Early Bactericidal Activity Trial of Nitazoxanide for Pulmonary Tuberculosis.

This study was conducted in treatment-naive adults with drug-susceptible pulmonary tuberculosis in Port-au-Prince, Haiti, to assess the safety, bactericidal activity, and pharmacokinetics of nitazoxanide (NTZ). This was a prospective phase II clinical trial in 30 adults with pulmonary tuberculosis. Twenty participants received 1 g of NTZ orally twice daily for 14 days. A control group of 10 participants received standard therapy over 14 days. The primary outcome was the change in time to culture positivity (TTP) in an automated liquid culture system. The most common adverse events seen in the NTZ group were gastrointestinal complaints and headache. The mean change in TTP in sputum over 14 days in the NTZ group was 3.2 h ± 22.6 h and was not statistically significant (P = 0.56). The mean change in TTP in the standard therapy group was significantly increased, at 134 h ± 45.2 h (P < 0.0001). The mean NTZ MIC for Mycobacterium tuberculosis isolates was 12.3 µg/ml; the mean NTZ maximum concentration (Cmax) in plasma was 10.2 µg/ml. Negligible NTZ levels were measured in sputum. At the doses used, NTZ did not show bactericidal activity against M. tuberculosis Plasma concentrations of NTZ were below the MIC, and its negligible accumulation in pulmonary sites may explain the lack of bactericidal activity. (This study has been registered at ClinicalTrials.gov under identifier NCT02684240.).

Diagnostic yield of active case finding for tuberculosis at human immunodeficiency virus testing in Haiti.

SETTING: The Groupe Haïtien d'étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) Centres, Port-au-Prince, Haiti, facilitate "test and treat" strategies by screening all patients for tuberculosis (TB) at human immunodeficiency virus (HIV) testing.OBJECTIVE: 1) To determine the proportion of patients with chronic cough at HIV testing diagnosed with TB, stratified by HIV test results; and 2) to evaluate the additional diagnostic yield of Xpert® MTB/RIF vs. sputum microscopy.DESIGN: We conducted a retrospective cohort analysis including all adults tested for HIV at GHESKIO from August 2014 to July 2015.RESULTS: Of 29 233 adult patients tested for HIV, 2953 (10%) were diagnosed as HIV-positive. Chronic cough lasting ≥2 weeks was reported by 1116 (38%) HIV-positive patients; 984 (88%) were tested and 265 (27%) were diagnosed with TB. Chronic cough was reported by 5985 (23%) HIV-negative patients; 5654 (94%) were tested and 1179 (21%) were diagnosed with TB. Of all bacteriologically confirmed cases, 27% were smear-negative and Xpert-positive. Among all TB patients, 81% were HIV-negative.CONCLUSIONS: Screening for TB at HIV testing was high-yield, among both HIV-infected and HIV-negative individuals. Testing for both diseases should be conducted among patients who present with chronic cough at HIV testing.

Diagnostic yield of active case finding for tuberculosis and HIV at the household level in slums in Haiti.

SETTING: Haiti has the highest burden of tuberculosis (TB) in the Americas, with an estimated prevalence of 254 per 100 000 population. The Haitian Group for the Study of Kaposi's Sarcoma and Opportunistic Infections (Groupe Haïtien d'Etude du Sarcome de Kaposi et des Infections Opportunistes, GHESKIO) conducted active case finding (ACF) for TB at the household level in nine slums in Port-au-Prince. OBJECTIVE: We report on the prevalence of undiagnosed TB detected through GHESKIO's ACF campaign. DESIGN: From 1 August 2014 to 31 July 2015, we conducted a retrospective cohort analysis using GHESKIO's ACF campaign data. All individuals who reported chronic cough (cough 2 weeks) were tested for TB at GHESKIO, and those aged 10 years were included in the analyses. RESULTS: Of 104 097 individuals screened in the community, 5598 (5%) reported chronic cough and satisfied the study inclusion criteria. A total of 1110 (20%) were diagnosed with active TB disease (prevalence of 1066/100 000). Of the 5472 (98%) patients tested for human immunodeficiency virus (HIV), 528 (10%) were HIV-positive; 143 (3%) patients were diagnosed with both diseases. CONCLUSION: Household-level screening for cough with TB and HIV testing for symptomatic patients was a high-yield strategy, leading to the detection of a prevalence of undiagnosed disease exceeding national estimates by more than four-fold for TB, and by five-fold for HIV.

CD4 deficit and tuberculosis risk persist with delayed antiretroviral therapy: 5-year data from CIPRA HT-001.

SETTING: Port-au-Prince, Haiti. OBJECTIVE: To determine long-term effects of early vs. delayed initiation of antiretroviral therapy (ART) on immune recovery and tuberculosis (TB) risk in human immunodeficiency virus (HIV) infected individuals. DESIGN: Open-label randomized controlled trial of immediate ART in HIV-infected adults with CD4 counts between 200 and 350 cells/mm(3) vs. deferring ART until the CD4 count was <200 cells/mm(3). The primary comparisons were CD4 counts over time and risk for incident TB, with 5 years of follow-up. RESULTS: A total of 816 participants were enrolled, with 408 in each treatment arm. The early treatment group started ART within 2 weeks, while the deferred treatment group started ART a median of 1.3 years after enrollment. After 5 years, the mean CD4 count in the early treatment group was significantly higher than in the deferred treatment group (496 cells/mm(3), 95% confidence interval [CI] 477-515 vs. 373 cells/mm(3), 95%CI 357-389; P < 0.0001). TB risk was higher in the deferred treatment group (unadjusted HR 2.41, 95%CI 1.56-3.74; P < 0.0001) and strongly correlated with lower CD4 counts in time-dependent multivariate analysis. CONCLUSION: Delays in ART initiation for HIV-infected adults with CD4 counts of 200-350 cells/mm(3) can result in long-term immune dysfunction and persistent increased risk for TB.

Stepwise implementation of a new diagnostic algorithm for multidrug-resistant tuberculosis in Haiti.

SETTING: The uptake of tests endorsed by the World Health Organization to detect and appropriately confirm multidrug-resistant tuberculosis (MDR-TB) in low-income countries remains insufficient. OBJECTIVE: To validate the implementation of line-probe assays (LPA) and liquid culture to develop an algorithm to detect MDR-TB in the challenging setting of Haiti. METHODS: Through an EXPAND-TB (Expanding Access to New Diagnostics for TB) partnership, proficiency testing and validation of 221 acid-fast bacilli positive specimens were performed. Sensitivity, cost and processing time were analysed. RESULTS: Using liquid vs. solid culture shortened the turnaround time from 54 to 19 days, with a sensitivity of 100% vs. 98.6% and a total cost reduction of 13%. LPA detected all TB and MDR-TB cases at a lower cost than culture, in a mean time of 7.5 days. CONCLUSION: The combined use of molecular and liquid culture techniques accelerates the accurate diagnosis of TB and susceptibility testing against first-line drugs in a significantly shorter time, and is less expensive. The implementation of this new algorithm could significantly and accurately improve the screening and treatment follow-up of patients affected with TB and MDR-TB.

Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation.

Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes. In C3 plants, NADP-MDH is a part of the 'malate valve' that controls the export of reducing equivalents in the form of malate to the cytosol. NADP-MDH is completely inactive in the dark and is activated in the light with reduced thioredoxin. Compared with its permanently active NAD-linked counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide. Upon reduction of the C-terminal disulphide, the enzyme active site becomes accessible for the substrate. Reduction of the N-terminal disulphide promotes a conformational change advantageous for catalysis. To trace the evolutionary development of this intricate regulation mechanism, we isolated cDNA clones for NADP-MDH from the mossfern Selaginella and from two unicellular green algae. While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine pattern of the higher plant enzyme, the sequences from the green algae are devoid of the N-terminal regulatory disulphide. Phylogenetic analysis of new sequences and of those available in the databases led to the conclusion that the chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via duplication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages. Redox-sensitive NADP-MDH activity was detected only in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms. The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis.

A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1. 1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Cloning and sequence analysis of cDNAs encoding plant cytosolic malate dehydrogenase.

Here we report the first complete sequence of plant cytosolic malate dehydrogenase (EC 1.1.1.37). The phylogenetic relationships between malate dehydrogenases from different cell compartments are discussed. The constructed phylogenetic tree shows that cytosolic NAD-MDH and chloroplast NADP-MDH have evolved through gene duplication of the pre-existing nuclear gene.

Redox equilibria between the regulatory thiols of light/dark-modulated chloroplast enzymes and dithiothreitol: fine-tuning by metabolites.

Three light/dark-modulated chloroplast enzymes, namely NADP-dependent malate dehydrogenase (EC 1.1.1.82), D-fructose 1,6-bisphosphatase (EC 3.1.3.11), and phosphoribulokinase (EC 2.7.1.19) were purified to apparent homogeneity from spinach leaves. Equilibrium constants for the covalent modification of the regulatory disulfide bonds of these enzymes in dithiothreitol (DTT)-redox buffer were determined according to a previously published method in the literature (Clancey and Gilbert (1987) J. Biol. Chem. 262, 13545-13549). The thiol/disulfide-redox potential (Kox) was defined as the ratio of reduced to oxidized dithiothreitol at which 50% of the maximal enzyme activity was observed after equilibrium had been established. All Kox values were very high, comparable to those of extracellular disulfide containing proteins: 0.23 +/- 0.02 for NADP-malate dehydrogenase, 0.59 +/- 0.17 for phosphoribulokinase, and 0.70 +/- 0.16 for D-fructose 1,6-bisphosphatase. The equilibrium constants for the reactions between these enzymes and the redox buffers were also determined in the presence of various concentrations of specific metabolites known to influence the rates of reduction and oxidation. Increasing concentrations of D-fructose 1,6-bisphosphate in the presence of Ca2+ shift the equilibrium constant between D-fructose 1,6-bisphosphatase and the DTT-redox buffer to much lower values. A decreasing NADPH/(NADP + NADPH) ratio increases the Kox of NADP-malate dehydrogenase in the redox buffer to very high values. For PRK, low concentrations of ATP result in a slight decrease of the Kox that is not further affected by higher ATP concentrations. The differences of the equilibrium constants of NADP-malate dehydrogenase and D-fructose 1,6-bisphosphatase as dependent upon the NADPH/(NADP + NADPH) ratio and the concentration of D-fructose 1,6-bisphosphate, respectively, reflect a mechanism of feed-back and feed-forward regulation by the product NADP and the substrate D-fructose 1,6-bisphosphate, respectively. Thus the actual activation state of these two key enzymes of chloroplast metabolism are determined in an independent manner. The relatively small effect of the ATP concentration upon the redox potential of phosphoribulokinase indicates that fine-regulation at this step might be achieved on another level (e.g., catalysis or aggregation state).

Cysteines of chloroplast NADP-malate dehydrogenase form mixed disulfides.

Chloroplast NADP-malate dehydrogenase (NADP-MDH) from pea and from spinach was N-terminally truncated by limited proteolysis with Staphylococcus aureus protease V8. The resulting monomeric enzymes lacking, respectively, the 37 and 38 N-terminal amino acids were inactive. Reduction and addition of low concentrations of guanidine-HCl (50-100 mM) resulted in a highly active enzyme of 850 units per mg protein. Equilibration of the truncated enzyme with various glutathione (GSH) redox buffers and assaying its activity in the presence of guanidine-HCl was used to establish the existence of protein-GSH mixed disulfides. This finding was further confirmed using incorporation of radioactively labelled thiol. The possible function of such cysteine modifications under oxidative stress and their regeneration by the thioredoxin system in the light is discussed.

Effects of N-terminal truncations upon chloroplast NADP-malate dehydrogenases from pea and spinach.

Using the purification procedure of Fickenscher and Scheibe (Biochim. Biophys. Acta 749 (1983), 249-254) and a modification of the method, we produced a series of NADP-MDH forms from spinach and pea-leaf extracts that were characterized by a stepwise shortening of the N-terminal sequences. Limited proteolysis of the enzymes resulted in the generation of even shorter forms. Immunoprecipitation of the NADP-MDH from crude extracts revealed that the sequences of the intact enzymes from pea, spinach and maize started at a position (Ser) identical with that established for the Sorghum enzyme (Crétin, C., et al. (1990) Eur. J. Biochem. 192, 299-303). Spinach NADP-MDH isolated by conventional methods was shown to represent the intact form. Thus, the kinetic, regulatory and structural properties of the various truncated forms could be compared with those of an intact form. Removal of 5 or 11 amino acids, as occurred during isolation of the pea NADP-MDH, was without any significant effect. The enzymes were all dimeric and still exhibited the characteristic redox-regulatory properties. However, removal of 31 and 37 amino acids using aminopeptidase K resulted in the formation of active monomers characterized by only slightly lowered affinities towards the substrates, a shift of their pH optimum from 8 to 7, the loss of oxaloacetate inhibition and an increased maximal velocity. Although these forms lacked most or all of the N-terminal extra-peptide, including the 2 cysteines involved in redox-modification, they were still sensitive to the redox-potential. However, the low concentration of thiol required for immediate and complete restoration of any lost activity (40 mM beta-mercaptoethanol) suggested that this reaction might not be relevant for redox-regulation in vivo.