Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil

New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Deciphering the contribution of lipid droplets in leprosy: multifunctional organelles with roles in Mycobacterium leprae pathogenesis

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves, presenting a singular clinical picture. Across the leprosy spectrum, lepromatous leprosy (LL) exhibits a classical hallmark: the presence of a collection of M. leprae-infected foamy macrophages/Schwann cells characterised by their high lipid content. The significance of this foamy aspect in mycobacterial infections has garnered renewed attention in leprosy due to the recent observation that the foamy aspect represents cells enriched in lipid droplets (LD) (also known as lipid bodies). Here, we discuss the contemporary view of LD as highly regulated organelles with key functions in M. leprae persistence in the LL end of the spectrum. The modern methods of studying this ancient disease have contributed to recent findings that describe M. leprae-triggered LD biogenesis and recruitment as effective mycobacterial intracellular strategies for acquiring lipids, sheltering and/or dampening the immune response and favouring bacterial survival, likely representing a fundamental aspect of M. leprae pathogenesis. The multifaceted functions attributed to the LD in leprosy may contribute to the development of new strategies for adjunctive anti-leprosy therapies.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis(Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.