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Biomedical subjects

R Frothingham

Publications and source records attributed to R Frothingham.

At least 37 records · Page 2Linked to original sources
PubMed1997

The risks and benefits of childhood bacille Calmette-Guérin immunization among adults with AIDS. International MAC study groups.

OBJECTIVE: To define the risks of disseminated bacille Calmette-Guérin (BCG) or disseminated Mycobacterium tuberculosis in adults with AIDS who were immunized with BCG in childhood. DESIGN: HIV-infected patients with CD4 < 200 x 10(6)/l were enrolled from five study sites (New Hampshire, Boston, Finland, Trinidad and Kenya). Prior BCG immunization was determined and blood cultures for mycobacteria were obtained at study entry and at 6 months. Acid-fast bacilli were identified as Mycobacterium tuberculosis complex (MTBC) using DNA probes. MTBC isolates were then typed by both IS6110 restriction fragment length polymorphism and polymerase chain reaction/restriction enzyme analysis. SETTING: Most patients in New Hampshire and Finland were outpatients; most patients in Trinidad were inpatients with terminal illness; and most patients in Kenya were outpatients, although 44 were inpatients with terminal illness. PARTICIPANTS: A total of 566 patients were enrolled, including 155 with childhood BCG immunization; 318 patients had a single study visit and culture, and 248 patients had two study visits and cultures. MAIN OUTCOME MEASURES: Isolation and identification of mycobacteria from blood cultures. RESULTS: Blood cultures were positive for MTBC in 21 patients; none were positive for M. bovis BCG, and 21 were M. tuberculosis-positive. In Trinidad, seven (87%) out of eight isolates of M. tuberculosis were indistinguishable by IS6110 typing; BCG immunization was associated with a decreased risk of bacteremic infection with M. tuberculosis (P = 0.05). CONCLUSIONS: The risk of disseminated BCG among adult AIDS patients with childhood BCG immunization is very low. Childhood BCG immunization is associated with protection against bacteremia with M. tuberculosis among adults with advanced AIDS in Trinidad.

AIDS-Related Opportunistic Infections↗
PubMed1997

Subspecific differentiation of Mycobacterium avium complex strains by automated sequencing of a region of the gene (hsp65) encoding a 65-kilodalton heat shock protein.

To develop a strategy for rapid species assignment and strain differentiation of Mycobacterium avium complex (MAC) organisms, the sequence of a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein was determined for 56 isolates, including 21 patient isolates and 35 reference strains. Eleven hsp65 alleles were identified, and there was no sharing of alleles between strains classified as M. avium and Mycobacterium intracellulare based on serovar and species-specific DNA hybridization probes. Phylogenetic analysis showed that 30 strains had one of two hsp65 alleles which were found in known M. avium organisms, 23 strains had one of six alleles allied with known M. intracellulare organisms, and three MAC isolates had one of three hsp65 alleles that differed substantially from the consensus M. avium and M. intracellulare hsp65 sequences. Estimates of strain relationships based on the sequences of hsp65 and the 16S-23S ribosomal DNA internal transcribed spacer were similar. Automated DNA sequencing of a 360-bp region of the hsp65 gene from MAC organisms provides a rapid and unambiguous marker system for strain differentiation and permits specific assignment of these acid-fast organisms for diagnostic purposes.

Alleles↗
PubMed1997

PCR identification of Mycobacterium bovis BCG.

The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG.

Animals↗
PubMed1996

Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

Amidohydrolases↗
PubMed1995

Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG.

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Seven strains of the M. tuberculosis complex were sequenced in a region of about 300 bp which contains multiple 15-bp tandem repeats and which is part of a 1,551-bp open reading frame. Four distinct sequences were obtained, each defining a sequevar. A sequevar includes the strain or strains with a given sequence. The type strain M. tuberculosis TMC 102 (H37Rv) was designated sequevar MED-G. When compared to MED-G, sequevar LONG had an insertion of one 15-bp tandem repeat and sequevar SHORT had a deletion of one tandem repeat. Sequevar MED-C had a G-->C substitution, coding for the conservative change Ser-->Thr. BanI cuts only sequevar MED-C at the site of the substitution. PCR-restriction enzyme analysis was used to determine the sequevars of 92 M. tuberculosis complex strains. All 23 M. bovis BCG strains belonged to sequevar MED-C. The M. africanum type strain was sequevar SHORT. The remaining 68 strains of M. tuberculosis, M. bovis (not BCG), and M. microti were sequevars LONG (3 strains) or MED-G (65 strains). PCR-restriction enzyme analysis was applied to reference strains and clinical isolates with a worldwide distribution. This method provides rapid, sensitive, and specific identification of the important vaccine strain M. bovis BCG.

Amino Acid Sequence↗
PubMed1994

Molecular phylogeny of the Mycobacterium avium complex demonstrates clinically meaningful divisions.

Phylogenetic analysis of nucleotide sequence data is widely used for viral epidemiology. To explore its use in bacterial strain differentiation, the variable 16S-23S rDNA internal transcribed spacer (ITS) in 24 clinical isolates originally identified as Mycobacterium avium complex (MAC) was sequenced. Three isolates had an identical sequence that differed greatly from the rest. They belonged to the recently described Mycobacterium celatum. The 21 MAC clinical isolates gave 6 ITS sequences, each defining a sequevar. Thirteen isolates from 11 AIDS patients with disseminated MAC disease belonged to 2 sequevars, which differed in ITS sequence by 1 nucleotide. In contrast, 7 pulmonary-source MAC isolates were genetically more diverse. They belonged to 4 sequevars, which differed from each other by 6-20 nucleotides and from the disseminated disease-associated sequevars by at least 12 nucleotides. The single urine MAC isolate had the same sequence as 1 of the pulmonary isolates. Because the disseminated disease-associated MAC strains were distinct by ITS sequence analysis, it should be possible to develop a molecular assay to detect them directly in clinical specimens or in environmental samples. Molecular phylogeny at the strain level may be widely useful in studies of bacterial epidemiology and virulence.

Adult↗
PubMed1994

Extensive DNA sequence conservation throughout the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex includes the four species M. tuberculosis, M. bovis, M. africanum, and M. microti. We sequenced 13 M. tuberculosis complex strains in the 16S-to-23S rDNA internal transcribed spacer (ITS). The ITS has a high rate of nucleotide substitution. Previous reports found three nucleotide substitutions in the ITS between two M. tuberculosis complex strains. In contrast, we found the same ITS sequence in all 13 M. tuberculosis complex strains (including all four species and M. bovis BCG). This finding confirms the conservation of 16S rDNA sequence and the high DNA-DNA relatedness found in previous studies. By the usual criteria, the four species of the M. tuberculosis complex would be considered a single species. In a phylogenetic analysis based on the ITS sequence, the four species of the M. tuberculosis complex were distinct from nontuberculous mycobacteria. The ITS contains at least seven potential sites for oligonucleotide probes with specificity for the M. tuberculosis complex.

Base Sequence↗
PubMed1993

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.

Base Sequence↗
PubMed1992

Acyclovir-resistant herpes simplex virus keratouveitis after penetrating keratoplasty.

PURPOSE: A case of acyclovir-resistant herpes simplex virus keratouveitis after penetrating keratoplasty is reported. METHODS: Resistance to acyclovir was evident clinically and was confirmed by in vitro susceptibility testing. The susceptibility of the herpes simplex isolates to acyclovir and foscarnet was determined by a dye uptake assay that measured cytopathic effect, and thymidine kinase activity was measured by a plaque autoradiography technique. RESULTS: The viral isolate from postoperative day 22 was susceptible to acyclovir and foscarnet, and showed normal thymidine kinase activity. Isolates from postoperative days 29 and 32 (coinciding with deterioration in clinical appearance) were resistant to acyclovir, susceptible to foscarnet, and deficient in thymidine kinase activity. CONCLUSION: Practitioners should be aware of the potential for the emergence of resistance in this setting; prophylaxis and rational alternate therapies are discussed.

Acyclovir↗
PubMed1992

UV absorption complicates PCR decontamination.

UV irradiation is widely used to inactivate contaminating DNA in PCR. Highly UV-absorbent deoxyribonucleoside triphosphates in PCR mixtures reduce the efficiency of UV decontamination. Optimal decontamination may be achieved by irradiating the PCR mixture without the deoxyribonucleoside triphosphates.

Artifacts↗
PubMed1991

Phylogeny of the Whipple's-disease-associated bacterium.

Efforts to culture and identify the intracellular bacteria associated with Whipple's disease have been unsuccessful. Nucleotide sequencing and amplification by the polymerase chain reaction was done on the bacterial 16 S ribosomal DNA present in a small-bowel biopsy specimen taken from a patient with Whipple's disease. A search by computer for similar rRNA sequences filed in databases showed the Whipple's-associated organism to be most similar to bacteria of the Rhodococcus, Streptomyces, and Arthrobacter genera, and more weakly related to mycobacteria. The biopsy specimen was estimated to contain around 10(7) cells of the organism. The probable aetiological agent for our patient's illness has not been identified previously in a patient with Whipple's disease.

Aged↗
PubMed1991

Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.

Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.

Autoradiography↗