Co-existence of HHV-6 and <i>Chlamydia</i> in cervical smears.

<p>Samples with detectable HHV-6 DNA as well as chlamydial (Ctr) DNA in cervical smears were grouped in this table. HHV-6 DNA load in cervical smears and in total blood from the respective samples are shown together with serological data for <i>Chlamydia</i> IgG, IgM and IgA. Ctr IgG values less than 32 (<32) indicate IgG negative. ND, not detected; POS, positive test result; NEG, negative test result.</p

Two-way Chi-square test to study the association between <i>Chlamydia</i> and HHV-6 load in cervical smear of all patients.

*<p>Samples have been arbitrarily divided into 4 sub groups (group 1, 2, 3 and 4) depending on the HHV-6 viral load. Respective HHV-6 DNA load is mentioned within brackets.</p>**<p>Similarly, all the samples have been arbitrarily divided into 3 sub groups (group A, B and C) depending on the chlamydial (Ctr) DNA load. Group A: more than 25,000 copies of Ctr/1000 cells, Group B: between 100–25,000 copies/1000 cells, Group C: Below 100 copies of Ctr (not detectable)/1000 cells.</p

Quantitative real time PCR assay to detect HHV-6 and chlamydial load in cervical swabs and total blood samples.

<p>(<b>A–C</b>) Standard curves for HHV-6 and <i>C. trachomatis</i> quantification. Ten fold serial dilutions of U94 HHV-6A (<b>A</b>), <i>C. trachomatis</i> (<b>B</b>) and PI15 (<b>C</b>) plasmids were tested in triplicates in qPCR reaction. Mean Ct values were plotted against the copy numbers. Correlation coefficients and slope values are mentioned in each figure. (<b>D</b>) Scatter plot showing quantitative distribution of HHV-6 and <i>C. trachomatis</i> in cervical swabs of patients (n = 65) with suspected <i>C. trachomatis</i> infection as determined by real-time qPCR assay. Samples with no detectable HHV-6 and <i>C. trachomatis</i> are not included. Samples with HHV-6 DNA below detection limit (5 copies/10³ cells) were arbitrarily specified as 4 copies/10³ cells. Similarly, samples with less than 100 copies/10³ cells were arbitrarily specified as 50 copies/10³ cells. Sensitivity of PCR was marked as detection limits.</p

HHV-6 and chlamydial load in the <i>Chlamydia</i> negative control group.

<p>Samples with negative NAAT results were grouped in this table. HHV-6 DNA load in cervical smears as detected in qPCR assays is shown together with the HHV-6 DNA load in total blood from the respective samples. Serological data for <i>Chlamydia</i> (Ctr) IgG, IgM and IgA has also been included. Ctr IgG values less than 32 (<32) indicate IgG negative. ND, not detected; NEG, negative test result.</p

<i>Chlamydia trachomatis</i> infection induces CiHHV-6 DNA replication.

<p>(<b>A</b>) qPCR analysis of viral DNA in HSB-ML cells after 2 days of <i>C. trachomatis</i> infection. HSB-ML cells were infected with <i>C. trachomatis</i> (Ctr) for 2 days. In parallel, the same number of HSB-ML cells was cultured in the presence of 1 µM hydrocortisone or 80 ng/ml of TSA for 2 days. As TSA was dissolved in DMSO, a DMSO control was also included in the study. Data represent the mean ± SEM of 5 independent experiments. (<b>B</b>) qPCR analysis of viral DNA in HeLa cells carrying latent HHV-6A after 2 days of <i>C. trachomatis</i> infection. Cells were infected with <i>C. trachomatis</i> or were treated with TSA or hydrocortisone as mentioned in (A). Data represent the mean ± SEM of 5 independent experiments. (<b>C, D, E</b>) qPCR analysis of viral DNA in HSB-ML (C), JL-LCL (D) and PL-LCL (E) cells after 9 days of <i>C. trachomatis</i> infection. Cells were infected with <i>C. trachomatis</i> at an MOI of 2–5 for 2 days after which cells were washed thoroughly and grown in presence of fresh media with 1 µg/ml of doxycycline for another 7 days. Total DNA was isolated and used for qPCR assay. Data represent the mean ± SEM of three independent experiments. (<b>F</b>) qPCR analysis of viral DNA in freshly isolated blood cells from CiHHV-6 individuals after 5 days of <i>C. trachomatis</i> infection. Fresh PBMCs were isolated from total blood and were infected with <i>C. trachomatis</i> for 2 days at an MOI of 5 after which cells were washed thoroughly and grown in presence of fresh media with 1 µg/ml of doxycycline for another 3 days. In parallel, the same numbers of PBMCs were cultured in presence of 80 ng/ml of TSA for 5 days. Total leukocytes were separated from monocyte-derived macrophages and were infected with <i>C. trachomatis</i> in parallel. Heat inactivated <i>C. trachomatis</i> (hiCtr) were added to total PBMCs in a separate well and were included as negative control. Data represent the mean ± SEM of results from blood samples of 5 different CiHHV-6 individuals. (<b>G</b>) qPCR analysis of viral DNA in total leukocytes and monocyte-derived macrophages from CiHHV-6 individuals after 5 days of <i>C. trachomatis</i> infection. Total leukocytes were separated from monocyte-derived macrophages and were infected with <i>C. trachomatis</i> for 2 days at an MOI of 5 after which cells were washed thoroughly and grown in presence of fresh media with 1 µg/ml of doxycycline for another 3 days. Data represent the mean ± SEM of three independent experiments performed at the same time from one CiHHV-6 individual.</p

Study drug adherence and loss to follow-up.

<p><b>A, B</b>: Study drug adherence calculated from pill counts of returned, unused study medication in female (A) and male (B) study participants. P-values were calculated by 2way ANOVA <b>C</b>: Loss to follow-up during the two-year study. P value was calculated by log-rank (Mantel-Cox) analysis.</p

Effects of prednisolone on CD4 counts and CD4/CD8 ratio.

<p><b>A</b>: Mean CD4 ± S.D. P-values were determined by 2way ANOVA. <b>B</b>: Mean CD4 changes ± S.D. relative to baseline and linear regression of the data. 2-year gains/losses were calculated from slope of linear regression. <b>C</b>: Mean ± S.D. CD4/CD8-ratio and linear regression. B, C: P values on the right indicate non-zero hypotheses for the slope. P-value on the left for difference between the two data sets was calculated by 2way ANOVA. <b>D-F</b> and <b>G-I</b>: Post-hoc analyses for female study participants (D-F) and for male study participants (H-I) for absolute CD4 counts (D, G), CD4 relative CD4 changes (E, H), and CD4/CD8 ratio (F, I).</p

Study participants at risk.

<p>Number of study participants at risk within the intent-to-treat (ITT) population (<b>A</b>) or the per protocol (PP) population (<b>B</b>). Separate analyses for female study participants (C, D) and for male study participants (E, F).</p

Progression to HAART as treated.

<p>Kaplan-Meyer estimate of progression to HAART as treated within the intent-to-treat (ITT) population (<b>A</b>) or the per protocol (PP) population (<b>B</b>). Separate analyses for female study participants (B, E) and for male study participants (C, F) for progression to HART as treated. P values were calculated by log-rank (Mantel-Cox) analysis.</p

Effects of prednisolone on HIV disease progression: Primary Study Endpoint.

<p>Kaplan-Meyer estimate of progression to combined study endpoint (onset of CDC stage-C-condition or drop of CD4 counts below 200) within the intent-to-treat (ITT) population (<b>A</b>) or the per protocol (PP) population (<b>B</b>). <b>C</b>: Progression to AIDS-defining condition. All study participants who received HAART, but did not reach the study endpoint were censored for the KM analysis. <b>D-F</b> and <b>G-I</b>: Post-hoc analyses for female study participants (D-F) and for male study participants (H-I) for progression to combined endpoint within the ITT population (D, G), progression to combined endpoint within the PP population (E, H), and progression to AIDS-defining condition (F, I). A-I: P values were calculated by log-rank (Mantel-Cox) analysis.</p