New anti-SARS-CoV-2 aminoadamantane compounds as antiviral candidates for the treatment of COVID-19.

Here, the antiviral activity of aminoadamantane derivatives were evaluated against SARS-CoV-2. The compounds exhibited low cytotoxicity to Vero, HEK293 and CALU-3 cells up to a concentration of 1,000 µM. The inhibitory concentration (IC50) of aminoadamantane was 39.71 µM in Vero CCL-81 cells and the derivatives showed significantly lower IC50 values, especially for compounds 3F4 (0.32 µM), 3F5 (0.44 µM) and 3E10 (1.28 µM). Additionally, derivatives 3F5 and 3E10 statistically reduced the fluorescence intensity of SARS-CoV-2 protein S from Vero cells at 10 µM. Transmission microscopy confirmed the antiviral activity of the compounds, which reduced cytopathic effects induced by the virus, such as vacuolization, cytoplasmic projections, and the presence of myelin figures derived from cellular activation in the face of infection. Additionally, it was possible to observe a reduction of viral particles adhered to the cell membrane and inside several viral factories, especially after treatment with 3F4. Moreover, although docking analysis showed favorable interactions in the catalytic site of Cathepsin L, the enzymatic activity of this enzyme was not inhibited significantly in vitro. The new derivatives displayed lower predicted toxicities than aminoadamantane, which was observed for either rat or mouse models. Lastly, in vivo antiviral assays of aminoadamantane derivatives in BALB/cJ mice after challenge with the mouse-adapted strain of SARS-CoV-2, corroborated the robust antiviral activity of 3F4 derivative, which was higher than aminoadamantane and its other derivatives. Therefore, aminoadamantane derivatives show potential broad-spectrum antiviral activity, which may contribute to COVID-19 treatment in the face of emerging and re-emerging SARS-CoV-2 variants of concern.

Serum biomarkers in patients with unilateral or bilateral active pulmonary tuberculosis: Immunological networks and promising diagnostic applications.

The present observational study was designed to characterize the integrative profile of serum soluble mediators to describe the immunological networks associated with clinical findings and identify putative biomarkers for diagnosis and prognosis of active tuberculosis. The study population comprises 163 volunteers, including 84 patients with active pulmonary tuberculosis/(TB), and 79 controls/(C). Soluble mediators were measured by multiplexed assay. Data analysis demonstrated that the levels of CCL3, CCL5, CXCL10, IL-1ß, IL-6, IFN-γ, IL-1Ra, IL-4, IL-10, PDGF, VEGF, G-CSF, IL-7 were increased in TB as compared to C. Patients with bilateral pulmonary involvement/(TB-BI) exhibited higher levels of CXCL8, IL-6 and TNF with distinct biomarker signatures (CCL11, CCL2, TNF and IL-10) as compared to patients with unilateral infiltrates/(TB-UNI). Analysis of biomarker networks based in correlation power graph demonstrated small number of strong connections in TB and TB-BI. The search for biomarkers with relevant implications to understand the pathogenetic mechanisms and useful as complementary diagnosis tool of active TB pointed out the excellent performance of single analysis of IL-6 or CXCL10 and the stepwise combination of IL-6 â†’ CXCL10 (Accuracy = 84 %; 80 % and 88 %, respectively). Together, our finding demonstrated that immunological networks of serum soluble biomarkers in TB patients differ according to the unilateral or bilateral pulmonary involvement and may have relevant implications to understand the pathogenetic mechanisms involved in the clinical outcome of Mtb infection.

Sociodemographic and Clinical Factors Associated with Treatment Outcomes for Drug-resistant Tuberculosis.

Drug-resistant tuberculosis (DR-TB) continues to be a serious public health problem. The objective of this study was to evaluate the sociodemographic, radiological, clinical, and outcome characteristics and assess the determinants of unfavorable outcomes in DR-TB. The descriptive-analytical study was carried out in a reference outpatient clinic in Rio de Janeiro, Brazil, among DR-TB cases that received treatment between February 2016 and October 2020, using descriptive statistics, χ2 test, and logistic regression multivariate. Of the 148 cases, 12.2% were resistant to rifampicin, 12.2% were resistant to isoniazid, 18.2% were polyresistant, 56.1% multidrug resistant, and 1.3% were extensively drug resistant. Most of the patients were men, aged up to 44 years, with brown or black skin, having up to 8 years of schooling, unemployed or working in the informal economy, and of low income. Presenting with acquired resistance or positive sputum smear microscopy in the diagnosis, taking more than four drugs, and being unemployed were associated with unfavorable outcomes. Having no income or acquired resistance doubled the chances of unfavorable outcomes. There was a high proportion of unfavorable outcomes, thereby highlighting the need to concentrate efforts on planning and executing public policies that include the severity of DR-TB and its risk factors.

Cost analysis for patients with presumed pulmonary tuberculosis attended in the public health system of rio de janeiro, Brazil.

Background: In last years, few attention has given to the patient's prediagnostic costs when evaluating the introduction of new technologies for tuberculosis (TB) and in this context, this study evaluated patient's costs and cost-effectiveness incurred with TB diagnosis comparing BactecTMMGITTM960 system (MGIT) to the Löwestein-Jensen (LJ) culture in a health center and in a university hospital, in Rio de Janeiro City, Brazil. Methods: Patient's mean costs were evaluated during the diagnosis process and cost-effectiveness based on mean time in days for the adoption of appropriate clinical anti-TB treatment in two health units comparing culture by means LJ and MGIT. Results: The mean cost of LJ and MGIT in the health center was U. S. dollars (US$) 26.6 and US$ 45.13, respectively, and in university hospital was US$ 206.87 and US$ 285.48, respectively. Comparing the two approaches for TB diagnosis incurred by the patients, the incremental cost-effectiveness of MGIT compared to LJ was US$ 0.88 and US$ 4.03 per patient, respectively, to reduce the average time to adopt appropriate treatment. Conclusions: The culture method directly impacts patient costs while waiting for the correct diagnosis and contributing to aggravating costs with patients with TB.

Characterization of Mycobacterium tuberculosis heteroresistance by genotyping.

Background: Heteroresistance is the coexistence of susceptible and resistant strains in the same individual, considered the preliminary step for total resistance, and can stem from mixed infection or clonal heterogeneity. The aim of this study was to evaluate the heteroresistance of Mycobacterium tuberculosis to rifampicin and isoniazid and its characterization. Method: GenoType MTBDRplus®; Sanger sequencing of the rpoB, katG, and inhA genes; and Mycobacterial Interspersed Repetitive Unit - Variable Number Tandem Repeat (MIRU-VNTR) were performed. Results: In a total of 654 isolates, 530 were resistant, 124 were susceptible, and 29 were heteroresistant to a first-line drug. GenoType MTBDRplus® detected heteroresistance in the rpoB gene in 26/29 (89.6%), as compared to 5/29 (17.2%) in the katG gene and 2/29 (6.8%) in the inhA gene. Four isolates showed heteroresistance in these genes. The Sanger sequencing detected heteroresistance in the rpoB gene in 7/29 (24.1%), as compared to 3/29 (10.3%) in the katG gene. In one isolate, heteroresistance was concomitant in both the rpoB and katG genes. MIRU-VNTR detected mixed infection in three heteroresistant isolates, while four isolates showed clonal heterogeneity. Conclusions: GenoType MTBDRplus® detected more cases of heteroresistance when compared to sequencing. It was also possible to characterize mixed infection and clonal heterogeneity by MIRU-VNTR.

The activity-based cost of drug-susceptibility test of Mycobacterium tuberculosis through Kit SIRE Nitratase® Plastlabor.

Background: Drug-resistant tuberculosis (TB) is an ongoing health threat, and the greatest challenge to adequate control of TB in many countries lies in the lack of proper laboratory drug-susceptibility test. The aim of this study was to evaluate the activity-based costs (ABC) of Kit SIRE Nitratase® (Kit SIRE) and compare its values with the conventional drug-susceptibility test. Methods: The ABC was calculated for three different approaches: Kit SIRE (clinical samples and cultures), proportion methods in Lowenstein Jensen (PM-LJ), and the Bactec™ MGIT™ 960 system based on Mycobacterial Research Laboratory's routine. Results: The ABC of Kit SIRE from cultures was US$ 148.54, while from clinical samples was US$ 136.12. In the case of conventional tests, the ABC of Bactec™ MGIT™ 960 was US$ 227.63 and of the PM-LJ was US$ 132.64. The Kit SIRE has a lower ABC when clinical samples are used instead of cultures. Compared to conventional tests, the ABC is similar to the PM-LJ and lower the Bactec™ MGIT™ 960. Conclusion: The Kit SIRE should be used as a screening method in clinical specimens and in culture for laboratories that do not have Bactec™ MGIT™ 960. Therefore, it can be incorporated into the routine of laboratories in countries with low resources and a high burden of TB and drug-resistant TB.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides.

BACKGROUND: Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES: To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS: Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS: The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS: qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Mapping the tuberculosis scientific landscape among BRICS countries: a bibliometric and network analysis.

BACKGROUND: The five BRICS (Brazil, Russian, Indian, China, and South Africa) countries bear 49% of the world's tuberculosis (TB) burden and they are committed to ending tuberculosis. OBJECTIVES: The aim of this paper is to map the scientific landscape related to TB research in BRICS countries. METHODS: Were combined bibliometrics and social network analysis techniques to map the scientific publications related to TB produced by the BRICS. Was made a descriptive statistical data covering the full period of analysis (1993-2016) and the research networks were made for 2007-2016 (8,366 records). The bubble charts were generated by VantagePoint and the networks by the Gephi 0.9.1 software (Gephi Consortium 2010) from co-occurrence matrices produced in VantagePoint. The Fruchterman-Reingold algorithm provided the networks' layout. FINDINGS: During the period 1993-2016, there were 38,315 peer-reviewed, among them, there were 11,018 (28.7%) articles related by one or more authors in a BRICS: India 38.7%; China 23.8%; South Africa 21.1%; Brazil 13.0%; and Russia 4.5% (The total was greater than 100% because our criterion was all papers with at least one author in a BRICS). Among the BRICS, there was greater interaction between India and South Africa and organisations in India and China had the highest productivity; however, South African organisations had more interaction with countries outside the BRICS. Publications by and about BRICS generally covered all research areas, especially those in India and China covered all research areas, although Brazil and South Africa prioritised infectious diseases, microbiology, and the respiratory system. MAIN CONCLUSIONS: An overview of BRICS scientific publications and interactions highlighted the necessity to develop a BRICS TB research plan to increase efforts and funding to ensure that basic science research successfully translates into products and policies to help end the TB epidemic.

Multicenter evaluation of TB-SPRINT 59-Plex Beamedex®: accuracy and cost analysis.

BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.

Frequency of the Mycobacterium tuberculosis RDRio genotype and its association with multidrug-resistant tuberculosis.

BACKGROUND: In recent decades, Mycobacterium tuberculosis with the RDRio genotype, frequently isolated from tuberculosis patients in Rio de Janeiro, has become part of the Latin American - Mediterranean (LAM) family and has been associated with multidrug-resistant tuberculosis (MDR-TB). The aim of this study was to investigate the frequency of M. tuberculosis RDRio in the state of Minas Gerais, Brazil, and its relationship with MDR-TB. METHODS: For convenience, 172 susceptible and 63 MDR M. tuberculosis isolates were taken from pulmonary samples from patients diagnosed between January 2007 and December 2011. The DNA extracted from these isolates was analyzed by spoligotyping, PCR-RFLP to characterize fbpC103/Ag85C103, multiplex PCR to detect RDRio and RD174, and MIRU-VNTR 24 loci. RESULTS: Among the 235 isolates, the RDRio pattern was identified in 122 (51.9%) isolates (IC 0.45-0.58), with 100 (42.5%) wild-type and 13 (5.5%) mixed pattern isolates, whereas RD174 was identified in 93 of the 122 RDRio positive samples (76.3%). The LAM family and the LAM9 lineage were the most frequently identified among the isolates in this study. Among the 63 MDR isolates, 41 (65.1%) were RDRio and 28 (44.4%) RD174. CONCLUSION: The association of both deletions with MDR proved to be statistically significant, corroborating the few reports that have associated RDRio with MDR.

Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil.

BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.

In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety.

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.

In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety. / In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety.

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3

, 100

, 100

and 89.7

, respectively whereas accuracy was 90.7

. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.

Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: preliminary study.

BACKGROUND: Developments in molecular detection and strain differentiation of members of Mycobacterium tuberculosis complex have proved to be useful. The DNA extraction method influences the amplification efficiency, causing interference on the sensitivity and respective inhibitors. The aim of this study was to standardize a simple and fast DNA extraction method, providing DNA amplification by IS6110-PCR effectively free from undue interferences. FINDINGS: The efficiency of the six different protocols tested in M. tuberculosis cultures has varied from 75% to 92.5%. This preliminary study evaluating the IS6110 PCR sensitivity and specificity was developed in DNA extracted from microscope slides, and achieved 100% of efficiency. CONCLUSIONS: DNA extraction by Chelex + NP-40 method from both, cultures of M. tuberculosis and smear slides, resulted in good quantity of interference free DNA, especially in samples with low concentrations of genetic material; therefore, such technique may be used for the molecular diagnosis of tuberculosis.