Schematic overview of allele-specific real-time PCR primers design and setting the standard curve analysis.

<p>In presence of K103N-AAC (A1) and K103-AAT (A2) mutants, only the specific mutant amplicons will be amplified when using mutant specific forward (MSFP) and a common reverse primer (CRP), respectively. The total population of sequences in the reaction are amplified using non-specific forward primer (NSFP) and the CRP (A3). An intentional mismatch at the penultimate base (indicated with I) was introduced in the allele-specific real-time PCR primers in order to increase the specificity and minimize the risk for amplification of the wild type allele. Mutant specific (Sp) and non-specific (NSp) standard curves of K103N AAC allele (B1), K103N AAT allele (B2) and Y181C TGT allele (B3) are in parallel with each experiment. The copy number of each mutant specific and total population of sequences amplifications of clinical samples were determined using such standard curves that has been run in duplicate, parallel with each sample. The quantity of the patients' mutant specific and the total population of sequences (amplified with non-specific primer) was then determined by comparing the samples Ct value with those of the specific and non-specific standard curves derived from the standard plasmid controls using the corresponding primers. The percentage of mutant specific sequences was then determined by dividing the quantity of mutant specific sequence by the quantity of the total sequences and multiplying by 100. Positive samples were repeated at least twice. Correlation coefficients (r²) were higher than 0.99. Sp: mutant specific amplification. MSFP, mutant-specific forward primer; CRP, common reverse primer; NSFP, non-specific forward primer. NSp: non-specific amplification (amplify the total population of sequences).</p

Characteristics of treatment naïve HIV-1 infected patients included in the study.

a<p>IQR, interquartile range;</p>b<p>IVDU, intravenous drug use;</p>c<p>DRM, drug resistant mutation.</p><p>Characteristics of treatment naïve HIV-1 infected patients included in the study.</p

Minority Drug-Resistant HIV-1 Variants in Treatment Naïve East-African and Caucasian Patients Detected by Allele-Specific Real-Time PCR

<div><p>Objective</p><p>To assess the presence of two major non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutations (DRMs), Y181C and K103N, in minor viral quasispecies of treatment naïve HIV-1 infected East-African and Swedish patients by allele-specific polymerase chain reaction (AS-PCR).</p><p>Methods</p><p>Treatment naïve adults (n = 191) with three epidemiological backgrounds were included: 92 Ethiopians living in Ethiopia; 55 East-Africans who had migrated to Sweden; and 44 Caucasians living in Sweden. The <i>pol</i> gene was analysed by standard population sequencing and by AS-PCR for the detection of Y181C and K103N.</p><p>Results</p><p>The Y181C was detected in the minority quasispecies of six Ethiopians (6.5%), in two Caucasians (4.5%), and in one East-African (1.8%). The K103N was detected in one East- African (1.8%), by both methods. The proportion of mutants ranged from 0.25% to 17.5%. Additional DRMs were found in all three treatment naïve patient groups by population sequencing.</p><p>Conclusions</p><p>Major NNRTI mutations can be found by AS-PCR in minor quasispecies of treatment naïve HIV-1 infected Ethiopians living in Ethiopia, in East-African and Caucasian patients living in Sweden in whom population sequencing reveal wild-type virus only. Surveys with standard sequencing are likely to underestimate transmitted drug resistance and the presence of resistant minor quasispecies in treatment naïve patients should be topic for future large scale studies.</p></div

DataSheet_2_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.xlsx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p

DataSheet_1_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.docx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p

Detection frequency of mutant K103N and Y181C minority variants.

<p>Ninety-two treatment naive HIV patients plasma were analysed by allele-specific PCR, detecting the two major NNRTI mutations K103N and Y181C in the reverse transcriptase gene. The limit of detection for the K103 mutations (AAC and AAT) is indicated by a dashed line. The limit of detection for the Y181C mutation (TGT) is indicated by a solid line. Crosses indicate two patients who died within three months. Shaded area indicates the assay background, which was determined as a mean Ct value from eight independent runs of 100% wild type template with mutant specific primers plus three standard deviations.</p

Characteristics of East-African, Caucasian and Ethiopian patients with drug resistance mutations.

a<p>ND, not done.</p><p>Characteristics of East-African, Caucasian and Ethiopian patients with drug resistance mutations.</p

Example of allele-specific real-time PCR amplification curves of cloned wild type and mutant TGT plasmid DNAs at different frequencies (raw data).

<p>The specificity and accuracy of the assay was determined by running allele-specific real-time PCR of mixtures of mutant and wild type DNA standards ranging from 0.01 to 100% and amplified with the mutant specific primers in the background of wild type sequence. Amplification of the total population results always in the same Ct values, regardless of the amount of mutant DNAs present in the reaction. WT, wild type.</p

Efficacy and Safety of Antiretroviral Therapy Initiated One Week after Tuberculosis Therapy in Patients with CD4 Counts < 200 Cells/μL: TB-HAART Study, a Randomized Clinical Trial

<div><p>Background</p><p>Given the high death rate the first two months of tuberculosis (TB) therapy in HIV patients, it is critical defining the optimal time to initiate combination antiretroviral therapy (cART).</p><p>Methods</p><p>A randomized, open-label, clinical trial comparing efficacy and safety of efavirenz-based cART initiated one week, four weeks, and eight weeks after TB therapy in patients with baseline CD4 count < 200 cells/μL was conducted. The primary endpoint was all-cause mortality rate at 48 weeks. The secondary endpoints were hepatotoxicity-requiring interruption of TB therapy, TB-associated immune reconstitution inflammatory syndrome, new AIDS defining illnesses, CD4 counts, HIV RNA levels, and AFB smear conversion rates. All analyses were intention-to-treat.</p><p>Results</p><p>We studied 478 patients with median CD4 count of 73 cells/μL and 5.2 logs HIV RNA randomized to week one (n = 163), week four (n = 160), and week eight (n = 155). Sixty-four deaths (13.4%) occurred in 339.2 person-years. All-cause mortality rates at 48 weeks were 25 per 100 person-years in week one, 18 per 100 person-years in week four and 15 per 100 person-years in week eight (P = 0.2 by the log-rank test). All-cause mortality incidence rate ratios in subgroups with CD4 count below 50 cells/μL versus above were 2.8 in week one (95% CI 1.2–6.7), 3.1 in week four (95% CI 1.2–8.6) and 5.1 in week eight (95% CI 1.8–16). Serum albumin < 3gms/dL (adjusted HR, aHR = 2.3) and CD4 < 50 cells/μL (aHR = 2.7) were independent predictors of mortality. Compared with similar subgroups from weeks four and eight, first-line TB treatment interruption was high in week one deaths (P = 0.03) and in the CD4 subgroup <50 cells/μL (P = 0.02).</p><p>Conclusions</p><p>Antiretroviral therapy one week after TB therapy doesn’t improve overall survival. Despite increased mortality with CD4 < 50 cells/μL, we recommend cART later than the first week of TB therapy to avoid serious hepatotoxicity and treatment interruption.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01315301" target="_blank">NCT 01315301</a></p></div

Frequency distribution of demographic and clinical variables, Addis Ababa, Ethiopia, August 2004-March 2005

<p>Frequency distribution of demographic and clinical variables, Addis Ababa, Ethiopia, August 2004-March 2005</p