Martin Llewelyn

Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study

BACKGROUND Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M. tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis. METHODS This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients. FINDINGS 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2.9 [95% CI 1.3-6.5], p=0.008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9.9 [1.3-76.2], p=0.009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2.8 [1.2-6.5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5.1 [1.4-18.4]). INTERPRETATION 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.

Superantigens: microbial agents that corrupt immunity

Microbial superantigens are a family of protein exotoxins that share the ability to trigger excessive and aberrant activation of T cells. The best characterised are the staphylococcal enterotoxins and the streptococcal pyrogenic exotoxins that trigger the staphylococcal and streptococcal toxic shock syndromes. It is now apparent that superantigens have a wider role in the pathology of infectious diseases than has previously been appreciated. Staphylococcus aureus and Streptococcus pyogenes together produce 19 different superantigens. The range of microorganisms known to produce superantigens has expanded to include Gram negative bacteria, mycoplasma, and viruses. Research is beginning to shed light on the more subtle parts these molecules play in causing disease and to produce some real possibilities for specific treatment of superantigen-induced toxicity. We aim to highlight these new developments and review the science behind these fascinating molecules.

Clinical management of Staphylococcus aureus bacteraemia

Staphylococcus aureus bacteraemia is one of the most common serious bacterial infections worldwide. In the UK alone, around 12,500 cases each year are reported, with an associated mortality of about 30%, yet the evidence guiding optimum management is poor. To date, fewer than 1500 patients with S aureus bacteraemia have been recruited to 16 controlled trials of antimicrobial therapy. Consequently, clinical practice is driven by the results of observational studies and anecdote. Here, we propose and review ten unanswered clinical questions commonly posed by those managing S aureus bacteraemia. Our findings define the major areas of uncertainty in the management of S aureus bacteraemia and highlight just two key principles. First, all infective foci must be identified and removed as soon as possible. Second, long-term antimicrobial therapy is required for those with persistent bacteraemia or a deep, irremovable focus. Beyond this, the best drugs, dose, mode of delivery, and duration of therapy are uncertain, a situation compounded by emerging S aureus strains that are resistant to old and new antibiotics. We discuss the consequences on clinical practice, and how these findings define the agenda for future clinical research.

Prediction of Staphylococcus aureus Antimicrobial Resistance by Whole-Genome Sequencing

ABSTRACT Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organisms phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Whole-Genome Sequencing Shows That Patient-to-Patient Transmission Rarely Accounts for Acquisition of Staphylococcus aureus in an Intensive Care Unit

An assessment of Staphylococcus aureus acquisition among intensive care patients using serial sampling and whole-genome sequencing found less than a fifth of acquisitions resulted from patient-to-patient transmission. Whole-genome sequencing identified transmission more accurately than spa-typing and patient stay data.

HLA class II polymorphisms determine responses to bacterial superantigens

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ α-chain polymorphisms such that HLA-DQA1*01 α-chains show greater binding than DQA1*03/05 α-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vβ8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vβ specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

Superantigen-Induced Proliferation of Human CD4+CD25− T Cells Is Followed by a Switch to a Functional Regulatory Phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki’s disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25+FOXP3+) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3− T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non–Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non–Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25+FOXP3+ cells arose from CD25− T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25+FOXP3+ T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.

Tuberculosis diagnosed during pregnancy: a prospective study from London

BACKGROUND A study was undertaken to characterise the presentation of tuberculosis in pregnancy and the difficulties in diagnosis in an area of the UK with a high incidence of tuberculosis. METHODS A prospective case series was investigated at Northwick Park Hospital, a university affiliated district general hospital in Brent and Harrow health authority in north-west London which incorporates a regional infectious diseases unit. Patients diagnosed with tuberculosis over the study period were included if the onset of symptoms occurred during pregnancy. RESULTS Thirteen patients were diagnosed during a 30 month period from December 1995 to May 1998 during which 9069 mothers were delivered, a prevalence of 143.3/100 000 deliveries. Symptoms began at a median of 22 weeks gestation (range 9–40 weeks). All patients were recent immigrants of Indian subcontinent or Somali origin and their median duration of residence in the UK was 31 months (range 1–72). Prevalence broken down for racial origin of mothers was 466.3/100 000 for mothers of black African origin and 239.1/100 000 for mothers of Indian origin. Nine of the 13 patients had extrapulmonary tuberculosis. Four patients with widely disseminated disease had a negative Mantoux response and five with localised disease had a strongly positive Mantoux response. HIV co-infection was absent. The median delay between the onset of symptoms and diagnosis was seven weeks (range 2–30). The response to standard treatment was excellent and all patients were cured. CONCLUSIONS Tuberculosis occurring in pregnancy is common in recent immigrants. Diagnosis during pregnancy is delayed because the disease is frequently extrapulmonary with few symptoms.

Predictors of Death after Clostridium difficile Infection: A Report on 128 Strain-Typed Cases from a Teaching Hospital in the United Kingdom

We assessed the relationship between strain type, clinical factors, and outcome in 128 patients with Clostridium difficile infection. Strain type was not associated with any outcome measure. On multivariate analysis, ischemic heart disease and hypoalbuminemia predicted death. Metronidazole treatment in severe disease was associated with a higher rate of treatment failure and death.

Identifying lineage effects when controlling for population structure improves power in bacterial association studies.

Bacteria pose unique challenges for genome-wide association studies because of strong structuring into distinct strains and substantial linkage disequilibrium across the genome1,2. Although methods developed for human studies can correct for strain structure3,4, this risks considerable loss-of-power because genetic differences between strains often contribute substantial phenotypic variability5. Here, we propose a new method that captures lineage-level associations even when locus-specific associations cannot be fine-mapped. We demonstrate its ability to detect genes and genetic variants underlying resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection, recombination and penetrance confer high power to recover known antimicrobial resistance mechanisms and reveal a candidate association between the outer membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method pinpoints locus-specific effects where possible and boosts power by detecting lineage-level differences when fine-mapping is intractable.