Performance of a simple flow cytometric assay in diagnosing active tuberculosis.

A flow cytometric assay measuring Mycobacterium tuberculosis-specific CD4 T-cell responses using co-expression of CD25/CD134 (OX40 assay) was explored as a diagnostic tool for active tuberculosis (TB) in a Thai population with and without HIV infection. Peripheral blood mononuclear cells (PBMC) obtained from 133 participants at TB diagnosis were cryopreserved. Seventy-six participants had a clinical diagnosis of TB which were confirmed by a positive culture. CD4 T-cell responses were measured after stimulation with a pool of overlapping peptides covering RD-1 antigens: CFP-10 + ESAT-6. The performance of the assay was also compared to the Xpert MTB/RIF assay. The overall sensitivity of the OX40 assay was 94.7% (95%CI 87.1-98.5); its specificity was 71.9% (95%CI, 58.5-83). The sensitivity of the OX40 assay among HIV-infected participants was 100% (95%CI, 88.8-100) with a specificity of 92.9% (95%CI, 66.1-99.8). OX40 assay performed particularly well in those with active TB and HIV infection.

Utility of urine lipoarabinomannan (LAM) in diagnosing tuberculosis and predicting mortality with and without HIV: prospective TB cohort from the Thailand Big City TB Research Network.

OBJECTIVES: To evaluate the applicability and accuracy of the urine lipoarabinomannan (LAM) test in tuberculosis (TB)/HIV co-infected patients and HIV-negative patients with disseminated TB. METHODS: Frozen urine samples obtained at baseline from patients in the TB research cohort with proven culture-positive TB were selected for blinded urine LAM testing. One hundred and nine patients were categorized into four groups: (1) HIV-positive patients with TB; (2) HIV-negative patients with disseminated TB; (3) HIV-negative immunocompromised patients with TB; and (4) patients with diseases other than TB. The sensitivity of urine LAM testing for culture-positive TB, specificity of urine LAM testing for patients without TB, positive predictive value (PPV), and negative predictive value (NPV) were assessed. RESULTS: The sensitivity of the urine LAM test in group 1 patients with a CD4 T-cell count of >100, ≤100, and ≤50 cells/mm3 was 38.5%, 40.6%, and 45%, respectively. The specificity and PPV of the urine LAM test were >80%. The sensitivity of the test was 20% in group 2 and 12.5% in group 3, and the specificity and PPV were 100% for both groups. A positive urine LAM test result was significantly associated with death. CONCLUSIONS: This promising diagnostic tool could increase the yield of TB diagnosis and may predict the mortality rate of TB infection, particularly in TB/HIV co-infected patients.

Incomplete restoration of Mycobacterium tuberculosis-specific-CD4 T cell responses despite antiretroviral therapy.

OBJECTIVES: Despite antiretroviral therapy (ART), HIV-infected persons have increased risk of active tuberculosis (TB). PPD and combined ESAT-6 and CFP-10-specific-CD4 (EC-Sp-CD4) responses were examined over 96 weeks. METHODS: HIV-infected, ART-naive Thai adults with CD4 T cell count ≤350 cells/µL starting ART were assessed at baseline, wk4, 8, 12, 24, 48 and 96. PPD and EC-Sp-CD4 T cells were detected by CD25/CD134 co-expression after stimulation with antigens. RESULTS: Fifty subjects were enrolled, 39 were male, median age 32 yrs, median baseline CD4 T cell count 186 cells/µL and plasma HIV-viral-load 4.9log10 copies/mL. Seventeen were TB-sensitised. At baseline, 25 had positive PPD and 15 had positive EC-Sp-CD4 response. CD4 T cell count <100 cells/µL was less (P = 0.005) and TB-sensitisation was more likely (P = 0.013) to be associated with positive baseline PPD-Sp-CD4 response. At wk4, the number of subjects with positive PPD-Sp-CD4 response rose to 35 (P = 0.021). Mean PPD-Sp-CD4 T cells increased at wk4 (P = 0.017) in patients not classified as TB-sensitised. The number of subjects with positive EC-Sp-CD4 response did not change significantly post ART. In TB-sensitised patients, mean EC-Sp-CD4 T cells declined to below baseline from wk12 (P = 0.010) onwards. EC-Sp-CD4 responses were undetectable in 3 out of 17 TB-sensitised patients. CONCLUSIONS: Restoration of responses to TB-antigens was incomplete and inconsistent under the employed experimental conditions and may account for persistent increased risk of TB despite ART.

Restoration of CMV-specific-CD4 T cells with ART occurs early and is greater in those with more advanced immunodeficiency.

OBJECTIVES: Restoration of Cytomegalovirus-specific-CD4 T cell (CMV-Sp-CD4) responses partly accounts for the reduction of CMV-disease with antiretroviral-therapy (ART), but CMV-Sp-CD4 may also drive immune activation and immunosenescence. This study characterized the dynamics of CMV-Sp-CD4 after ART initiation and explored associations with CD4 T cell recovery as well as frequency of naïve CD4 T cells at week 96. METHODS: Fifty HIV-infected, ART-naïve Thai adults with CD4 T cell count ≤ 350 cells/µL and starting ART were evaluated over 96 weeks (ClinicalTrials.gov identifier NCT01296373). CMV-Sp-CD4 was detected by co-expression of CD25/CD134 by flow cytometry after CMV-antigen stimulation. RESULTS: All subjects were CMV sero-positive, 4 had quantifiable CMV-DNA (range 2.3-3.9 log10 copies/mL) at baseline but none had clinically apparent CMV-disease. Baseline CMV-Sp-CD4 response was positive in 40 subjects. Those with CD4 T cell count < 100 cells/µL were less likely to have positive baseline CMV-Sp-CD4 response (P=0.003). Positive baseline CMV-Sp-CD4 response was associated with reduced odds of quantifiable CMV-DNA (P=0.022). Mean CD4 T cell increase at week 96 was 213 cells/µL. This was associated positively with baseline HIV-VL (P=0.001) and negatively with age (P=0.003). The frequency of CMV-Sp-CD4 increased at week 4 (P=0.008), then declined. Those with lower baseline CMV-Sp-CD4 (P=0.009) or CDC category C (P<0.001) had greater increases in CMV-Sp-CD4 at week 4. At week 96, CD4 T cell count was positively (P<0.001) and the frequency of CMV-Sp-CD4 was negatively (P=0.001) associated with the percentage of naïve CD4 T cells. CONCLUSIONS: Increases in CMV-Sp-CD4 with ART occurred early and were greater in those with more advanced immunodeficiency. The frequency of CMV-Sp-CD4 was associated with reduced naïve CD4 T cells, a marker associated with immunosenescence.

Association of APOBEC3G genotypes and CD4 decline in Thai and Cambodian HIV-infected children with moderate immune deficiency.

INTRODUCTION: Human APOBEC3G is a host defense factor that potently inhibits HIV replication. We hypothesize that HIV-infected children with a genetic variant of APOBEC3G will have a more rapid disease progression. METHODS: Antiretroviral therapy (ART)-naïve children, aged 1-12 years old with CD4 15-24% and without severe HIV-related symptoms were enrolled. The children had CD4% and absolute CD4 counts every 12 weeks and HIV-RNA every 24 weeks until 144 weeks. ART was started when CD4% declined to < 15% or AIDS-related events developed.APOBEC3G genetic variants were performed by PCR-based restriction fragment length polymorphism techniques from peripheral blood mononuclear cells. Random-effect linear regression analysis was performed to correlate APOBEC3G genotypes and disease progression. RESULTS: 147 children, 35% male, with a median (IQR) age of 6.5 (4.3-8.8) years were enrolled. CDC N:A:B were 1:63:36%. Median baseline values were 20% for CD4% 605 cells/mm3 for CD4 count and 4.7 log10copies/mL for HIV-RNA.The frequencies of APOBEC3G genotypes AA (186H/H), AG (186H/R), GG (186R/R) were 86%, 12%, and 2% respectively. The APOBEC3G genotype GG was associated with a significant decline in CD4% -5.1% (-8.9 to -1.2%), p<0.001, and CD4 counts -226 (-415 to -34) cells/mm3, p<0.001 by random-effect liner regression analysis. No significant associations of APOBEC3G genotypes with HIV-RNA changes overtime (p=0.16) or progression to CDC B and C (p=0.49) were observed. CONCLUSIONS: APOBEC3G genotype GG was significantly associated with a more rapid decline in CD4. APOBEC3G's antiviral effects on HIV disease progression in children should be further explored.

A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays.

A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.