Syntheses and conformation of synthetic peptide substrates of protocollagen lysyl hydroxylase

Hydroxylation of specific lysyl residues by lysyl hydroxylase is an important posttranslational modification process in collagen biosynthesis. The main objective of this work was to investigate the conformational requirement for the enzymic reaction. -- Eight lysine-containing peptides which had amino acid sequences comparable to amino acid sequences around hydroxylysine or lysine in collagen were synthesized by solution-phase techniques. The peptides varied in length from three to seven amino acid residues. The structures of these peptides were investigated through circular dichroism (CD) and infrared (IR) spectroscopic methods. -- Lysyl hydroxylase was partially purified from chicken embryos using the established procedures. Seven of the synthetic peptides were tested for their ability to act as substrates of partially purified lysyl hydroxylase. The hydroxylation reaction was assayed by a technique involving measurement of ¹⁴CO₂ released stoichiometrically from 2-[1-¹⁴C]oxoglutarate and/or by a specific chemical procedure for hydroxylysine. -- Five peptides with the -Lys-Gly- sequence were hydroxylated to varying degrees, the degree of hydroxylation increasing with increasing chain length. Examination of these hydroxylated peptides by CD and IR spectral measurements revealed that the tripeptides NαtBocIleLysGlyOH and NαtBocAlaLysGlyOH adopt a γ-turn in which lysine occupies the second position of this structure. The tetrapeptide (NαtBocAlaLysGlySerOH) adopts both a β- and γ-turn and is more hydroxylated than the precursor tripeptide. This increase in the degree of hydroxylation may be attributed to the presence of the β-turn which may stabilize the γ-turn formed by the AlaLysGlyOH segment. The hexapeptide (NαtBocLeuHyPGlyAlaLysGlyOH) adopts a consecutive β- and γ-turn and is more hydroxylated than the tetrapeptide. This increase in hydroxylation may be attributable to the Gly³-Ala⁴ segment which may increase the binding of the enzyme to the substrate thereby enhancing hydroxylation. The heptapeptide (NαtBocLeuHyPGlyAlaLysGlySerOH) is hydroxylated more than the precursor hexapeptide. CD and model building studies have shown that NαtBocLeuHyPGlyAlaLysGlySerOH adopts two consecutive β-turns and a γ-turn. The second β-turn which is similar to that found in the tetrapeptide (NαtBocAlaLysGlySerOH) may be responsible for the increase in hydroxylation in comparison with the hexapeptide. -- All the hydroxylated peptides have one structural feature in common, namely the γ-turn with lysine in the second position. In contrast, two peptides (NαtBocAlaGlyLysOH and NαtBocAlaGlyLysHyPOH) which have the Gly-Lys sequence were not hydroxylated. Interestingly, both peptides adopt a γ-turn but the lysine is found in the third position of this structure. These data indicate that lysyl hydroxylase recognizes specific secondary structure(s) in its substrates. The nature of the amino acid around lysine and the chain length of the peptide may be the critical determinants in the synthesis of hydroxylysine by lysyl hydroxylase

Comparative performance characteristics of the urine lipoarabinomannan strip test and sputum smear microscopy in hospitalized HIV-infected patients with suspected tuberculosis in Harare, Zimbabwe

BackgroundIn Zimbabwe, sputum smear microscopy (SSM) is the routinely used TB diagnostic tool in hospitalised HIV-infected patients. However, SSM has poor sensitivity in HIV-infected patients. We compared performance of urine lipoarabinomannan strip test (LAM) and SSM among hospitalized HIV-infected patients with suspected TB.MethodsHospitalized HIV-infected patients with suspected TB were randomized to LAM plus SSM or SSM alone groups as part of a larger multi-country parent study. Here we present a comparison of LAM versus SSM performance from the Zimbabwe study site. LAM analyses (grade 2 cut-off) were conducted using (i) a microbiological reference standard (MRS; culture positivity for M.tb and designated definite TB) and (ii) a composite reference standard (CRS; definite TB plus probable TB i.e. patients with clinical TB excluded from the culture negative group). CRS constituted the primary analysis.Results82/457 (18%) of the patients randomized to the LAM group were M.tuberculosis culture positive. Using CRS, sensitivity (%, 95 % CI) of LAM was significantly higher than SSM [49.2 (42.1-56.4) versus 29.4(23.2-36.3); p 100 cells/μL. The combined sensitivity of LAM and SSM was higher than SSM alone being highest at CD4 counts 97% in all the 3 CD4 strata.ConclusionAmong hospitalized HIV-infected patients with suspected TB, the sensitivity of LAM is significantly higher than that of SSM, especially at low CD4 counts. LAM and SSM are complimentary tests for diagnosis of TB in HIV-infected patients. We recommend a combination of LAM and SSM for TB diagnosis in HIV-infected patients with low CD4 counts in HIV/TB co-endemic countries, where alternative methods are unavailable

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis.

BACKGROUND: Xpert MTB/RIF, the most widely used automated nucleic acid amplification test for tuberculosis, is available in more than 130 countries. Although diagnostic accuracy is well documented, anticipated improvements in patient outcomes have not been clearly identified. We performed an individual patient data meta-analysis to examine improvements in patient outcomes associated with Xpert MTB/RIF. METHODS: We searched PubMed, Embase, ClinicalTrials.gov, and the Pan African Clinical Trials Registry from inception to Feb 1, 2018, for randomised controlled trials (RCTs) comparing the use of Xpert MTB/RIF with sputum smear microscopy as tests for tuberculosis diagnosis in adults (aged 18 years or older). We excluded studies of patients with extrapulmonary tuberculosis, and studies in which mortality was not assessed. We used a two-stage approach for our primary analysis and a one-stage approach for the sensitivity analysis. To assess the primary outcome of cumulative 6-month all-cause mortality, we first performed logistic regression models (random effects for cluster randomised trials, with robust SEs for multicentre studies) for each trial, and then pooled the odds ratio (OR) estimates by a fixed-effects (inverse variance) or random-effects (Der Simonian Laird) meta-analysis. We adjusted for age and gender, and stratified by HIV status and previous tuberculosis-treatment history. The study protocol has been registered with PROSPERO, number CRD42014013394. FINDINGS: Our search identified 387 studies, of which five RCTs were eligible for analysis. 8567 adult clinic attendees (4490 [63·5%] of 7074 participants for whom data were available were HIV-positive) were tested for tuberculosis with Xpert MTB/RIF (Xpert group) versus sputum smear microscopy (sputum smear group), across five low-income and middle-income countries (South Africa, Brazil, Zimbabwe, Zambia, and Tanzania). The primary outcome (reported in three studies) occurred in 182 (4·5%) of 4050 patients in the Xpert group and 217 (5·3%) of 4093 patients in the smear group (pooled adjusted OR 0·88, 95% CI 0·68-1·14 [p=0·34]; for HIV-positive individuals OR 0·83, 0·65-1·05 [p=0·12]). Kaplan-Meier estimates showed a lower rate of death (12·73 per 100 person-years in the Xpert group vs 16·38 per 100 person-years in the sputum smear group) for HIV-positive patients (hazard ratio 0·76, 95% CI 0·60-0·97; p=0·03). The risk of bias was assessed as reasonable and the statistical heterogeneity across studies was low (I2<20% for the primary outcome). INTERPRETATION: Despite individual patient data analysis from five RCTs, we were unable to confidently rule in nor rule out an Xpert MTB/RIF-associated reduction in mortality among outpatients tested for tuberculosis. Reduction in mortality among HIV-positive patients in a secondary analysis suggests the possibility of population-level impact. FUNDING: US National Institutes of Health

Higher serum 25-hydroxyvitamin D concentrations are associated with active pulmonary tuberculosis in hospitalised HIV infected patients in a low income tropical setting: a cross sectional study

Abstract Background The inherent risk of developing tuberculosis (TB) in HIV- infected individuals is further enhanced by hypovitaminosis D. Interventions that offset HIV-associated immune deterioration potentially arrest disease progression and incidence of opportunistic infections including TB. Despite conflicting reports on association between vitamin D deficiency (VDD) and risk of TB, vitamin D (VD) supplementation remains a promising intervention. Methods We conducted a comparative cross-sectional study on 145 HIV+/pulmonary TB+ (PTB) and 139 HIV+/PTB− hospitalised patients to investigate association of vitamin D status and risk of PTB. Stratified random sampling was used to select archived serum specimens from participants enrolled in a randomised controlled trial (RCT) conducted to investigate the impact of using a point-of-care urine lipoarabinomannan strip test for TB diagnosis. PTB status was confirmed using sputum smear microscopy, culture or GeneXpert MTB/RIF. Serum 25-hydroxyvitamin D [25(OH) D] concentrations were assayed by competitive chemiluminescent immunoassay prior to commencement of anti-TB treatment. Effect of VD status on duration of hospital stay and patient outcomes on follow up at 8 weeks were also investigated. Median serum 25(OH) D concentrations were compared using Mann-Whitney test and covariates of serum VD status were assessed using logistic regression analysis. Results Overall VDD prevalence in the cohort was 40.9% (95% CI: 35.1–46.8). Median serum 25(OH)D concentrations were significantly higher in HIV+/PTB+ group (25.3 ng/ml, IQR:18.0–33.7) compared to the HIV+/PTB− group (20.4 ng/ml, IQR:14.6–26.9), p = 0.0003. Patients with serum 25(OH) D concentration ≥ 30 ng/ml were 1.9 times more likely to be PTB+ compared to those with serum 25(OH) D concentrations < 30 ng/ml (odds ratio (OR) 1.91; 95% CI 1.1–3.2). PTB-related death was associated with higher odds of having 25(OH) D levels≥30 ng/ml. Age, gender, CD4+ count, combination antiretroviral therapy (cART) status, efavirenz based cART regimen and length of hospital stay were not associated with vitamin D status. Conclusions The finding of an association between higher serum 25(OH) D concentrations and active PTB and TB-related mortality among hospitalised HIV-infected patients in the present study is at variance with the commonly reported association of hypovitaminosis and susceptibility to TB. Our findings though, are in concordance with a small pool of reports from other settings

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis.

BACKGROUND:Xpert MTB/RIF, the most widely used automated nucleic acid amplification test for tuberculosis, is available in more than 130 countries. Although diagnostic accuracy is well documented, anticipated improvements in patient outcomes have not been clearly identified. We performed an individual patient data meta-analysis to examine improvements in patient outcomes associated with Xpert MTB/RIF. METHODS:We searched PubMed, Embase, ClinicalTrials.gov, and the Pan African Clinical Trials Registry from inception to Feb 1, 2018, for randomised controlled trials (RCTs) comparing the use of Xpert MTB/RIF with sputum smear microscopy as tests for tuberculosis diagnosis in adults (aged 18 years or older). We excluded studies of patients with extrapulmonary tuberculosis, and studies in which mortality was not assessed. We used a two-stage approach for our primary analysis and a one-stage approach for the sensitivity analysis. To assess the primary outcome of cumulative 6-month all-cause mortality, we first performed logistic regression models (random effects for cluster randomised trials, with robust SEs for multicentre studies) for each trial, and then pooled the odds ratio (OR) estimates by a fixed-effects (inverse variance) or random-effects (Der Simonian Laird) meta-analysis. We adjusted for age and gender, and stratified by HIV status and previous tuberculosis-treatment history. The study protocol has been registered with PROSPERO, number CRD42014013394. FINDINGS:Our search identified 387 studies, of which five RCTs were eligible for analysis. 8567 adult clinic attendees (4490 [63·5%] of 7074 participants for whom data were available were HIV-positive) were tested for tuberculosis with Xpert MTB/RIF (Xpert group) versus sputum smear microscopy (sputum smear group), across five low-income and middle-income countries (South Africa, Brazil, Zimbabwe, Zambia, and Tanzania). The primary outcome (reported in three studies) occurred in 182 (4·5%) of 4050 patients in the Xpert group and 217 (5·3%) of 4093 patients in the smear group (pooled adjusted OR 0·88, 95% CI 0·68-1·14 [p=0·34]; for HIV-positive individuals OR 0·83, 0·65-1·05 [p=0·12]). Kaplan-Meier estimates showed a lower rate of death (12·73 per 100 person-years in the Xpert group vs 16·38 per 100 person-years in the sputum smear group) for HIV-positive patients (hazard ratio 0·76, 95% CI 0·60-0·97; p=0·03). The risk of bias was assessed as reasonable and the statistical heterogeneity across studies was low (I2&lt;20% for the primary outcome). INTERPRETATION:Despite individual patient data analysis from five RCTs, we were unable to confidently rule in nor rule out an Xpert MTB/RIF-associated reduction in mortality among outpatients tested for tuberculosis. Reduction in mortality among HIV-positive patients in a secondary analysis suggests the possibility of population-level impact. FUNDING:US National Institutes of Health