A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis.

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.

Red fox viromes in urban and rural landscapes.

The Red fox (Vulpes vulpes) has established large populations in Australia's urban and rural areas since its introduction following European settlement. The cryptic and highly adaptable nature of foxes allows them to invade cities and live among humans whilst remaining largely unnoticed. Urban living and access to anthropogenic food resources also influence fox ecology. Urban foxes grow larger, live at higher densities, and are more social than their rural counterparts. These ecological changes in urban red foxes are likely to impact the pathogens that they harbour, and foxes could pose a disease risk to humans and other species that share these urban spaces. To investigate this possibility, we used a meta-transcriptomic approach to characterise the virome of urban and rural foxes across the Greater Sydney region in Australia. Urban and rural foxes differed significantly in virome composition, with rural foxes harbouring a greater abundance of viruses compared to their urban counterparts. We identified ten potentially novel vertebrate-associated viruses in both urban and rural foxes, some of which are related to viruses associated with disease in domestic species and humans. These included members of the Astroviridae, Picobirnaviridae, Hepeviridae, and Picornaviridae as well as rabbit haemorrhagic disease virus-2. This study sheds light on the viruses carried by urban and rural foxes and emphasises the need for greater genomic surveillance of foxes and other invasive species at the human-wildlife interface.

Mapping of a novel susceptibility locus suggests a role for MC3R and CTSZ in human tuberculosis.

RATIONALE: Tuberculosis remains a major cause of morbidity and mortality in the developing world. A better understanding of the mechanisms of disease protection could allow novel strategies to disease management and control. OBJECTIVES: To identify human genomic loci with evidence of linkage to tuberculosis susceptibility and, within these loci, to identify individual genes influencing tuberculosis susceptibility. METHODS: Affected sibling pair analysis in South African and Malawian populations. Independent case-control study in West Africa. MEASUREMENTS AND MAIN RESULTS: Two novel putative loci for tuberculosis susceptibility are identified: chromosome 6p21-q23 and chromosome 20q13.31-33--the latter with the strongest evidence for any locus reported to date in human tuberculosis (single point LOD score of 3.1, P = 10(-4), with a maximum likelihood score [MLS] of 2.8). An independent, multistage genetic association study in West African populations mapped this latter region in detail, finding evidence that variation in the melanocortin 3 receptor (MC3R) and cathepsin Z (CTSZ) genes play a role in the pathogenesis of tuberculosis. CONCLUSIONS: These results demonstrate how a genomewide approach to the complex phenotype of human tuberculosis can identify novel targets for further research.

Uracil-DNA glycosylase (UNG) influences the melting temperature (T(m)) of herpes simplex virus (HSV) hybridization probes.

Prior to PCR amplification, uracil-DNA glycosylase (UNG) can be incorporated to prevent amplicon contamination. Melting temperature (T(m)) analysis of herpes simplex virus (HSV) hybridization probes was used to demonstrate a concentration-dependent decrease in T(m) values when heat stabile or heat labile UNG was added.

A study of the prevalence of cytotoxic and non-cytotoxic Klebsiella oxytoca fecal colonization in two patient populations.

BACKGROUND: Klebsiella oxytoca is a cause of antibiotic-associated hemorrhagic colitis. Few reports of the occurrence of K oxytoca within stool exist and there is no gold standard method for its isolation. METHODS: MacConkey agar was modified to culture K oxytoca. Ampicillin was added and adonitol was substituted for lactose. Rectal swabs from 200 patients being screened for vancomycin-resistant enterococci (VRE) and stool specimens from 429 patients who tested negative for Clostridium difficile cytotoxin were cultured. K oxytoca isolates were evaluated for cytotoxicity to HEp-2 cells. Available charts of K oxytoca-positive patients and a convenience sample of 93 K oxytoca-negative patients who underwent testing for C difficile cytotoxicity were reviewed retrospectively for documentation of bloody stool. RESULTS: K oxytoca was isolated from 14 of 200 patients (7.0%) being screened for VRE; only one of the 14 isolates (7.1%) was cytotoxic. The organism was isolated from 42 of 429 patients (9.8%) tested for C difficile cytotoxicity; 10 isolates (23.8%) were cytotoxic. Differences in isolation and cytotoxicity rates between groups were not statistically significant. Two of 13 (15.4%) K oxytoca-positive patients screened for VRE, three of 27 (11.1%) K oxytoca-positive patients tested for C difficile cytotoxicity, and 11 of 93 (11.8%) patients from the convenience sample had documented bloody stool. CONCLUSIONS: A medium that greatly facilitates isolation of K oxytoca was developed. Occurrence of K oxytoca colonization was similar in the two patient populations studied and isolation of cytotoxic K oxytoca was not usually associated with hematochezia. Current understanding of the occurrence and causality of antibiotic-associated hemorrhagic colitis is insufficient for clinical laboratories to begin culturing K oxytoca and testing for cytotoxicity.

Variants in the SP110 gene are associated with genetic susceptibility to tuberculosis in West Africa.

The sst1 locus has been identified in a mouse model to control resistance and susceptibility of Mycobacterium tuberculosis infection. Subsequent studies have now identified Ipr1 (intracellular pathogen resistance 1) to be the gene responsible. Ipr1 is encoded within the sst1 locus and is expressed in the tuberculosis lung lesions and macrophages of sst1-resistant, but not sst1-susceptible mice. We have therefore examined the closest human homologue of Ipr1, SP110, for its ability to control susceptibility to M. tuberculosis infection in humans. In a study of families from The Gambia we have identified three polymorphisms that are associated with disease. On examination of additional families from Guinea-Bissau and the Republic of Guinea, two of these associations were independently replicated. These variants are in strong linkage disequilibrium with each other and lie within a 31-kb block of low haplotypic diversity, suggesting that a polymorphism within this region has a role in genetic susceptibility to tuberculosis in humans.

Polymorphism within the interferon-gamma/receptor complex is associated with pulmonary tuberculosis.

RATIONALE: Interferon-gamma (IFN-gamma) is of central interest in the study of tuberculosis. A number of single-gene mutations have been identified in the IFN-gamma signaling pathway that predispose to severe mycobacterial disease, but the relevance of polymorphism within these genes to the common phenotype of tuberculosis remains unclear. METHODS: A total of 1,301 individuals were included in a large, detailed study of West African populations with pulmonary tuberculosis. We investigated disease association with the genes encoding IFN-gamma and its receptor subunits (IFNG, IFNGR1, and IFNGR2). RESULTS: Within the IFNG gene, two promoter variants showed evidence of novel disease association: -1616GG (odds ratio [OR], 1.49; 95% confidence interval [CI], 1.11-2.00; p = 0.008) and +3234TT (OR, 1.40; 95% CI, 1.09-1.80; p = 0.009). The +874AA genotype was not significantly more frequent among cases over control subjects (OR, 1.16; 95%CI, 0.89-1.51; p = 0.25). In addition, novel disease association was also found with the -56CC genotype of the IFNGR1 promoter (OR, 0.75; 95% CI, 0.57-0.99; p = 0.041). No disease association was seen with the IFNGR2 locus. CONCLUSIONS: These results provide evidence of a significant role for genetic variation at the IFNG locus and provide detailed understanding of the genetic mechanisms underlying this association. The disease association with IFNGR1 is novel, and together these findings support the hypothesis that genetically determined variation in both IFN-gamma production and responsiveness influences the risk of developing tuberculosis.

Association of a polymorphism in the P2X7 gene with tuberculosis in a Gambian population.

Adenosine triphosphate (ATP) ligation of P2X(7) receptors expressed on human macrophages that are infected with mycobacteria induces cell death and subsequent loss of intracellular bacterial viability. Marked heterogeneity observed in cell donor ATP responsiveness suggests that this antimycobacterial mechanism may be genetically regulated. Five single-nucleotide polymorphisms (SNPs) previously identified in a putative 1.8-kb promoter region upstream of P2RX7 exon 1 were screened for associations with clinical tuberculosis. The frequencies of these promoter SNPs and a polymorphism in P2RX7 exon 13 at position 1513 were compared among >300 Gambian patients with tuberculosis and >160 ethnically matched control subjects by sequence-specific oligonucleotide hybridization and ligation detection reaction analysis. A significant protective association against tuberculosis was found for 1 promoter SNP, at nucleotide position -762 (odds ratio [OR] for variant C allele, 0.70; 95% confidence interval [CI], 0.54-0.89; P=.003; OR for CC genotype, 0.545; 95% CI, 0.318-0.934; P=.027). This association supports a role for ATP/P2X(7)-mediated host regulation of Mycobacterium tuberculosis infection.

Vitamin D receptor polymorphisms and susceptibility to tuberculosis in West Africa: a case-control and family study.

Vitamin D receptor (VDR) gene polymorphisms have been implicated in susceptibility to tuberculosis (TB), but reports have been inconsistent. We genotyped the VDR single-nucleotide polymorphisms (SNPs) FokI, BsmI, ApaI, and TaqI in 1139 case patients and control subjects and 382 families from The Gambia, Guinea, and Guinea-Bissau. The transmission-disequilibrium test on family data showed a significant global association of TB with SNP combinations FokI-BsmI-ApaI-TaqI and FokI-ApaI that were driven by the increased transmission to affected offspring of the FokI F and ApaI A alleles in combination. The ApaI A allele was also transmitted to affected offspring significantly more often than expected. Case-control analysis showed no statistically significant association between TB and VDR variants. BsmI, ApaI, and TaqI showed strong linkage disequilibrium. The significance of the family-based associations found between TB and FokI-BsmI-ApaI-TaqI and the FA haplotype supports a role for VDR haplotypes, rather than individual genotypes, in susceptibility to TB.

Detecting recent positive selection in the human genome from haplotype structure.

The ability to detect recent natural selection in the human population would have profound implications for the study of human history and for medicine. Here, we introduce a framework for detecting the genetic imprint of recent positive selection by analysing long-range haplotypes in human populations. We first identify haplotypes at a locus of interest (core haplotypes). We then assess the age of each core haplotype by the decay of its association to alleles at various distances from the locus, as measured by extended haplotype homozygosity (EHH). Core haplotypes that have unusually high EHH and a high population frequency indicate the presence of a mutation that rose to prominence in the human gene pool faster than expected under neutral evolution. We applied this approach to investigate selection at two genes carrying common variants implicated in resistance to malaria: G6PD and CD40 ligand. At both loci, the core haplotypes carrying the proposed protective mutation stand out and show significant evidence of selection. More generally, the method could be used to scan the entire genome for evidence of recent positive selection.

Variants of the CD40 ligand gene are not associated with increased susceptibility to tuberculosis in West Africa.

Evidence for linkage between tuberculosis and human chromosomal region Xq26 has previously been described. The costimulatory molecule CD40 ligand, encoded by TNFSF5 and located at Xq26.3, is a promising positional candidate. Interactions between CD40 ligand and CD40 are involved in the development of humoral- and cell-mediated immunity, as well as the activation of macrophages, which are the primary host and effector cells for Mycobacterium tuberculosis. We hypothesised that common variation within TNFSF5 might affect susceptibility to tuberculosis disease and, thus, might be responsible for the observed linkage to Xq26. Sequencing 32 chromosomes from a Gambian population identified nine common polymorphisms within the coding, 3' and 5' regulatory sequences of the gene. Six single nucleotide polymorphisms (SNPs) and a 3' microsatellite were genotyped in 121 tuberculosis patients and their available parents. No association with tuberculosis was detected for these variants using a transmission disequilibrium test, although one SNP at -726 showed some evidence of association in males. This finding, however, did not replicate in a separate case control study of over 1,200 West African individuals. We conclude that common genetic variation in TNFSF5 is not likely to affect tuberculosis susceptibility in West Africa and the linkage observed in this region is not due to variation in TNFSF5.