Development and evaluation of a multiple-locus variable-number tandem-repeats analysis assay for subtyping Salmonella Typhi strains from sub-Saharan Africa.

PURPOSE: Molecular epidemiological investigations of the highly clonal Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) are important in outbreak detection and in tracking disease transmission. In this study, we developed and evaluated a multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) assay for characterization of S. Typhi isolates from sub-Saharan Africa. METHODOLOGY: Twelve previously reported VNTR loci were evaluated and an MLVA assay consisting of five polymorphic loci was adopted. The MLVA assay was developed for use on capillary electrophoresis systems by testing a collection of 50 S. Typhi isolates. This S. Typhi strain panel consisted of six outbreak related isolates and 44 epidemiologically unlinked isolates. Amongst these were nine S.Typhi haplotype H58 isolates. RESULTS: The MLVA assay characterized the 50 isolates into 47 MLVA profiles while PFGE analysis of the same isolates revealed 34 pulsotypes. MLVA displayed higher discriminatory power (Simpson's index of diversity (DI) 0.998 [95 % confidence interval (CI) 0.995-1.000)] as compared to pulsed-field gel electrophoresis [Simpson's DI 0.984 (95 % CI 0.974-0.994)]. CONCLUSION: The MLVA assay presented in this study is a simple, rapid and more accessible tool that serves as a good alternative to other molecular subtyping methods for S. Typhi.

Drug susceptibility testing using molecular techniques can enhance tuberculosis diagnosis.

BACKGROUND: Sputum samples were collected from tuberculosis patients in a high tuberculosis incidence area in the Western Cape, South Africa. The aim of this study was to evaluate the performance and time to diagnosis of a genotypic drug susceptibility testing method. METHODOLOGY: During June 2000 and November 2003, a total of 1,540 samples were sent for drug susceptibility testing (DST) to the national health laboratory services, and of those, a phenotypic DST result was obtained for 1,373 samples whereas a genotypic DST result was obtained for 1,301 of 1,540 samples. Performance-based calculations were done on 1,244 samples for which both a phenotypic and genotypic DST result was available. RESULTS: The reproducibility of the genotypic and phenotypic DST methods was 97% and 95%, respectively. The sensitivity and specificity of the genotypic DST method was 68% and 99% for Isoniazid and 87% and 99% for Rifampicin, respectively. Smear gradation was found to influence the performance of the genotypic DST method. The genotypic DST method gave accurate DST results for 75% of the samples within 20 days (range, 15-25), whereas the phenotypic DST results were only available for 75% of the samples after 38 days (range, 26-115) (p<0.001). CONCLUSION: This study showed that the genotypic DST could improve tuberculosis control by rapid diagnosis of drug resistant tuberculosis. This finding may have important implications for the control of drug resistant tuberculosis as it may reduce the chance for further transmission events.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis.

The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (Delta19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce Delta19::pSMT181. The Delta19 strain multiplied less well than Delta19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with Delta19. Delta19 induced lower interleukin 1beta (IL-1beta) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1beta, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.

Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2.

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the alpha-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Deltaacr2) was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.