Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCG-vaccinated populations.

T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.

High-sensitive and rapid detection of Mycobacterium tuberculosis infection by IFN-gamma release assay among HIV-infected individuals in BCG-vaccinated area.

BACKGROUND: An accurate test for Mycobacterium tuberculosis infection is urgently needed in immunosuppressed populations. The aim of this study was to investigate the diagnostic power of enzyme-linked immunospot (ELISPOT)-based IFN-gamma release assay in detecting active and latent tuberculosis in HIV-infected population in bacillus Calmette-Guerin (BCG)-vaccinated area. A total of 100 HIV-infected individuals including 32 active tuberculosis patients were recruited. An ELISPOT-based IFN-gamma release assay, T-SPOT.TB, was used to evaluate the M. tuberculosis ESAT-6 and CFP-10 specific IFN-gamma response. Tuberculin skin test (TST) was performed for all recruited subjects. RESULTS: The subjects were divided into group HIV+ATB (HIV-infected individuals with active tuberculosis, n = 32), group HIV+LTB (HIV-infected individuals with positive results of T-SPOT.TB assay, n = 46) and group HIV only (HIV-infected individuals with negative results of T-SPOT.TB assay and without evidence of tuberculosis infection, n = 22). In group HIV+ATB and HIV+LTB, T-SPOT.TB positive rate in subjects with TST <5 mm were 50% (16/32) and 41.3% (19/46), respectively. Individuals in group HIV+ATB and HIV+LTB with CD4+ T cells <500/microl, T-SPOT.TB showed a higher sensitivity than TST (64.5% vs. 22.6% and 62.2% vs. 29.7%, respectively, both P < 0.0001). In addition, the sensitivity of T-SPOT.TB assay in group HIV+ATB increased to >85% in patients with TB treatment for less than 1 month and CD4+ T cells > or = 200/microl, while for patients treated for more than 3 months and CD4+ T cells <200/microl, the sensitivity was decreased to only 33.3%. Furthermore, the results could be generated by T-SPOT.TB assay within 24 hours, which was more rapid than TST with 48-72 hours. CONCLUSION: ELISPOT-based IFN-gamma release assay is more sensitive and rapid for the diagnosis of TB infection in Chinese HIV-infected individuals with history of BCG vaccination, and could be an effective tool for guiding preventive treatment with isoniazid in latently infected people and for TB control in China.

Evolution of hepatitis B virus polymerase mutations in a patient with HBeAg-positive chronic hepatitis B virus treated with sequential monotherapy and add-on nucleoside/nucleotide analogues.

BACKGROUND: Nucleoside/nucleotide analogues are a fundamental tool for the treatment of chronic hepatitis B virus (HBV). Sequential anti-HBV treatment might lead to the selection of mutations. OBJECTIVE: This report aimed to analyze the genetic evolution of the reverse-transcriptase (RT) gene of viral quasispecies in a patient with hepatitis B e antigen (HBeAg)-positive chronic HBV who received, sequentially, lamivudine (LAM), adefovir dipivoxil (ADV), and ADV + telbivudine (LDT) combination treatment over a total of 108 weeks. METHODS: A 20-year-old Chinese man presented to Huashan Hospital, Fudan University, Shanghai, People's Republic of China, with hepatitis B surface antigen-positive and HBeAg-positive chronic HBV and was sequentially treated with LAM 100 mg/d for 18weeks,ADV 10mg/d for 68weeks, and ADV 10mg/d + LDT 600 mg/d combination treatment for 22 weeks. Compliance was monitored every 4 weeks using a pill count. For genotypic analysis, the RT region of the polymerase gene from the serum of this patient was amplified, cloned, and sequenced. Fifty clones with HBV insert were selected for sequencing at weeks 0 (baseline), 18, 22, 60, 70, 86, and 108. RESULTS: The rtM204V/L LAM-resistance mutation was detected in 4.4% (2/45) of clones prior to LAM treatment. At week 18 during LAM treatment, the rtM204I mutation became predominant, being present in 79.5% (35/44) of clones. The rtM204I mutation was associated with compensatory mutations (rtL180M and rtT184L). A total of 9.1% (4/44) of the clones harbored the rtL180M + rtT184L + rtM204I mutations. Two new mutations, rtL229V and rtV191I, were detected in 75.0% (33/44) and 11.4% (5/44) of clones, respectively. At week 22 during ADV treatment, LAM-resistance mutations (rtL180M, rtT184L, rtM204I, rtV191I, and rtL229V) were not detected. At week 86 during ADV therapy, the rtN236T ADV-resistance mutation was detected in 58.8% (20/34) of clones. A total of 20.6% (7/34) of the clones harbored the rtK212T + rtM250L mutation, and rtA181V was found in 2.9% (1/34) of the clones. At week 108, after the patient had been receiving ADV + LDT combination therapy for 22 weeks, rtS202G and rtI269T had emerged, representing 28.9% (13/45) and 8.9% (4/45), respectively, of the viral population during ADV + LDT combination treatment. We also detected several polymorphic sites,including rtF221Y, rtS223A, rtI224V, rtN238H, rtL267Q, and rtQ271M, during the sequential treatment. After 22 weeks of combination treatment, HBV DNA count was decreased to less than the lower limit of quantitation (<200 copies/mL). CONCLUSIONS: This report identified HBV mutations that escaped the antiviral pressure of LAM, ADV, and ADV + LDT in this patient and provided insight into the process of mutation selection through genotypic analysis during antiviral treatment. Mutations selected under sequential treatments of LAM, ADV, and ADV + LDT can lead to a series of compensatory mutations, which partially restore the level of viral replication. ADV administered in combination with LDT appeared to be effective in this selected case with clinical or virologic resistance to sequential treatment with LAM and ADV.

Potent immune responses of Ag-specific Vgamma2Vdelta2+ T cells and CD8+ T cells associated with latent stage of Mycobacterium tuberculosis coinfection in HIV-1-infected humans.

OBJECTIVE: To investigate immune responses of peptide-specific CD4+ and CD8+ T cells, and nonpeptide-specific Vgamma2Vdelta2+ T cells during clinical quiescence of latent Mycobacterium tuberculosis coinfection in HIV-1-infected humans. METHODS: One hundred HIV-1-infected individuals who had HIV infection only [HIV+tuberculosis-(TB-)], latent Mycobacterium tuberculosis coinfection (HIV + LTB), or active tuberculosis (HIV + TB) were recruited to measure mycobacterium purified protein derivative (PPD)-specific IFNgamma+ CD4+ and CD8+ T cells, and phosphoantigen HMBPP-specific IFNgamma+ Vgamma2Vdelta2+ T cells using enzyme-linked immunospot and intracellular cytokine staining assays. RESULTS: Both HIV + TB and HIV + LTB groups had low levels of PPD-specific IFNgamma+ CD4+ T cells regardless of CD4+ peripheral blood lymphocytes counts. However, numbers of PPD-specific IFNgamma+ CD8+ T cells in the HIV + LTB group were significantly greater than those in the HIV + TB group. Surprisingly, numbers of phosphoantigen hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells in the HIV + LTB group were much greater than those in the HIV + TB group (P < 0.001). This difference was present in the subgroups of HIV + LTB whatever the levels of CD4+ T-cell counts more than 200/microl or less than 200/microl. Numbers of hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells were even five times greater than those of PPD-specific IFNgamma+ CD8 T cells within the HIV + LTB group. CONCLUSION: Potent immune responses of hydroxy-3-methyl-but-2-enyl pyrophosphate-specific IFNgamma+ Vgamma2Vdelta2+ T cells and PPD-specific IFNgamma+ CD8+ T cells were detected in HIV + LTB persons but not HIV + TB patients. The robust immune responses of Vgamma2Vdelta2+ and CD8+ T effector cells were associated with the latent stage of Mycobacterium tuberculosis coinfection in HIV-1-infected humans.