Search for Antimicrobial Activity Among Fifty-Two Natural and Synthetic Compounds Identifies Anthraquinone and Polyacetylene Classes That Inhibit Mycobacterium tuberculosis.

Drug-resistant tuberculosis threatens to undermine global control programs by limiting treatment options. New antimicrobial drugs are required, derived from new chemical classes. Natural products offer extensive chemical diversity and inspiration for synthetic chemistry. Here, we isolate, synthesize and test a library of 52 natural and synthetic compounds for activity against Mycobacterium tuberculosis. We identify seven compounds as antimycobacterial, including the natural products isobavachalcone and isoneorautenol, and a synthetic chromene. The plant-derived secondary metabolite damnacanthal was the most active compound with the lowest minimum inhibitory concentration of 13.07 µg/mL and a favorable selectivity index value. Three synthetic polyacetylene compounds demonstrated antimycobacterial activity, with the lowest MIC of 17.88 µg/mL. These results suggest new avenues for drug discovery, expanding antimicrobial compound chemistries to novel anthraquinone and polyacetylene scaffolds in the search for new drugs to treat drug-resistant bacterial diseases.

Whole genome sequencing of drug resistant Mycobacterium tuberculosis isolates from a high burden tuberculosis region of North West Pakistan.

Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81 M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG, ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA. Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18 M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulness of whole genome sequencing as a diagnostic tool for characterizing M. tuberculosis isolates, which will assist future epidemiological studies and disease control activities in Pakistan.

Performance of centralized versus decentralized tuberculosis treatment services in Southern Brazil, 2006-2015.

BACKGROUND: Tuberculosis (TB) control programs face the challenges of decreasing incidence, mortality rates, and drug resistance while increasing treatment adherence. The Brazilian TB control program recommended the decentralization of patient care as a strategy for combating the disease. This study evaluated the performance of this policy in an area with high default rates, comparing epidemiological and operational indicators between two similar municipalities. METHODS: This study analyzed epidemiological and operational indicators on new cases of pulmonary tuberculosis reported in the Brazilian Notifiable Diseases Information System between 2006 and 2015. In addition, to characterize differences between the populations of the two studied municipalities, a prospective cohort study was conducted between 2014 and 2015, in which patients with new cases of culture-confirmed pulmonary tuberculosis were interviewed and monitored until the disease outcome. A descriptive analysis, the chi-square test, and a Poisson regression model were employed to compare TB treatment outcomes and health care indicators between the municipalities. RESULTS: Two thousand three hundred nine cases were evaluated, of which 207 patients were interviewed. Over the 2006-2015 period, TB incidence per 100,000 population in the municipality with decentralized care was significantly higher (39%, 95% CI 27-49%) in comparison to that of the municipality with centralized care. TB treatment default rate (45%, 95% CI 12-90%) was also higher in the municipality with decentralized care. During the two-year follow-up, significant differences were found between patients in centralized care and those in decentralized care regarding treatment success (84.5 vs. 66.1%), treatment default (10.7 vs. 25.8%), illicit drug use (27.7 vs. 45.9%), and homelessness (3.6 vs. 12.9%). The operational indicators revealed that the proportion of control smear tests, medical imaging, and HIV tests were all significantly higher in the centralized care. However, a significantly higher proportion of patients started treatment in the early stages of the disease in the municipality with decentralized care. CONCLUSIONS: These data showed a low success rate in TB treatment in both municipalities. Decentralization of TB care, alone, did not improve the main epidemiological and operational indicators related to disease control when compared to centralized care. Full implementation of strategies already recommended is needed to improve TB treatment success rates.

Transcriptional Profiling Mycobacterium tuberculosis from Patient Sputa.

The emergence of drug resistance threatens to destroy tuberculosis control programs worldwide, with resistance to all first-line drugs and most second-line drugs detected. Drug tolerance (or phenotypic drug resistance) is also likely to be clinically relevant over the 6-month long standard treatment for drug-sensitive tuberculosis. Transcriptional profiling the response of Mycobacterium tuberculosis to antimicrobial drugs offers a novel interpretation of drug efficacy and mycobacterial drug-susceptibility that likely varies in dynamic microenvironments, such as the lung. This chapter describes the noninvasive sampling of tuberculous sputa and techniques for mRNA profiling M. tb bacilli during patient therapy to characterize real-world drug actions.

From stable disease to acute-on-chronic liver failure: Circulating cytokines are related to prognosis in different stages of cirrhosis.

INTRODUCTION: Although both pro- and anti-inflammatory circulating cytokines are known to be elevated in liver cirrhosis, its clinical significance is not completely recognized. Our aim was to evaluate the prognostic significance of circulating cytokines interleukin (IL)-6, IL-17 and IL-10 in different stages of cirrhosis. METHODS: This prospective study included two cohorts: (1) stable cirrhosis attended in the Outpatient Clinic (n=118), and (2) subjects hospitalized for acute decompensation (AD) (n=130). Thirty healthy subjects served as control group. RESULTS: Patients with cirrhosis exhibited higher levels of cytokines as compared to controls. In stable cirrhosis, during a median follow-up of 17months, liver-related events occurred in 26 patients. Higher IL-10 levels and Child-Pugh B/C were independently associated with reduced event-free survival. In AD cohort, death after 90days of follow-up occurred in 39 patients and was independently associated with ascites, higher IL-6 and model for end-stage liver disease. IL-6 levels also showed higher AUROC than CRP for predicting bacterial infection in the AD cohort (0.831±0.043vs. 0.763±0.048, respectively). IL-17 decreased at third day of hospitalization only in patients who progressed to death. Higher IL-6 levels were observed in acute-on-chronic liver failure (ACLF) patients even in the absence of bacterial infection whereas IL-10 was higher only in subjects with infection-related ACLF. Higher IL-10 and IL-17 levels were associated with progression to death in ACLF. CONCLUSIONS: The pattern of immune response seems to vary according to the phase of cirrhosis and is related to prognosis, from stable disease to ACLF.

Prognostic significance of insulin-like growth factor-I serum levels in acute decompensation of cirrhosis.

CONTEXT: IGF-I serum levels are suppressed in cirrhosis, but its prognostic significance is unknown. OBJECTIVES: To investigate the prognostic value of IGF-I in patients admitted for acute decompensation of cirrhosis. MATERIALS AND METHODS: Cohort study that included 103 patients. IGF-I was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Ninety-day mortality was 26.2% and it was independently associated with MELD, age and IGF-I. The Kaplan-Meier survival probability at 90 days was 94.3% in patients with IGF-I ≥13 ng/mL and 63.2% for patients with IGF-I <13 ng/mL (p = .001). DISCUSSION AND CONCLUSION: IGF-I levels are independently associated with mortality in acute decompensation of cirrhosis.

Circulating insulin-like growth factor-binding protein 3 as prognostic biomarker in liver cirrhosis.

AIM: To investigate the prognostic significance of insulin-like growth factor-binding protein 3 (IGFBP-3) in patients with cirrhosis. METHODS: Prospective study that included two cohorts: outpatients with stable cirrhosis (n = 138) and patients hospitalized for acute decompensation (n = 189). Development of complications, mortality or liver transplantation was assessed by periodical phone calls and during outpatient visits. The cohort of stable cirrhosis also underwent clinical and laboratory evaluation yearly (2013 and 2014) in predefined study visits. In patients with stable cirrhosis, IGFBP-3 levels were measured at baseline (2012) and at second re-evaluation (2014). In hospitalized subjects, IGFBP-3 levels were measured in serum samples collected in the first and in the third day after admission and stored at -80 °C. IGFBP-3 levels were measured by immunochemiluminescence. RESULTS: IGFBP-3 levels were lower in hospitalized patients as compared to outpatients (0.94 mcg/mL vs 1.69 mcg/mL, P < 0.001) and increased after liver transplantation (3.81 mcg/mL vs 1.33 mcg/mL, P = 0.008). During the follow-up of the stable cohort, 17 patients died and 11 received liver transplantation. Bivariate analysis showed that death or transplant was associated with lower IGFBP-3 levels (1.44 mcg/mL vs 1.74 mcg/mL, P = 0.027). The Kaplan-Meier transplant-free survival probability was 88.6% in patients with IGFBP-3 ≥ 1.67 mcg/mL and 72.1% for those with IGFBP3 < 1.67 mcg/mL (P = 0.015). In the hospitalized cohort, 30-d mortality was 24.3% and was independently associated with creatinine, INR, SpO2/FiO2 ratio and IGFBP-3 levels in the logistic regression. The 90-d transplant-free survival probability was 80.4% in patients with IGFBP-3 ≥ 0.86 mcg/mL and 56.1% for those with IGFBP3 < 0.86 mcg/mL (P < 0.001). CONCLUSION: Lower IGFBP-3 levels were associated with worse outcomes in patients with cirrhosis, and might represent a promising prognostic tool that can be incorporated in clinical practice.

The performance of prognostic models as predictors of mortality in patients with acute decompensation of cirrhosis.

BACKGROUND: Although several prognostic models have been proposed for cirrhotic patients listed for transplantation, the performance of these scores as predictors of mortality in patients admitted for acute decompensation of cirrhosis has not been satisfactorily investigated. AIMS: To study MELD, MELD-Na, MESO, iMELD, Refit-MELD and Refit MELD-Na models as prognostic predictors in cirrhotic patients admitted for acute decompensation, and to compare their performance between admission and 48 hours of hospitalization to predict in-hospital mortality. MATERIAL AND METHODS: This cohort study included cirrhotic patients admitted to hospital due to complications of the disease. Individuals were evaluated on admission and after 48 h of hospitalization, and mortality was evaluated during the present admission. RESULTS: One hundred and twenty-three subjects with a mean age of 54.26 ± 10.79 years were included; 76.4% were male. Mean MELD score was 16.43 ± 7.08 and 52.0% of patients were Child-Pugh C. Twenty-seven patients (22.0%) died during hospitalization. Similar areas under the curve (AUROCs) for prognosis of mortality were observed when different models were compared on admission (P > 0.05) and after 48 h of hospitalization (P > 0.05). When models executed after 48 h of hospitalization were compared to their corresponding model calculated on admission, significantly higher AUROCs were obtained for all models (P < 0.05), except for MELD-Na (P = 0.075) and iMELD (P = 0.119). CONCLUSION: The studied models showed similar accuracy as predictors of in-hospital mortality in cirrhotic patients admitted for acute decompensation. However, the performance of these models was significantly better when applied 48 h after admission when compared to their calculation on admission.

Factors associated with 25-hydroxyvitamin D levels in patients with liver cirrhosis.

INTRODUCTION: Lower 25-hydroxyvitamin D [25(OH)D] levels have been observed in cirrhotic patients and have been related to disease severity. However, most previous studies included patients with very advanced disease, lacking an adequate control for other variables that could interfere with vitamin D levels. We sought to investigate the prevalence of hypovitaminosis D and the factors related to its occurrence. MATERIAL AND METHODS: This cross-sectional study included 133 cirrhotic patients and 30 healthy controls. Bivariate and multivariate analyses were performed to determine factors associated with 25(OH)D levels below the lower tertile. Thirty patients who had been recently hospitalized were compared in two time points. RESULTS: Mean 25(OH)D levels were 32.34 ± 11.38 in controls and 27.03 ± 6.22 ng/mL in patients (P = 0.018). 25(OH)D levels were < 30 ng/mL in 69.9% and < 20 ng/mL in 14.3% of the sample. Levels of 25(OH)D below the lower tertile (< 24 ng/mL) were independently associated with higher triceps skinfold and non-Caucasian race. Parathyroid hormone above the reference value (65 pg/mL) was found in 24.6% of patients without association with 25(OH)D or severity of liver disease. Significantly lower levels of 25(OH)D were found at the time of acute decompensation of cirrhosis. CONCLUSIONS: In conclusion, hypovitaminosis D was prevalent in cirrhotics and it was associated with adiposity and non-Caucasian race in stable patients with relatively well preserved liver function. However, significantly lower levels were observed during admission for acute decompensation suggesting an impact of systemic inflammation or liver dysfunction on 25(OH)D levels.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria.

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Micobactérias: epidemiologia e diagnóstico / Mycobacteria: epidemiology and diagnosis

Estima-se que um terço da população mundial esteja infectada com Mycobacterium tuberculosis, oque resulta em 2 milhões de mortes anualmente. No mundo, são registrados mais de 8 milhões de novos casos de tuberculose (TB) por ano e o Brasil ocupa o décimo nono lugar dentre os 23 países detentores da maior carga de TB. Os fatores determinantes para o controle desta doença incluem a detecção rápida, a terapia adequada e os meios para que sejam evitadas futuras transmissões. O diagnósticoconvencional (baciloscopia e cultura do microrganismo) apresenta limitações quanto ao tempo de execução e à operacionalidade, visto que o resultado pode levar até 60 dias para ser liberado.Portanto, a importância da detecção precoce do bacilo se torna fundamental para o bloqueio da cadeia de transmissão da TB. Já as micobacterioses, doenças causadas pelas micobactérias não tuberculosas, também estão provocando um importante impacto em razão do aumento dos surtos de infecções cirúrgicas. A identificação rápida e específica destes microrganismos é importante para o diagnóstico e a consequente escolha do tipo de tratamento do paciente, que está diretamente relacionada com a espécie. Portanto, o conhecimento dos agentes etiológicos das doenças causadaspelas micobactérias e o diagnóstico sensível e específico permitem o tratamento adequado e,consequentemente, o bloqueio da cadeia de transmissão da TB e o controle dos surtos relacionados às micobactérias não tuberculosas.

It is estimated that one third of the world population is infected with Mycobacterium tuberculosis, resulting in 2 million deaths annually. More than 8 million new cases of tuberculosis (TB) are registered per year worldwide, and Brazil ranks 19th among the top 23 countries with the highest rates of TB. The determining factors for the control of this disease include early detection, appropriate therapy and measuresfor avoiding transmission. The conventional diagnoses (smear and microorganism culture) have time limitations for implementation and operation, since the result may take up to 60 days to be released. Therefore, early detection is critical for blockingthe chain of TB transmission. Mycobacterial diseases caused by nontuberculous mycobacteria are also having a major impact due to increased outbreaks of surgical infections. Thereby, the rapid and specific identification of microorganisms is important for the diagnosis, which will determine the type of treatment (treatment according to species). Knowledge of etiologic agents of mycobacterial diseases, as well as sensitive and specific diagnosis allows proper treatment by blocking the chain of TB transmission and controlling nontuberculous mycobacteria outbreaks.