Richard Frothingham

Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats

Genetic loci containing variable numbers of tandem repeats (VNTR loci) form the basis for human gene mapping and identification, forensic analysis and paternity testing. The variability of bacterial tandem repeats has not been systematically studied. Eleven tandem repeat loci in the M. tuberculosis genome were analysed. Five major polymorphic tandem repeat (MPTR) loci contained 15-bp repeats with substantial sequence variation in adjacent copies. Six exact tandem repeat (ETR) loci contained large DNA repeats with identical sequences in adjacent repeats. These 11 loci were amplified in 48 strains to determine the number of tandem repeats at each locus. The strains analysed included 25 wild-type strains of M. tuberculosis, M. bovis, M. africanum and M. microti and 23 substrains of the attenuated M. bovis BCG vaccine. One of the five MPTR loci and all six ETR loci had length polymorphisms corresponding to insertions or deletions of tandem repeats. Most ETR loci were located in intergenic regions where copy number may influence expression of downstream genes. Each ETR locus had multiple alleles in the panel. Combined analysis identified 22 distinct allele profiles in 25 wild-type strains of the M. tuberculosis complex and five allele profiles in 23 M. bovis BCG substrains. Allele profiles were reproducible and stable, as demonstrated by analyses of multiple isolates of particular reference strains obtained from different laboratories. VNTR typing may be generally useful for strain differentiation and evolutionary studies in bacteria.

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

Efforts to culture and identify the intracellular bacteria associated with Whipples 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 Whipples disease. A search by computer for similar rRNA sequences filed in databases showed the Whipples-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 patients illness has not been identified previously in a patient with Whipples disease.

Disseminated Bacille Calmette-Guérin Disease After Vaccination: Case Report and Review

The attenuated bacille Calmette-Guérin (BCG) vaccine is administered to prevent tuberculosis. Complications of vaccination are uncommon. We report a new case of disseminated BCG disease and review 27 additional cases identified from a review of > 5,000 reports published between 1980 and 1996. Twenty-four of the 28 total cases were associated with an immune deficiency, including nine cases of AIDS. Seventy-one percent of the cases occurred in children younger than 2 years old. Sixty-eight percent of the patients were male. About one-half of the patients were vaccinated in a developed nation, but 85% of the cases were reported from a developed nation. Response to therapy was poor, with an overall mortality rate of 71%. We made two new observations. Disseminated BCG disease has historically been a disease of infants, but cases now occur in adults and older children coinfected with human immunodeficiency virus. Cases also occur after revaccination of individuals who were anergic following the initial administration of BCG vaccine. Disseminated BCG disease is an uncommon but devastating complication of vaccination that should be considered in the appropriate clinical setting. Immunocompromised infants and patients with late-stage AIDS are at greatest risk and respond poorly to standard therapies.

Efficacy and safety of live attenuated persistent and rapidly cleared Mycobacterium tuberculosis vaccine candidates in non-human primates.

Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc(2)6020 (DeltalysA DeltapanCD) and mc(2)6030 (DeltaRD1 DeltapanCD) in cynomolgus macaques. In murine models, mc(2)6020 is rapidly cleared while mc(2)6030 persists. Both mc(2)6020 and mc(2)6030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc(2)6020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc(2)6030 vaccinates, but survival did not differ among the groups.

Complex Transmission Dynamics of Clonally Related Virulent Mycobacterium tuberculosis Associated with Barhopping by Predominantly Human Immunodeficiency Virus-Positive Gay Men

Limited data suggest that measures to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Molecular epidemiologic methods (analysis of IS6110 patterns, spoligotypes, variable numbers of tandem DNA repeats, and automated DNA sequence data) identified a cohort of 48 persons who were infected with progeny of the same Mycobacterium tuberculosis strain. Epidemiologic investigation documented that a large proportion of the patients were gay white human immunodeficiency virus-positive men. Most practiced barhopping, an activity that involved patronizing many bars in the same neighborhood each night. Few subjects were directly linked to more than 1 or 2 other persons by conventional investigation methods, which shows that the transmission dynamics were unusually complex compared with most previously described episodes of strain spread. The data support the concept that identification of locations where pathogen dissemination likely occurs may provide additional strategies for targeted tuberculosis control.

Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors

It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm‐dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm‐independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis–C. elegans pathogenesis system to show that previously known and unknown virulence‐related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis–C. elegans pathogenesis system that is described here can be used to ide.jpgy and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.

Rapid identification of laboratory contamination with Mycobacterium tuberculosis using variable number tandem repeat analysis.

ABSTRACT Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study ofMycobacterium tuberculosis isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.

Glucose Homeostasis Abnormalities Associated with Use of Gatifloxacin

BACKGROUND More than 20 published case reports have described an association between the use of gatifloxacin and hypoglycemia or hyperglycemia. We compare the rates of glucose homeostasis abnormality (GHA) adverse event reports (AERs) associated with the use of gatifloxacin and comparator quinolones. METHODS We obtained spontaneous AERs associated with the use of ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin from the US Food and Drug Administration that were reported between November 1997 and September 2003. We removed duplicate and foreign cases. We used specific coding terms to identify GHA AERs. We calculated GHA AER rates, using either the total number of AERs or estimated retail prescriptions as denominators. RESULTS The use of ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin was associated with 10,025 unique AERs in the United States, including 568 GHA AERs, 25 of which had fatality. Use of gatifloxacin was associated with 453 GHA AERs (80%) and 17 GHA AERs with fatality (68%). GHA AERs comprised 24% of all AERs associated with gatifloxacin, compared with ciprofloxacin (1.3%), levofloxacin (1.6%), and moxifloxacin (1.3%) (P<.0001 for each comparison). Use of gatifloxacin was associated with 477 GHA AERs per 10(7) retail prescriptions, compared with ciprofloxacin (4 GHA AERs), levofloxacin (11 GHA AERs), and moxifloxacin (39 GHA AERs) (P<.0001 for each comparison). Patients with GHA AERs were older and more likely to be receiving concomitant treatment for diabetes. Limitations of the study include the use of spontaneous adverse event reporting, which is incomplete and potentially biased. This analysis cannot be used alone to demonstrate causality. CONCLUSIONS Use of gatifloxacin is associated with a much higher rate of GHA AERs than are comparator quinolones. This analysis is consistent with the results of in vitro analyses, animal studies, human volunteer studies, case reports, and a large randomized trial. Alternatives to gatifloxacin should be used in patients with diabetes.

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.

Identification and Evolution of an IS6110 Low-Copy-Number Mycobacterium tuberculosis Cluster

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.