High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers.

AIMS: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. METHODS AND RESULTS: Forty-seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi-locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. CONCLUSIONS: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.

A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracis.

The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.

Distinctive effects of CCR5, CCR2, and SDF1 genetic polymorphisms in AIDS progression.

The Genetics of Resistance to Infection by HIV-1 (GRIV) cohort represents 200 nonprogressor/slow-progressor (Slowprog) and 90 fast-progressor (Fastprog) HIV-1-infected patients. Using this unique assembly, we performed genetic studies on three recently discovered polymorphisms of CCR5, CCR2, and SDF1, which have been shown to slow the rate of disease progression. The increased prevalence of mutant alleles among Slowprogs from the GRIV cohort was significant for CCR5 (p < .0001) but not for CCR2 (p = .09) or SDF1 (p = . 12), emphasizing the predominant role of CCR5 as the major HIV-1 coreceptor. However, the prevalence of the CCR2 mutant allele (64I) was significantly increased among Slowprogs homozygous for wild-type CCR5 compared with Fastprogs (p = .04). The prevalence of double mutants SDF1-3'A/3'A genotypes was also increased among Slowprogs homozygous for wild-type CCR5 compared with Fastprogs (p = .05). The effects of the CCR2 and SDF1 mutations are overshadowed by the protective effects of the CCR5 deletion. Predictive biologic markers such as CD4 cell counts or viral load in the Slowprog population did not show significant differences between Slowprog groups with wild-type or mutant alleles for the three genes. Thus, our data suggest that the effects of these genes are exerted earlier in infection and no longer evident in the Slowprog of the GRIV cohort whose average duration of HIV infection is 12 years. We conclude that these genes, whose products serve as viral coreceptors or their ligands, may play a role early in infection and delay the onset of disease. However, among Slowprogs, whose duration of infection is >8 years, they are no longer influential for maintenance of their longterm nonprogression status. Other genetic determinants may be responsible for late protective effects.

Development and implementation of cost-effective guidelines in the laboratory investigation of diarrhea in a community hospital.

BACKGROUND: Fecal cultures are often inappropriately requested in the investigation of diarrhea. OBJECTIVES: To develop and determine the efficacy of practice guidelines for the ordering and processing of stool cultures that are submitted for the diagnosis of community-acquired diarrhea. METHODS: The results of stool cultures that were submitted to the microbiology laboratory of a tertiary care nonteaching community hospital were retrospectively reviewed. Following the implementation of guidelines, the efficacy was evaluated by comparison of fecal culture results in a prospective manner. RESULTS: Analysis of results of stool cultures that were obtained from 3072 patients during a 3-year period revealed that (1) the sensitivity (40%) and predictive value (20%) of finding neutrophils in smear preparations were too low to be clinically useful, (2) routine cultures from patients with nosocomial diarrhea were uniformly negative, and (3) multiple specimens from a patient rarely provided additional information. Based on these findings, new guidelines were developed and implemented with the cooperation of clinical staff. Three-month follow-up results showed that the total number of specimens, the number of specimens from patients with nosocomial diarrhea, and multiple specimens declined by 37.7%, 70.6%, and 50%, respectively. However, the isolation rate of pathogens increased from 11.7% to 18.7%. CONCLUSIONS: The application of practice guidelines that include elimination of smear examination of rectal swabs, exclusion of routine cultures from patients with nosocomial diarrhea, and rejection of repeated cultures can result in significant cost saving without adversely affecting patient care.