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Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and, in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae–Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M. abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1–2 nt differences in rpoB and internal transcribed spacer (ITS) sequences. Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values >70 % confirmed that the five isolates belong to the same species, while values M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results, demonstrated that they share characteristics with M. chelonae–M. abscessus members, but constitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM 10906T ( = CCUG 66554T = LMG 28586T = INCQS 0733T).

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoBin RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S–23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to ‘Mycobacterium franklinii’ DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae–Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA–DNA hybridization demonstrated that ‘M. franklinii’ DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae–M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524T ( = ATCC BAA-2149T) is the type strain.

Purpose Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. Methods In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. Results Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. Conclusions Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

The Tuberculosis (TB) notification rates are 5 to 81 times higher in prisons worldwide when compared to the general population. The state of Santa Catarina (SC) has few epidemiological data regarding TB in prisons. The aim of this study was to evaluate the molecular epidemiology of circulating strains in prisons of SC. The study comprised 95 clinical samples from six prisons. Among the cases included, all subjects were male, predominantly caucasians, and young adults, with low education level. The positive smear in the TB diagnosis comprised 62.0% of cases. About 50% of subjects had some condition associated with TB. The Spoligotyping results showed that the most frequent lineages were LAM (50.7%), T (22.2%) and S (11.6%). The 12-loci MIRU generated 62 different genotypes. The MSTs showed evolutionary relationships between Mycobacterium tuberculosis spoligotypes from SC and evolutionary relationships between the prison isolates and studied parameters. This first study on TB in prison units of SC highlighted the predominance of SIT216/LAM5, and SIT34/S. Interestingly, his profile was found to be different from that observed in a previous study performed with the state's general population. This data shows the need for continued surveillance of episodes of TB occurring among prison inmates in an emerging country like Brazil.

Isolates of the Mycobacterium chelonae-M. abscessus complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA (R) Mycobacterium spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as M. chelonae (14 isolates), M. franklinii (2 isolates) and M. salmoniphium (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that rpoB alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the M. chelonae-M. abscessus complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.

Objectives The availability of point-of-care (POC) tests for infectious diseases has revolutionised the provision of healthcare for remote rural populations without access to laboratories. However, quality assurance for POC tests has been largely overlooked. We have evaluated the use and stability of dry tube specimens (DTS) for External Quality Assurance (EQA) for HIV and syphilis screening in remote indigenous populations in the Amazon region of Brazil. Methods All healthcare workers (HCWs) participating in the community-screening were trained. We used HIV and syphilis DTS panels developed by the reference laboratory, containing samples with negative and positive results at different antibody concentrations, for both infections. DTS panels were distributed to HCWs in the communities for reconstitution and testing using POC HIV and syphilis tests. The results of testing were sent to the reference laboratory for marking and remedial action taken where necessary. Results In total 268 HCWs tested 1607 samples for syphilis and 1608 samples for HIV. Results from HCWs showed a concordance rate of 90% for syphilis and 93% for HIV (κ coefficients of 0.74 and 0.78, respectively) with reference laboratories. Most false negatives were in samples of very low antibody concentration. DTS syphilis specimens produced the expected results after storage at 2–8°C or at 18–24°C for up to 3 weeks. Conclusions The results show that POC tests for syphilis and HIV give valid results in environments where traditional tests do not work, but errors in the interpretation of POC test results were identified by the EQA programme using DTS. EQA using DTS can help to improve the quality of screening programmes using POC tests in remote regions.

Introduction. Bacterial infection is a frequent complication in patients with decompensated liver cirrhosis and is related to high mortality rates during follow-up of these individuals. We sought to evaluate the diagnostic value of C-reactive protein (CRP) and procalcitonin (PCT) in diagnosing infection and to investigate the relationship between these biomarkers and mortality after hospital admission.Material and methods. Prospective study that included cirrhotic patients admitted to the hospital due to complications of the disease. The diagnostic accuracy of CRP and PCT for the diagnosis of infection was evaluated by estimating the sensitivity and specificity and by measuring the area under the receiver operating characteristics curve (AUROC).Results. A total of 64 patients and 81 hospitalizations were analyzed during the study. The mean age was 54.31 ± 11.87 years with male predominance (68.8%). Significantly higher median CRP and PCT levels were observed among infected patients (P < 0.001). The AUROC of CRP and PCT for the diagnosis of infection were 0.835 ± 0.052 and 0.860 ± 0.047, respectively (P = 0.273). CRP levels > 29.5 exhibited sensitivity of 82% and specificity of 81% for the diagnosis of bacterial infection. Similarly, PCT levels > 1.10 showed sensitivity of 67% and specificity of 90%. Significantly higher levels of CRP (P = 0.026) and PCT (P = 0.001) were observed among those who died within three months after admission.Conclusion. CRP and PCT were reliable markers of bacterial infection in subjects admitted due to complications of liver cirrhosis and higher levels of these tests are related to short-term mortality in those patients.

Background. IGF-I and IGFBP-3 are part of IGF system and, due to their predominantly hepatic synthesis, they seem to correlate with hepatic dysfunction intensity. Aims. To investigate the significance of IGF-I and IGFBP-3 in patients with decompensated liver disease. Material and methods. Cross-sectional study that included cirrhotic patients admitted to hospital due to complications of the disease, in whom IGF-I and IGFBP-3 serum levels were measured by chemiluminescence. Results. Seventy-four subjects with a mean age of 53.1 ± 11.6 years were included in the study, 73% were males. IGF-I levels were positively correlated with IGFBP-3 and albumin, and negatively correlated with Child-Pugh, MELD, creatinine, INR and aPTT ratio. IGFBP-3 levels were positively correlated with IGF-I and albumin, and negatively correlated with Child-Pugh, MELD, creatinine, INR, total bilirubin and aPTT ratio. Significantly lower scores of IGF-I and IGFBP-3 were observed in patients with higher MELD values and higher Child-Pugh classes (P < 0.05). Conclusions. In cirrhotic patients admitted to hospital due to complications of the disease, IGF-I and IGFBP-3 serum levels were associated with variables related to liver dysfunction and to more advanced liver disease. The levels of these markers seem to undergo little influence from other clinical and laboratory variables, therefore mainly reflecting hepatic functional status.

The interaction between ghrelin and adiponectin is still controversial. We investigated the effect of cafeteria diet and pioglitazone on body weight, insulin resistance, and adiponectin/ghrelin levels in an experimental study on male Wistar rats. The animals were divided into four groups of 6 rats each, and received balanced chow with saline (CHOW-O) or pioglitazone (CHOW-P), or a cafeteria diet with saline (CAFE-O) or pioglitazone (CAFE-P). The chow/cafeteria diets were administered for 35 days, and saline/pioglitazone (10 mg · kg body weight(-1) · day(-1)) was added in the last 14 days prior to euthanasia. CAFE-O animals had a higher mean final weight (372.5 ± 21.01 g) than CHOW-O (317.66 ± 25.11 g, P = 0.017) and CHOW-P (322.66 ± 28.42 g, P = 0.035) animals. Serum adiponectin levels were significantly higher in CHOW-P (55.91 ± 20.62 ng/mL) than in CHOW-O (30.52 ± 6.97 ng/mL, P = 0.014) and CAFE-O (32.54 ± 9.03 ng/mL, P = 0.027) but not in CAFE-P. Higher total serum ghrelin levels were observed in CAFE-P compared to CHOW-P animals (1.65 ± 0.69 vs 0.65 ± 0.36 ng/mL, P = 0.006). Likewise, acylated ghrelin levels were higher in CAFE-P (471.52 ± 195.09 pg/mL) than in CHOW-P (193.01 ± 87.61 pg/mL, P = 0.009) and CAFE-O (259.44 ± 86.36 pg/mL, P = 0.047) animals. In conclusion, a cafeteria diet can lead to a significant weight gain. Although CAFE-P animals exhibited higher ghrelin levels, this was probably related to food deprivation rather than to a direct pharmacological effect, possibly attenuating the increase in adiponectin levels.

Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria.

Molecular epidemiology of Mycobacterium tuberculosis is useful for understanding disease transmission dynamics, and to establish strategic measures for TB control and prevention. The aim of this study was to analyze clinical, epidemiological and molecular characteristics of MTBC clinical isolates from Santa Catarina state, southern Brazil. During one-year period, 406 clinical isolates of MTBC were collected from Central Laboratory of Public Health and typed by spoligotyping. Demographic and clinical data were collected from the Brazilian National Mandatory Disease Reporting System. The majority of cases occurred in highest population densities regions and about 50% had some condition associated with TB. Among all isolates, 5.7% were MDR, which showed association with drug addiction. LAM was the most predominant lineage with 47.5%, followed by the T superfamily with 25.9% and Haarlem with 12.3%. The MST showed two major groups: the first was formed mainly by the LAM lineage and the second was mainly formed by the T and Haarlem lineages. Others lineages were distributed in peripheral positions. This study provides the first insight into the population structure of M. tuberculosis in SC State. Spoligotyping and other genotyping analyses are important to establish strategic measures for TB control and prevention.

Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller–Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller–Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3–5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000–0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000–0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.

BACKGROUND Tuberculosis (TB), one of the major airborne infectious bacterial diseases, remains an important health problem worldwide. It is estimated that there are 1700 new cases per year in Santa Catarina State, Brazil. OBJECTIVE To improve polymerase chain reaction (PCR) sensitivity in detecting Mycobacterium tuberculosis in sputum samples. METHODS This study proposed the use of glass beads as a modification of the routine protocol for sputum preparation used in the Laboratory of Molecular Biology and Mycobacteria at the Federal University of Santa Catarina, Florianopolis, Brazil. The study comprised 120 sputum samples, 60 of which were treated with the routine protocol, while 60 were treated with the modified protocol using glass beads. RESULTS Samples treated with the routine protocol had a sensitivity of 56.7% (95%CI 44.1-69.2) in 16S rRNA PCR and 81.7% (95%CI 71.9-91.5) in insertion sequence (IS) 6110 PCR, compared with culture. Samples treated with the modified protocol had a sensitivity of 73.3% (95%CI 62.1-84.5) in 16S rRNA PCR and 100% in IS6110 PCR. CONCLUSION The modified protocol using glass beads greatly improved mycobacterial detection in sputum samples compared with the routine protocol.

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 measures for 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 blocking the 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.

Volume 52, no. 5, p. [1727–1729][1], 2014. Page 1727: Figure 1 contains gel images that were included in a previous publication from our group (S. C. Leao et al., PLoS One 8 (4):e60746, 2013, ). They were adapted and reproduced in the figure for