Ravesh Singh

Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a High Burden Setting: A Prospective Study

Vinod Patel and colleagues evaluate the sensitivity and specificity of quantitative PCR using Xpert MTB/RIF for diagnosis of TB meningitis in the high-burden setting of South Africa. Please see later in the article for the Editors Summary

Association of TRIM22 with the Type 1 Interferon Response and Viral Control during Primary HIV-1 Infection

ABSTRACT Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-α, IFN-β, myxovirus resistance protein A (MxA), human TRIM5α (huTRIM5α), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-β (P = 0.0005), MxA (P = 0.007), and TRIM22 (P = 0.01) and lower levels of huTRIM5α (P < 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5α correlated positively with type 1 IFN (IFN-α, IFN-β, and MxA) (all P < 0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P = 0.0418). Furthermore, TRIM22 but not huTRIM5α, IFN-α, IFN-β, or MxA showed a negative correlation with plasma viral load (P = 0.0307) and a positive correlation with CD4+ T-cell counts (P = 0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5α expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.

Cerebrospinal T-cell responses aid in the diagnosis of tuberculous meningitis in a human immunodeficiency virus- and tuberculosis-endemic population.

RATIONALEnCurrent tools for the rapid diagnosis of tuberculous meningitis (TBM) are suboptimal. We evaluated the clinical utility of a quantitative RD-1 IFN-gamma T-cell enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB), using cerebrospinal fluid cells for the rapid immunodiagnosis of TBM.nnnOBJECTIVESnTo evaluate the diagnostic utility of the RD1 antigen- specific ELISPOT assay for the diagnosis of tuberculous meningitis.nnnMETHODSnThe ELISPOT assay was evaluated in 150 patients with suspected TBM who were categorized as definite-TBM, probable-TBM, and non-TBM. Culture or polymerase chain reaction positivity for Mycobacerium tuberculosis served as the reference standard. To determine the diagnostic value of the ELISPOT assay, a clinical prediction rule was derived from baseline clinical and laboratory parameters using a multivariable regression model.nnnMEASUREMENTS AND MAIN RESULTSnA total of 140 patients (81% HIV-infected; median CD4 count, 160 cells/mm(3)) were included in the final analysis. When comparing the definite-TBM (n = 38) and non-TBM groups (n = 48), the ELISPOT assay (cut point of > or =228 spot-forming cells per 1 million mononuclear cells) was a useful rule-in test: sensitivity 58% (95% confidence interval [CI], 41-74); specificity 94% (95% CI, 83-99). However, ELISPOT outcomes improved when other rapid tests were concurrently used to exclude bacterial (Gram stain) and cryptococcal meningitis (latex-agglutination test) within the non-TBM group. Using this approach, the ELISPOT assay (cut point of > or =46 spot-forming cells) was an excellent rule-in test: sensitivity 82% (95% CI, 66-92); specificity 100% (95% CI, 78-100); positive predictive value, 100% (95% CI, 89-100); negative predictive value, 68% (95% CI, 45-86); area under the curve, 0.90. The ELISPOT assay had incremental diagnostic value compared with the clinical prediction rule.nnnCONCLUSIONSnThe RD-1 ELISPOT assay, using cerebrospinal fluid mononuclear cells and in conjunction with other rapid confirmatory tests (Gram stain and cryptococcal latex-agglutination test), is an accurate rapid rule-in test for TBM in a TB and HIV endemic setting.

Human TRIM5α expression levels and reduced susceptibility to HIV-1 infection.

BACKGROUNDnHuman TRIM5alpha (TRIM5alphahu), a member of the tripartite motif protein family, displays some anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro, although it is substantially less potent than its rhesus monkey counterpart (TRIM5alpharh). The effects of levels of TRIM5alphahu on prevention or control of HIV-1 infection in vivo are unknown.nnnMETHODSnWe used a quantitative real-time polymerase chain reaction (PCR) assay to measure levels of TRIM5alphahu expression in peripheral blood mononuclear cells (PBMCs) obtained from a cohort of individuals at high risk for HIV-1 infection in Durban, South Africa. Samples were available from 38 infected subjects (with all these samples obtained within 1 year of infection) and from 57 uninfected persons. Matched preinfection and postinfection samples were available from 13 individuals.nnnRESULTSnTRIM5alphahu messenger RNA levels were lower in the PBMCs of HIV-1-infected subjects than in those of uninfected subjects (P <.001). Seroconverters had lower preinfection levels of TRIM5alphahu than did nonseroconverters (P<.001). TRIM5alphahu levels did not change significantly after infection. There was no correlation between TRIM5alphahu levels and viral loads or CD4(+) T cell counts.nnnCONCLUSIONSnHigh expression of TRIM5alphahu is associated with reduced susceptibility to HIV-1 infection. Furthermore, infection is not associated with disregulation of TRIM5alphahu. TRIM5alphahu expression levels do not contribute to the control of primary HIV-1 viremia.

Comparison of a Clinical Prediction Rule and a LAM Antigen-Detection Assay for the Rapid Diagnosis of TBM in a High HIV Prevalence Setting

Background/Objective The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB®) using cerebrospinal fluid (CSF). Methods Patients with suspected TBM were classified as definite-TBM (CSF culture or PCR positive), probable-TBM and non-TBM. Results Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 countu200a=u200a132 [54; 241] cells/µl). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95%CI) was 31% (17;48) and 94% (85;99), respectively (cut-point ≥0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4<200 vs. >200 cells/µl; pu200a=u200a0.03). The sensitivity and specificity in those with a CD4<100 cells/µl was 50% (27;73) and 95% (74;99), respectively. A clinical-prediction rule ≥6 derived from multivariate analysis had a sensitivity and specificity (95%CI) of 47% (31;64) and 98% (90;100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47;68) and specificity remained high at 93% (82;98). Conclusions Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression.

Utility of a novel lipoarabinomannan assay for the diagnosis of tuberculous meningitis in a resource-poor high-HIV prevalence setting.

BackgroundIn Africa, tuberculous meningitis (TBM) is an important opportunistic infection in HIV-positive patients. Current diagnostic tools for TBM perform sub-optimally. In particular, the rapid diagnosis of TBM is challenging because smear microscopy has a low yield and PCR is not widely available in resource-poor settings.MethodsWe evaluated the performance outcome of a novel standardized lipoarabinomannan (LAM) antigen-detection assay, using archived cerebrospinal fluid samples, in 50 African TBM suspects of whom 68% were HIV-positive.ResultsOf the 50 participants 14, 23 and 13 patients had definite, probable and non-TBM, respectively. In the non-TB group there were 5 HIV positive patients who were lost to follow-up and in whom concomitant infection with Mycobacterium tuberculosis could not be definitively excluded. The test sensitivities and specificities were as follows: LAM assay 64% and 69% (cut-point 0.22), smear microscopy 0% and 100% and PCR 93% and 77%, respectively.ConclusionIn this preliminary proof-of-concept study, a rapid diagnosis of TBM could be achieved using LAM antigen detection. Although specificity was sub-optimal, the estimates provided here may be unreliable because of a classification bias inherent in the study design where it was not possible to exclude TBM in the presumed non-TBM cases owing to a lack of clinical follow-up. As PCR is largely unavailable, the LAM assay may well prove to be a useful adjunct for the rapid diagnosis of TBM in high HIV-incidence settings. These preliminary results justify further enquiry and prospective studies are now required to definitively establish the place of this technology for the diagnosis of TBM.

TRIM5α and TRIM22 are differentially regulated according to HIV-1 infection phase and compartment

ABSTRACT The antiviral role of TRIM E3 ligases in vivo is not fully understood. To test the hypothesis that TRIM5α and TRIM22 have differential transcriptional regulation and distinct anti-HIV roles according to infection phase and compartment, we measured TRIM5α, TRIM22, and type I interferon (IFN-I)-inducible myxovirus resistance protein A (MxA) levels in peripheral blood mononuclear cells (PBMCs) during primary and chronic HIV-1 infection, with chronic infection samples being matched PBMCs and central nervous system (CNS)-derived cells. Associations with biomarkers of disease progression were explored. The impact of IFN-I, select proinflammatory cytokines, and HIV on TRIM E3 ligase-specific expression was investigated. PBMCs from individuals with primary and chronic HIV-1 infection had significantly higher levels of MxA and TRIM22 than did PBMCs from HIV-1-negative individuals (P < 0.05 for all comparisons). PBMCs from chronic infection had lower levels of TRIM5α than did PBMCs from primary infection or HIV-1-uninfected PBMCs (P = 0.0001 for both). In matched CNS-derived samples and PBMCs, higher levels of MxA (P = 0.001) and TRIM5α (P = 0.0001) in the CNS were noted. There was a negative correlation between TRIM22 levels in PBMCs and plasma viral load (r = −0.40; P = 0.04). In vitro, IFN-I and, rarely, proinflammatory cytokines induced TRIM5α and TRIM22 in a cell type-dependent manner, and the knockdown of either protein in CD4+ lymphocytes resulted in increased HIV-1 infection. These data suggest that there are infection-phase-specific and anatomically compartmentalized differences in TRIM5α and TRIM22 regulation involving primarily IFN-I and specific cell types and indicate subtle differences in the antiviral roles and transcriptional regulation of TRIM E3 ligases in vivo. IMPORTANCE Type I interferon-inducible TRIM E3 ligases are a family of intracellular proteins with potent antiviral activities mediated through diverse mechanisms. However, little is known about the contribution of these proteins to antiviral immunity in vivo and how their expression is regulated. We show here that TRIM5α and TRIM22, two prominent members of the family, have different expression patterns in vivo and that the expression pattern depends on HIV-1 infection status and phase. Furthermore, expression differs in peripheral blood versus central nervous system anatomical sites of infection. Only TRIM22 expression correlated negatively with HIV-1 viral load, but gene silencing of both proteins enhances HIV-1 infection of target cells. We report subtle differences in TRIM5α and TRIM22 gene induction by IFN-I and proinflammatory cytokines in CD4+ lymphocytes, monocytes, and neuronal cells. This study enhances our understanding of antiviral immunity by intrinsic antiviral factors and how their expression is determined.

Biomarkers for Tuberculosis Based on Secreted, Species-Specific, Bacterial Small Molecules

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography-tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction-based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.

Comparative Utility of Cytokine Levels and Quantitative RD-1-Specific T Cell Responses for Rapid Immunodiagnosis of Tuberculous Meningitis

ABSTRACT The rapid diagnosis of tuberculous meningitis (TBM) is problematic. We found in 150 patients with suspected TBM that, similar to RD-1-specific quantitative cerebrospinal fluid (CSF) T-cell responses, unstimulated CSF gamma interferon (IFN-γ) levels when used together with other rapid confirmatory tests (Gram stain and cryptococcal latex agglutination test) may allow the accurate and rapid diagnosis of TBM in a setting in which tuberculosis (TB) and HIV are endemic. In resource-poor settings, a clinical prediction rule (CPR) may be useful to clinicians, and thus the IFN-γ assay may potentially need to be used only when the clinical score is below a prespecified threshold. These preliminary findings will need to be confirmed in further studies.

Comparison of Amplicor and GeneXpert MTB/RIF Tests for Diagnosis of Tuberculous Meningitis

ABSTRACT There are no data about the comparative accuracy of commercially available nucleic acid amplification tests (GeneXpert MTB/RIF and Roche Amplicor) for the diagnosis of tuberculous meningitis (TBM). A total of 148 patients with suspected TBM were evaluated, and cultures served as the reference standard. The sensitivities and specificities (95% confidence interval [CI]) for the Amplicor and Xpert MTB/RIF tests were similar: 46 (31–60) versus 50 (33–67) and 99 (93–100) and 94 (84–99), respectively.