex (MAI) isolates were studied by comparing the large restriction fragment (LRF) patterns produced by digesting their DNAs with infrequently cutting restriction endonucleases and separating the resultant large fragments by pulsed-field gel electrophoresis. Four reference strains and 35 randomly selected clinical MAI isolates gave highly diverse LRF patterns when their DNAs were digested with XbaI or AsnI. The LRF patterns of random isolates identified to be the same species by DNA probe analysis were not similar. The LRF patterns of random isolates of the same serotype were also different. In contrast, all isolates recovered from the same patient gave identical patterns. This included 28 isolates from nine patients. One isolate from sputum, one isolate from bone marrow, and two isolates from blood recovered over a 27-month period from a patient with AIDS were identical. Seven isolates recovered from the sputum of a second patient over 37 months also had identical patterns. The LRF patterns of unrelated MAI strains are highly polymorphic, appear to be strain specific, are relatively stable, and offer exciting promise as epidemiologic markers for the study of MAI infections.

DNA, Bacterial↗

PubMed1992

Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks.

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used to generate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacterium fortuitum biovariant fortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI, HpaI, AflII, DraI, NdeI, XbaI, SpeI, or SspI was used. Epidemiologically related isolates produced identical or minimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digested with XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiac surgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRF patterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In the remaining two outbreaks, isolates which could not be separated by prior typing methods were easily distinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patterns identical to those of the disease-producing strains, confirming that the local environment was the source of infection. Pulsed-field gel electrophoresis of LRFs of genomic DNA offers great promise as an epidemiologic tool for the study of M. fortuitum.

Cross Infection↗

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↗

PubMed1991

Chromosomal DNA fingerprint patterns produced with IS6110 as strain-specific markers for epidemiologic study of tuberculosis.

Mycobacterium tuberculosis isolates were studied by comparing chromosomal DNA fingerprint patterns produced by digestion of chromosomal DNA with BamHI, followed by agarose electrophoresis and hybridization with radiolabeled probes of insertion sequence IS6110. DNA fingerprints of 14 isolates from separate members of five households or closely associated individuals were compared. Marked differences were observed when unrelated isolates were compared. There were no or minimal differences in the restriction fragment patterns generated from isolates of any one household or associated group. Among related isolates, the only noticeable difference was an additional fragment of IS6110 in the fingerprint pattern of one isolate. Insertional activity was also suggested when restriction fragment patterns of H37Rv DNA isolated in 1987 and 1990 were compared. In a similar manner, M. tuberculosis reference strain Erdman was compared to a clinical isolate from an individual working with that strain. These isolates had identical DNA fingerprints which were distinct from all other isolates, verifying laboratory-acquired infection. Chromosomal DNA fingerprint patterns produced with IS6110 are excellent stain-specific markers for the epidemiologic study of tuberculosis.

DNA Fingerprinting↗

PubMed1991

Pneumonia in chronic obstructive lung disease.

Despite the apparent common occurrence of pneumonia in patients with chronic obstructive pulmonary disease (COPD), there are little firm data on incidence, etiology, diagnostic procedures, and therapy in these patients. It appears that traditional respiratory pathogens such as the pneumococcus are declining in importance while "new" pathogens such as Pseudomonas sp., Moraxella catarrhalis, and Legionella sp. are becoming more important. The diagnosis of a specific etiologic agent is difficult in COPD and can be aided by obtaining specimens bronchoscopically. Directed therapy is optimal; however, empiric therapy is frequently unavoidable.

Diagnosis, Differential↗