Francesca Zara

mmpL7 Gene of Mycobacterium tuberculosis Is Responsible for Isoniazid Efflux in Mycobacterium smegmatis

ABSTRACT The Mycobacterium tuberculosis mmpL7 gene, encoding a hypothetical resistance nodulation division transporter, confers a high resistance level to isoniazid when overexpressed in Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine and CCCP (carbonyl cyanide m-chlorophenylhydrazone). Energy-dependent efflux of isoniazid from M. smegmatis cells expressing the mmpL7 gene was observed.

Effect of antibiotic use on the prevalence of symptomatic vulvovaginal candidiasis

OBJECTIVE The purpose of the study was to evaluate the influence of antibiotic use on the prevalence of symptomatic vulvovaginal candidiasis. STUDY DESIGN This is a case-control study of 684 women with symptomatic vulvovaginal candidiasis who were enrolled at a vaginitis clinic and 901 control subjects who attended a cytologic screening service. RESULTS The prevalence of antibiotic use in the month preceding the visit was 19.3% (132/684) among patients with Candida infection compared with 11.9% (107/901) among control subjects (P <.001). After adjustment by logistic regression for potential confounders (age, marital status, and contraceptive method used), the odds ratio for vulvovaginal candidiasis among patients who reported antibiotic use in the month preceding the visit was 1. 75 (95% confidence interval 1.33 to 2.32). No differences in risk were found by type of antibiotic used. The likelihood of vulvovaginal candidiasis was directly related to the duration of antibiotic use and was higher in patients who had a history of repeated episodes of Candida vaginal infection. CONCLUSIONS Antibiotic use is a short-term risk factor for symptomatic vulvovaginal candidiasis, either as a first episode or in the form of recurrence. Increasing duration of antibiotic use is directly related with an increased prevalence of Candida vaginal infection.

Dominant Role of Host Selective Pressure in Driving Hepatitis C Virus Evolution in Perinatal Infection

ABSTRACT The dynamics of the genetic diversification of hepatitis C virus (HCV) populations was addressed in perinatal infection. Clonal sequences of hypervariable region 1 of the putative E2 envelope protein of HCV were obtained from four HCV-infected newborns (sequential samples spanning a period of 6 to 13 months after birth) and from their mothers (all samples collected at delivery). The data show that the variants detected between birth and the third month of life in samples from the four newborns were present in the HCV populations of their mothers at delivery. In the newborns, a unique viral variant (or a small group of closely related variants) remained stable for weeks despite active viral replication. Diversification of the intrahost HCV population was observed 6 to 13 months after birth and was substantially higher in two of the four subjects, as documented by the intersample genetic distance (GD) (P = 0.007). Importantly, a significant correlation between increasing GD and high values for the intersampleKa/Ks ratio (the ratio between antonymous and synonymous substitutions; an index of the action of selective forces) was observed, as documented by the increase of both parameters over time (P = 0.01). These data argue for a dominant role of positive selection for amino acid changes in driving the pattern of genetic diversification of HCV populations, indicate that the intrahost evolution of HCV populations is compatible with a Darwinian model system, and may have implications in the designing of future antiviral strategies.

Clinical Isolates of Mycobacterium tuberculosis in Four European Hospitals Are Uniformly Susceptible to Benzothiazinones

ABSTRACT The new antitubercular drug candidate 2-[2-S-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (BTZ043) targets the DprE1 (Rv3790) subunit of the enzyme decaprenylphosphoryl-β-d-ribose 2′-epimerase. To monitor the potential development of benzothiazinone (BTZ) resistance, a total of 240 sensitive and multidrug-resistant Mycobacterium tuberculosis clinical isolates from four European hospitals were surveyed for the presence of mutations in the dprE1 gene and for BTZ susceptibility. All 240 strains were susceptible, thus establishing the baseline prior to the introduction of BTZ043 in clinical trials.

Factors associated with nucleic acids related to human immunodeficiency virus type 1 in cervico-vaginal secretions

Objective To assess HIV‐related nucleic acids in cervico‐vaginal secretions and the factors associated with them.

Markers of local immunity in cervico-vaginal secretions of HIV infected women: implications for HIV shedding

Objectives: To link local proinflammatory cytokines with HIV related nucleic acids in cervico-vaginal secretions and the factors associated with them. Methods: An observational study on 60 HIV positive women attending the department of obstetrics and gynaecology, University of Pavia, Italy. HIV-1 RNA in plasma, proviral HIV-1-DNA, cell associated and cell free HIV-1 RNA in cervico-vaginal secretions were evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). IL-1β, IL-6, and TNF-α were measured by ELISA in cervico-vaginal lavages. Multiple regression analysis on ordinal categorical variables was used to test for the simultaneous associations of clinical and microbiological variables on quartiles of cytokine concentrations in lavage samples. Results: Proviral HIV-1 DNA, cell associated and cell free HIV-1 RNA were detected in 76.7% (46/60), 70% (42/60), and 71.7% (43/60) of the patients, respectively. IL-1β concentration was directly correlated with proviral HIV-DNA (Spearman rho = 0.35, p = 0.01) and cell associated HIV-RNA levels (Spearman rho = 0.263, p = 0.05). IL-1β concentration (153.9 pg/ml) was higher (p<0.05) among women with cytological squamous intraepithelial lesion (SIL) than negative controls (73.4 pg/ml). In women with vaginal infection both IL-1β (41.7 pg/ml) and IL-6 (10.2 pg/ml) were lower (p<0.05) in comparison to negative controls (144.9 pg/ml and 23.7 pg/ml, respectively). Women receiving stable antiretroviral therapy had significantly lower TNF-α (34.4 pg/ml versus 44.4 pg/ml, p = 0.04) and higher IL-6 (24.0 pg/ml versus 1.4 pg/ml, p = 0.004) levels in lavage samples compared to untreated women. The associations between the presence of SIL, antiretroviral treatment, vaginal infection and cytokine concentrations in cervico-vaginal secretions were confirmed in multiple regression analysis. Conclusions: Local immune activation may modulate HIV-1 shedding in cervico-vaginal secretion with possible influence on vaginal physiology and host defence. Pharmacological agents lowering HIV-1 replication cause a shift to a pattern of cytokine production which seems less favourable to the transmission of the disease.

Viral Excretion in Cervicovaginal Secretions of HIV-1-Infected Women Receiving Antiretroviral Therapy

Abstract A longitudinal study was conducted to evaluate the viral shedding present in cervicovaginal secretions of HIV-1-seropositive women receiving antiretroviral therapy. A total of 128 paired cervicovaginal and blood samples was obtained from 37 women during a median follow-up period of 21 months. A sensitive, competitive, polymerase chain reaction and a reverse transcription polymerase chain reaction were used for the simultaneous quantitation of HIV-1 proviral DNA and RNA in cervicovaginal cells and cell-free RNA in cervicovaginal secretions, as well as HIV-1 RNA in peripheral blood. The cumulative probability of detecting proviral DNA in genital secretions was significantly higher over time in women with detectable viremia than in women in whom HIV-1 RNA was persistently undetectable in plasma (<50 copies/ml) (P=0.028 by log-rank test). The presence and amount of proviral DNA, cell-associated RNA and cell-free RNA in the cervicovaginal secretions were positively correlated with the presence of detectable viremia or the number of HIV-1 RNA copies in plasma (Spearman rank correlation, 0.290, 0.279, and 0.305, respectively; all P<0.01), but no correlation was found with the CD4+ cell count. In addition, vaginal infections were positively correlated with the detection of proviral DNA in cervicovaginal secretions (odds ratio, 2.60; 95% confidence interval, 1.07–5.70). However, the positive correlation between the presence and amount of HIV in cervicovaginal secretions and the viral load in plasma provides no assurance that HIV shedding does not occur in the genital tract of women with undetectable HIV-RNA in plasma.

Human immunodeficiency virus type 1-related nucleic acids and papillomavirus DNA in cervicovaginal secretions of immunodeficiency virus-infected women.

Objective To evaluate simultaneous human immunodeficiency virus (HIV)-related nucleic acids and human papillomavirus (HPV)-DNA in cervicovaginal secretions of HIV-seropositive women. Methods We collected 47 paired blood and cervicovaginal lavage samples from 124 known HIV-1–seropositive women. Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA in cervicovaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (PCR) and reverse transcription PCR. Polymerase chain reaction and subsequent restriction fragment length polymorphism analysis of PCR products were used to detect HPV types 6, 11, 16, 18, 31, 33, 35, and 56. Results Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA were detected in 52.4% (65 of 124), 38.7% (48 of 124), and 33.9% (42 of 124) of lavage samples, respectively. Human papillomavirus-DNA in cervicovaginal secretions was detected in 64% (79 of 124) of participants. The rate of detection of HPV types of intermediate to high oncogenic risk was higher in HIV-positive women who tested positive for cell-associated (odds ratio [OR] 3.57, 95% confidence interval [CI] 1.17, 11.12) or cell-free (OR 4.63, 95% CI 1.42, 15.51) HIV-1 RNA in cervicovaginal secretions than their counterparts who tested negative. Logistic regression analysis showed that the association between HPV infection and the detection of HIV-1 RNA in cervicovaginal secretions persisted after adjustment for potential confounders such as CD4+ cell counts, HIV-1 RNA in plasma, use of antiretroviral drugs, vaginal infection, and regular condom use. In univariable and multivariable analysis, HPV-DNA detection was associated with amounts of cell-free and cell-associated HIV-1 RNA in cervicovaginal secretions (χ2 for trend 10.35, and 9.84, P = .001 and .002, respectively). Conclusions The rate of HPV detection in the genital tract of HIV-1–seropositive women is associated with the amount of cell-associated and cell-free HIV-1 RNA present in cervicovaginal secretions. The association does not appear to be attributable entirely to the effect of HIV-related immunodepression.

Synthesis and evaluation of α-ketotriazoles and α,β-diketotriazoles as inhibitors of Mycobacterium tuberculosis.

Two series of α-ketotriazole and α,β-diketotriazole derivatives were synthesized and evaluated for antitubercular and cytotoxic activities. Among them, two α,β-diketotriazole compounds, 6b and 9b, exhibited good activities (minimum inhibitory concentration = 7.6 μM and 6.9 μM, respectively) on Mycobacterium tuberculosis and multi-drug resistant M. tuberculosis strains and presented no cytotoxicity (IC₅₀ > 50 μM) on colorectal cancer HCT116 and normal fibroblast GM637H cell lines. These two compounds represent promising leads for further optimization.

Cervical intraepithelial neoplasia and cervicovaginal shedding of human immunodeficiency virus.

OBJECTIVE: Although human immunodeficiency virus (HIV) infection is a well-known risk factor for cervical intraepithelial neoplasia (CIN), the influence of CIN on cervicovaginal shedding of HIV is poorly understood. The purpose of this study was to evaluate the association between CIN and the shedding of HIV in cervicovaginal secretions. METHODS: Two hundred sixteen HIV-seropositive patients were followed up by Pap test, colposcopy, and targeted cervical biopsies for a median of 16 months (range 0–94). A diagnosis of low-grade CIN was made on the basis of Pap test and either colposcopy or cervical biopsy. High-grade CIN was diagnosed solely on the basis of cervical biopsy. At each follow-up visit, we measured HIV-1 RNA in plasma, proviral HIV-1 DNA, and cell-associated and cell-free HIV-1 RNA in cervicovaginal secretion by competitive polymerase chain reaction (cRT-PCR) and reverse transcriptase PCR. The univariable and multivariable associations between the occurrence of CIN and the presence of HIV-related nucleic acids in cervicovaginal secretions were evaluated with logistic generalized estimating equations. RESULTS: Overall, at enrollment and during the follow-up period, a diagnosis of either low-grade or high-grade CIN was made in 14.4% (99/689) and 6.7% (46/689) of the visits, respectively. The presence of measurable levels of plasma HIV-1 RNA was a significant risk factor for the detection of cervicovaginal HIV-1 DNA (odds ratio [OR] 1.86, 95% confidence interval [CI] 1.32–2.61, P < .001), cell-associated (OR 1.69, 95% CI 1.18–2.43, P = .004), and cell-free HIV-1 RNA (OR 1.84, 95% CI 1.28–2.63, P = .001). After the adjustment for the effect of plasma HIV-1 RNA, CD4+ positive cell counts less than 200 mm3, and bacterial vaginosis, the detection of cell-associated (OR 1.75, 95% CI 1.23–2.49, P = .006) and cell-free HIV-1 RNA (OR 2.0, 95% CI 1.39–2.87, P = .001) in cervicovaginal secretions was significantly associated with the diagnosis of CIN. CONCLUSION: The presence of CIN lesions is a significant risk factor for genital HIV shedding. Given the high prevalence of cervical disease among HIV-positive women, this finding could have important epidemiological implications in both heterosexual and perinatal transmission of HIV. LEVEL OF EVIDENCE: II-2