Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function.

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.

Urine lipoarabinomannan testing for diagnosis of pulmonary tuberculosis in children: a prospective study.

BACKGROUND: Urine tests for mycobacterial lipoarabinomannan might be useful for point-of-care diagnosis of tuberculosis in adults with advanced HIV infection, but have not been assessed in children. We assessed the accuracy of urine lipoarabinomannan testing for the diagnosis of pulmonary tuberculosis in HIV-positive and HIV-negative children. METHODS: We prospectively recruited children (aged ≤ 15 years) who presented with suspected tuberculosis at a primary health-care clinic and paediatric referral hospital in South Africa, between March 1, 2009, and April 30, 2012. We assessed the diagnostic accuracy of urine lipoarabinomannan testing with lateral fl ow assay and ELISA, with mycobacterial culture of two induced sputum samples as the reference standard. Positive cultures were identified by acid-fast staining and tested to confirm Mycobacterium tuberculosis and establish susceptibility to rifampicin and isoniazid. FINDINGS: 535 children (median age 42.5 months, IQR 19.1 ­ 66.3) had urine and two induced specimens available for testing. 89 (17%) had culture-confirmed tuberculosis and 106 (20%) had HIV. The lateral fl ow lipoarabinomannan test showed poor accuracy against the reference standard, with sensitivity of 48.3% (95% CI 37.6 ­ 59.2), specificity of 60.8% (56.1 ­ 65.3), and an area under the receiver operating characteristic curve of 0.53 (0.46 ­ 0.60) for children without HIV and 0.64 (0.51 ­ 0.76) for children with HIV. ELISA had poor sensitivity in children without HIV (sensitivity 3.0%, 95% CI 0.4 ­ 10.5) and children with HIV (0%, 0.0 ­ 14.3); overall specificity was 95.7% (93.4 ­ 97.4). INTERPRETATION: Urine lipoarabinomannan tests have insufficient sensitivity and specificity to diagnose HIV-positive and HIV-negative children with tuberculosis and should not be used in this patient population.

Incremental value of T-SPOT.TB for diagnosis of active pulmonary tuberculosis in children in a high-burden setting: a multivariable analysis.

INTRODUCTION: Interferon γ release assays (IGRAs) are increasingly used for tuberculosis (TB) infection, but their incremental value beyond patient demographics, clinical signs and conventional tests for active disease has not been evaluated in children. METHODS: The incremental value of T-SPOT.TB was assessed in 491 smear-negative children from two hospitals in Cape Town, South Africa. Bayesian model averaging was used to select the optimal set of patient demographics and clinical signs for predicting culture-confirmed TB. The added value of T-SPOT.TB over and above patient characteristics and conventional tests was measured using statistics such as the difference in the area under the receiver operating characteristic curve (AUC), the net reclassification improvement (NRI) and the integrated discrimination improvement (IDI). RESULTS: Cough longer than 2 weeks, fever longer than 2 weeks, night sweats, malaise, history of household contact and HIV status were the most important predictors of culture-confirmed TB. Binary T-SPOT.TB results did not have incremental value when added to the baseline model with clinical predictors, chest radiography and the tuberculin skin test. The AUC difference was 3% (95% CI 0% to 7%). Using risk cut-offs of <10%, 10-30% and >30%, the NRI was 7% (95% CI -8% to 31%) but the CI included the null value. The IDI was 3% (95% CI 0% to 11%), meaning that the average predicted probability across all possible cut-offs improved marginally by 3%. CONCLUSIONS: In a high-burden setting, the T-SPOT.TB did not have added value beyond clinical data and conventional tests for diagnosis of TB disease in smear-negative children.

Primary immunodeficiencies: a 27-year review at a tertiary paediatric hospital in Cape Town, South Africa.

INTRODUCTION: The epidemiology of primary immunodeficiencies (PID) is not well documented in Africa. The objective of this study was to describe the spectrum of PID at a tertiary paediatric centre in South Africa. METHODS: A retrospective study was conducted on 168 patients diagnosed with PID from 1983 to 2009. RESULTS: Over the study period, antibody deficiencies predominated (51%) followed by well-defined syndromes (24%). Common variable immunodeficiency was the commonest antibody deficiency. The mean age of diagnosis was 51 months overall but decreased significantly to 35 months over the last 9 years. Recurrent infections were the most common presenting complaint (74%). The overall mortality rate was 25% while combined immunodeficiencies accounted for 40% of the deaths. CONCLUSIONS: The spectrum of PID in South Africa was similar to international trends. The declining mean age of diagnosis indicated improved recognition of PID. Future research should focus on identifying children with PID more effectively.

Polymorphic variation in TIRAP is not associated with susceptibility to childhood TB but may determine susceptibility to TBM in some ethnic groups.

Host recognition of mycobacterial surface molecules occurs through toll like receptors (TLR) 2 and 6. The adaptor protein TIRAP mediates down stream signalling of TLR2 and 4, and polymorphisms in the TIRAP gene (TIRAP) have been associated with susceptibility and resistance to tuberculosis (TB) in adults. In order to investigate the role of polymorphic variation in TIRAP in childhood TB in South Africa, which has one of the highest TB incidence rates in the world, we screened the entire open reading frame of TIRAP for sequence variation in two cohorts of childhood TB from different ethnic groups (Xhosa and mixed ancestry). We identified 13 SNPs, including seven previously unreported, in the two cohorts, and found significant differences in frequency of the variants between the two ethnic groups. No differences in frequency between individual SNPs or combinations were found between TB cases and controls in either cohort. However the 558C-->T SNP previously associated with TB meningitis (TBM) in a Vietnamese population was found to be associated with TBM in the mixed ancestry group. Polymorphisms in TIRAP do not appear to be involved in childhood TB susceptibility in South Africa, but may play a role in determining occurrence of TBM.

X-linked hyper IgM (HIGM1) in an African kindred: the first report from South Africa.

BACKGROUND: The objective of this study was to describe the clinical and molecular features of the first South African family with X-linked hyper-IgM syndrome (HIGM1). METHODS: Diagnoses were based on immunoglobulin results and the absence of CD40 ligand (CD40L) expression on activated T-cells. Complete molecular characterisation involved CD40L cDNA sequencing, and genomic DNA analysis by polymerase chain reaction amplification, restriction enzyme digestion and sequencing. A PCR-based diagnostic assay was established for carrier detection and prenatal diagnosis in this family. RESULTS: There were originally six children, three males and three females. The eldest boy died after being diagnosed with hypogammaglobulinaemia, before HIGM1 was considered. This disorder was diagnosed in the second eldest boy at the age of 5 years, after failing to detect CD40L expression on his activated T-cells. A deficiency of CD40L was also confirmed in the youngest male at the age of 5 years. Both younger brothers have since died of infections relating to HIGM1. Molecular investigation showed that exon 3 was deleted from the CD40L mRNA of the affected males. Genomic DNA analysis identified a 1.5 kilobase deletion, spanning exon 3 and including extended flanking intronic sequence. Carrier status in the mother was confirmed by RT-PCR of her CD40L mRNA. Genetic analysis of the three female children was deferred because they were below the legal consenting age of 18 years. A PCR-based assay for genomic DNA was established for easy identification of female carriers and affected males in the future. CONCLUSIONS: This study confirmed the diagnosis of HIGM1 in the first South African family to be investigated and identified a novel mutation in the CD40L gene.