QuantiFERON-TB Gold Plus Assay in Patients With Latent vs. Active Tuberculosis in a Low Incidence Setting: Level of IFN-γ, CD4/CD8 Responses, and Release of IL-2, IP-10, and MIG.

Objectives: We analyzed the results of the QuantiFERON Glod Plus assay (QFT) and cytokine patterns associated with active tuberculosis (ATB) among patients with positive QFT. Methods: A total of 195 patients are QFT-positive, among which 24 had an ATB and 171 had a latent tuberculosis infection (LTBI). Interferon-gamma (IFN-γ) secretion was analyzed relative to interleukin-2 (IL-2), IFN-γ inducible protein or CXCL-10 (IP-10), and monokine induced by IFN-γ or CXCL-9 (MIG) secretion, and then compared between two sets of peptide antigens [tube 1 - cluster of differentiation 4 (CD4+) T cell stimulation; tube 2 - CD4+/CD8+ T cell response]. Results: Higher IFN-γ responses were measured in the ATB group (p = 0.0089). The results showed that there was a lower ratio of tube 1/tube 2 IFN-γ concentrations in the ATB group (p = 0.0009), and a median [interquartile ranges (IQR)] difference between the two sets at -0.82 IU/ml (-1.67 to 0.18) vs. -0.07 IU/ml (-0.035 to 0.11, p < 0.0001) in the ATB group compared to the LTBI group, respectively. In addition, patients with low ratios of IL-2/IFN-γ, IP-10/IFN-γ, and MIG/IFN-γ were much more likely to have ATB. Conclusion: High levels of IFN-γ secretion, preferential IFN-γ response in tube 2, and lower secretion of IL-2, IP-10, and MIG release relative to IFN-γ secretion were more likely observed in subjects with ATB. These features of T cell response may be helpful in low prevalence settings to suspect ATB in patients tested positive for IFN-γ release assays (IGRA).

Combined testing for herpes simplex virus and Mycobacterium tuberculosis DNA in cerebrospinal fluid of patients with aseptic meningitis in Burkina Faso, West Africa.

BACKGROUND: Little is known about the involvement of herpes simplex virus (HSV) or Mycobacterium tuberculosis (MTB) as potentially curable causes of central nervous system (CNS) infections in sub-Saharan Africa. OBJECTIVE: In this study, we developed a PCR assay dedicated to simultaneous testing of HSV1/HSV2 and MTB in Burkina Faso, a country where HSV is neglected as a cause of CNS infection and where TB prevalence is high. METHODS: A consensus HSV1/HSV2 set of primers and probe were designed and combined to primers and probe targeting the IS6110 repetitive insertion sequence of MTB. Analytical performances of the assay were evaluated on reference materials. Cerebrospinal fluid (CSF) collected from subjects with aseptic meningitis was tested for HSV1/HSV2 and MTB DNA. RESULTS: The UL29 gene was chosen as a highly conserved region targeted by the HSV1/HSV2 nucleic acid test. The lower limits of detection were estimated to be 2.45 copies/µL for HSV1, 1.72 copies/µL for HSV2, and 2.54 IS6110 copies per µL for MTB. The PCR was used in 202 CSF collected from subjects suspected of aseptic meningitis. Five samples (2.46%) tested positive, including two children positive for HSV1 (0.99%) and three adults tested positive for MTB (1.47%). CONCLUSION: Using an in-house real-time PCR assay, we showed that both HSV and MTB are etiologic pathogens contributing to aseptic meningitis in Burkina Faso. This molecular test may have clinical utility for early diagnosis for those treatable CNS infections.

Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens.

Background: When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum. Materials and Methods: The impact of targeting repetitive IS6110 sequences on the PCR sensitivity was evaluated across mycobacterium strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing <1.5 × 102 copies/ml ex vivo or after dilution. Results: The lower limit of detection (LOD) using the IS6110 PCR was 107 genome copies/ml (95% CI: 83-130) using MTB H37Rv as a reference strain, versus 741 genome copies/ml (95% CI: 575-1094) using the senX3 PCR. The proportion of recovered MTB DNA after lysis and extraction ranged from 35 to 82%. The Chelex® method appeared as a more efficient protocol among the six different protocols tested. The sensitivity and specificity in clinical sputum samples were 95.1% (95% CI: 90.7-99.6) and 100% (95% CI: 96.2-100.8), respectively. Among 40 samples with low MTB DNA concentration, 75% tested positive for IS6110 PCR, versus 55% using the Xpert MTB/RIF assay (p = 0.03). Conclusion: Laboratory assays based on an efficient MTB lysis and DNA extraction protocols combined with amplification of IS6110 repeat sequences appear as a sensitive diagnostic method to detect MTB DNA in sputum with low bacterial load.

Detection of four human polyomaviruses (MCPyV, HPyV6, HPyV7 and TSPyV) in cervical specimens from HIV-infected and HIV-uninfected women.

OBJECTIVES: To investigate the presence of recently discovered human polyomaviruses in cervical specimens collected from African and French women, in relation to HIV serostatus, high-risk human papillomavirus infection (HR-HPV) and cervical disease. METHODS: Cervical specimens were collected from 140 HIV-1-seropositive African women and 50 HIV-seronegative French women. Presence of Merkel cell polyomavirus (MCPyV), human polyomavirus 6 (HPyV6), human polyomavirus 7 (HPyV7) and trichodysplasia spinulosa-associated polyomavirus (TSPyV) was detected by real-time PCR, and presence of HR-HPV DNA by Hybrid Capture 2 assay with subsequent HPV genotyping using the INNO-LiPA HPV Genotyping Extra assay. Cervical biopsies were analysed by histopathology. RESULTS: The detection rates were 55.3%, 3.2%, 2.1% and 0% for MCPyV, HPyV6, HPyV7 and TSPyV, respectively, with no significant difference by population. The MCPyV viral load ranged from 14 to 210 DNA copies/106 cells (median, 80 DNA copies/106 cells), with no difference between women with and without cervical precancerous lesions. There was no association between detection of human polyomaviruses in cervical specimens and geographical origin/HIV serostatus, HR-HPV coinfection or precancerous cervical lesions. CONCLUSIONS: These observations argue against a possible role of MCPyV as a cofactor in HPV-induced carcinogenesis. MCPyV and, to a lesser extent, HPyV6 and HPyV7 might belong to the female genital tract microbiota.