Nitric oxide synthase 2A (NOS2A) polymorphisms are not associated with invasive pneumococcal disease

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>(pneumococcus) is responsible for over one million deaths per year, with young children, the elderly and immunocompromised individuals being most at risk. Approximately half of East African children have been reported to be asymptomatic carriers of pneumococcus with invasive infection occurring after the disruption of the respiratory membrane which is believed to be caused by the host immune response. Racial incidence of invasive pneumococcal disease (IPD) is higher in certain populations even after adjusting for environmental factors suggesting a genetic component to disease susceptibility. The nitric oxide synthase 2A (NOS2A) gene is responsible for the production of nitric oxide under pathological conditions including host defence against bacterial infection. Nitric oxide is a modulator of apoptotic and inflammatory cascades and endothelial permeability. We hypothesised that genetic variants within this gene may predispose to disease risk and survival.</p> <p>Methods</p> <p>A cohort of 299 children with IPD (221 meningitis, 41 pneumonia and 37 with bacteraemia) and 931 age matched controls from Malawi were used in this study. We investigated nine haplotype tagging single nucleotide polymorphisms within the NOS2A gene and compared the presence or absence of the minor alleles in cases and controls and survivors and non-survivors within the cases.</p> <p>Results</p> <p>We observed no significant associations between cases and controls or with survival in either all IPD cases or in the separate analysis of meningitis cases. A near significant association was obtained for the comparison of rs8078340 in cases and controls (p-value, 0.078). However, results were unadjusted for multiple testing.</p> <p>Conclusion</p> <p>Our results suggest that polymorphic variation within the NOS2A gene does not influence invasive pneumococcal disease susceptibility or survival.</p

The Diagnostic and Prognostic Accuracy of Five Markers of Serious Bacterial Infection in Malawian Children with Signs of Severe Infection

Early recognition and prompt and appropriate antibiotic treatment can significantly reduce mortality from serious bacterial infections (SBI). The aim of this study was to evaluate the utility of five markers of infection: C-reactive protein (CRP), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), CD163 and high mobility group box-1 (HMGB1), as markers of SBI in severely ill Malawian children.Children presenting with a signs of meningitis (n = 282) or pneumonia (n = 95), were prospectively recruited. Plasma samples were taken on admission for CRP, PCT, sTREM-1 CD163 and HMGB1 and the performance characteristics of each test to diagnose SBI and to predict mortality were determined. Of 377 children, 279 (74%) had SBI and 83 (22%) died. Plasma CRP, PCT, CD163 and HMGB1 and were higher in HIV-infected children than in HIV-uninfected children (p<0.01). In HIV-infected children, CRP and PCT were higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and PCT and CD163 were higher in non-survivors (p = 0.001, p = 0.05 respectively). In HIV-uninfected children, CRP and PCT were also higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and CD163 was higher in non-survivors (p = 0.05). The best predictors of SBI were CRP and PCT, and areas under the curve (AUCs) were 0.81 (95% CI 0.73–0.89) and 0.86 (95% CI 0.79–0.92) respectively. The best marker for predicting death was PCT, AUC 0.61 (95% CI 0.50–0.71).Admission PCT and CRP are useful markers of invasive bacterial infection in severely ill African children. The study of these markers using rapid tests in a less selected cohort would be important in this setting

PCR Improves Diagnostic Yield from Lung Aspiration in Malawian Children with Radiologically Confirmed Pneumonia

Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid.A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid.Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection.In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa

Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis

BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression. Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). CONCLUSION: We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis

Aetiology of radiologically confirmed pneumonia.

<p>NT: not tested, PcP: pneumocystis pneumonia, Hib: H<i>aemophilus influenzae b</i>, CMV: cytomegalovirus.</p><p><b>Bacteria:</b> 2 patients had mixed infection with <i>S.typhimurium</i> and <i>S.pneumoniae</i> ; 1 had <i>S.typhimurium</i> from blood culture and <i>S.pneumoniae</i> from blood PCR, and adenovirus from lung aspirate PCR, 1 had <i>S.typhimurium</i> from blood and lung aspirate culture and <i>S.pneumoniae</i> from lung aspirate PCR, 7 had <i>S.pneumoniae</i>/adenovirus, 1 had <i>S.pneumoniae</i>/Chlamydia, 1 case with <i>S.pneumoniae</i> had <i>M. tuberculosis</i> cultured from nasopharyngeal aspirate after induced sputum.</p><p><b>Pneumocystis:</b> 1 patient had PcP/Hib, 1 patient had PcP/CMV, 1 patient had PcP/adenovirus.</p><p><b>Viruses:</b> 1 adenovirus/CMV, 1 adenovirus/Chlamydia, 1 bocavirus/CMV.</p