Bioactive alkaloids from the venom of Dendrobatoidea Cope, 1865: a scoping review.

Bioactive compounds derived from secondary metabolism in animals have refined selectivity and potency for certain biological targets. The superfamily Dendrobatoidea is adapted to the dietary sequestration and secretion of toxic alkaloids, which play a role in several biological activities, and thus serve as a potential source for pharmacological and biotechnological applications. This article constitutes a scoping review to understand the trends in experimental research involving bioactive alkaloids derived from Dendrobatoidea based upon scientometric approaches. Forty-eight (48) publications were found in 30 journals in the period of 60 years, between 1962 and 2022. More than 23 structural classes of alkaloids were cited, with 27.63% for batrachotoxins, 13.64% for pyridinics, with an emphasis on epibatidine, 16.36% for pumiliotoxins, and 11.82% for histrionicotoxins. These tests included in vivo (54.9%), in vitro (39.4%), and in silico simulations (5.6%). Most compounds (54.8%) were isolated from skin extracts, whereas the remainder were obtained through molecular synthesis. Thirteen main biological activities were identified, including acetylcholinesterase inhibitors (27.59%), sodium channel inhibitors (12.07%), cardiac (12.07%), analgesic (8.62%), and neuromuscular effects (8.62%). The substances were cited as being of natural origin in the "Dendrobatidae" family, genus "Phyllobates," "Dendrobates," and seven species: Epipedobates tricolor, Phyllobates aurotaenia, Oophaga histrionica, Oophaga pumilio, Phyllobates terribilis, Epipedobates anthonyi, and Ameerega flavopicta. To date, only a few biological activities have been experimentally tested; hence, further studies on the bioprospecting of animal compounds and ecological approaches are needed.

Two names, one species: redescription and phylogenetic position of Schrankiana formosula Freitas, 1959 provides new insights into the evolutionary history of the Cosmocercidae.

Schrankiana are gastrointestinal parasites of anurans, distributed throughout Central and South America. Schrankiana formosula and Schrankiana inconspicata are some of the most commonly reported species parasitising anurans from Brazil, and the morphological differences between them are unclear. In the present study, we redescribed S. formosula based on a re-examination of type series and newly collected material from the frog Leptodactylus pentadactylus in the state of Amapá, Brazil. Additionally, we re-examined the type series of S. inconspicata, and propose it as a junior synonym of S. formosula. We provide detailed morphological and morphometric data with intraspecific variation analyses and new molecular data for S. formosula. In the present phylogeny, S. formosula formed a well-supported clade with Raillietnema sp. and Labeonema synodontisi. Based on molecular phylogenetic analyses and some morphological similarities, our findings support the hypothesis that Schrankiana is a member of the family Cosmocercidae, not Atractidae. Additionally, we provide the first ultrastructural descriptions of S. formosula, and establish the species' phylogenetic position from the Cosmocercidae.

Morphological and molecular characterization of Cosmocercoides amapari n. sp. (Nematoda: Cosmocercidae), parasitic in hylid frogs from the Brazilian Amazon.

Cosmocercoides Wilkie, 1930 are gastrointestinal parasites commonly found in amphibians and reptiles, with 4 species reported from the Neotropical region. In the present study, a new species of Cosmocercoides, namely Cosmocercoides amapari n. sp. is described using integrated approaches such as light and scanning microscopy, and DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. The specimens were collected from the large intestine of 3 species of hylid frogs in Amapá, Brazil. The new species can be distinguished from its congeners by morphological traits, including the pattern of caudal papillae, absence of the gubernaculum, 2 poorly sclerotized spicules, presence of lateral alae and somatic papillae along the body. In addition, our molecular analyses and phylogenetic reconstructions strongly support the status of the new taxon, which clustered poorly with a large clade of Cosmocerca spp. Cosmocercoides amapari n. sp. is the 29th species of the genus, the 5th from the Neotropical region, the third reported in Brazil, the second described from the Amazon region and the first belonging to the Neotropical region with molecular data.

Effect of Vehicle Composition on the Preparation of Different Types of Dapsone Crystals for Topical Drug Delivery.

Topical formulations composed of API-pure crystals have been increasingly studied, especially in regards to the impact of particle size in penetration efficiency. Less attention, however, has been devoted to the solid-state properties of drugs delivered to the skin. In this study, we address the effect of formulation composition on the crystal form existing in topical products. Dapsone (DAP) gel formulations were prepared by mixing an organic solution containing DAP with an aqueous solution containing polymers and preservatives. The organic solvent was chosen as ethoxydiglycol (DEGEE), polyethylene glycol (PEG), or 1-methyl-2-pirrolidone (MPR) to assess the impact of composition on DAP crystal form. Such solvent variations resulted in different particulate matter. In terms of crystalline nature, the presence of DEGEE in formulations induced the crystallization of DAP hydrate, while PEG cocrystal and a mixture of hydrate and MPR solvate crystallized from the same amounts of PEG and MPR, respectively. Microscopic analysis of the gels showed heterogeneous particles with different characteristics. The behavior of gels after application to the skin was also tested. Interestingly, the different formulations seemed to accumulate in different regions of the skin. This could be the result of the effect of vehicle composition/excipients on the characteristics of the skin, such as hydration. The site-specific accumulation, however, was more pronounced in crystal-loaded gels as opposed to blank formulations. These results indicate that future studies should consider the effect of formulation composition on the API crystal form landscape as part of the strategies used to successfully target drug delivery to the skin.

A New Saquinavir Mesylate-Sodium Decyl Sulfate Salt Discovered by Serendipity during an Anomalous Dissolution Test.

PURPOSE: To understand the anomalous behavior of Saquinavir Mesylate (SQVM) in sodium decyl sulfate (SDS) medium during a dissolution test through a crystallographic analysis of the crystal obtained. As a result, it will be possible to elucidate its crystal structure and carry out a complete solid-state characterization of the API. METHODS: The solid form obtained was characterized by a structural analysis through X-ray single crystal and powder diffraction. The crystallographic structures of the new salt and the SQVM were compared. In addition, a complete solid-state characterization of SQVM raw material was carried out by techniques such as diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), Raman spectroscopy, scanning electron microscopy and a dissolution method. RESULTS: A new salt consisting of SQVM and SDS was crystallized and its crystal structure was elucidated and reported herein for the first time. The anionic part of SDS interacts with the cationic segment of SQVM to obtain a new salt designated as SQV-DS, which precipitates. The main difference between the two structures occurs in the c-axis expansion, which increases from 15.966 (5) to 21.1924 (14), respectively. CONCLUSIONS: Some of the strategies to enhance the dissolution rate of poorly aqueous soluble APIs include the use of surfactants such as SDS in the dissolution medium, as well as in the formulated products. However, there have been constant reports of a dissolution rate slowdown by some surfactants. The interaction mechanisms between the APIs and the dissolution medium containing surfactants need to be carefully investigated in current pharmaceutical formulations. Graphical Abstract.

A new species of Rhabdias (Nematoda: Rhabdiasidae), a lung parasite of Pristimantis chiastonotus (Anura: Strabomantidae) from the Brazilian Amazon: description and phylogenetic analyses.

Rhabdias Stiles and Hassal, 1905 comprises approximately 90 species of parasitic lung nematodes of amphibians and reptiles that have a wide distribution, with 21 species occurring in the Neotropics. In the present study, we describe Rhabdias waiapi n. sp. found parasitizing the lungs of the anuran species Pristimantis chiastonotus from the Amazon Biome in the Amapá State, Northern Brazil. The new species is characterized by having an elongated body, expansions of the cuticular inflation in the anterior end that become more discrete along the body, an anterior end with a slight constriction at the level of the esophageal apex with four rounded subapical elevations of the body wall, six lips, four near the edge of the oral opening and two more distant lateral ones, and a gradually tapering elongated tail. In addition, molecular analyses and phylogenetic reconstructions were made, with sequences from the coding region of the mitochondrial cytochrome c oxidase subunit I gene. Those results strongly support the status of the new taxon, which formed a poorly supported clade with Rhabdias sp. 5 from Anolis brasiliensis from Northeast Brazil. Rhabdias waiapi n. sp. is the 19th species of the genus described in the Neotropics for amphibians, the 10th in Brazil, the second described from hosts of the family Strabomantidae from the Neotropical region, and the first amphibian nematode species described in the Amapá State.

Sensitive simultaneous voltammetric determination of the herbicides diuron and isoproturon at a platinum/chitosan bio-based sensing platform.

Phenylurea herbicides are persistent contaminants, which leads their transport to the surface and ground waters, affecting human and aquatic organisms. Different analytical methods have been reported for the detection of phenylureas; however, several of them are expensive, time-consuming, and require complex pretreatment steps. Here, we show a simple method for the simultaneous electrochemical determination of two phenylurea herbicides by differential pulse adsorptive stripping voltammetry (DPAdSV) using a modified platinum/chitosan electrode. The one-step synthesized platinum/chitosan PtNPs/CS was successfully characterized by TEM, XRPD, and FT-IR, and applied through the sensing platform designated as PtNPs/CS/GCE. This bio-based modified electrode is proposed for the first time for the individual and/or simultaneous electrochemical detection of the phenylurea herbicides diuron and isoproturon compounds extensively used worldwide that present a very similar chemical structure. Electrochemical and interfacial characteristics of the modified electrode were evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). It was found that the oxidation mechanism of diuron and isoproturon occurs in two different pathways, with a peak-to-peak definition of ca. 0.15 V. Under differential pulse adsorptive stripping voltammetry (DPAdSV) optimized conditions, the limit of detection (LOD) was estimated as 7 µg L-1 for isoproturon and 20 µg L-1 for diuron (Ed = +0.8 V; td = 100 s). The proposed method was successfully applied to the determination of both analytes in river water samples, at three different levels, with a recovery range of 90-110%. The employment of the bio-based sensing platform PtNPs/CS/GCE allows a novel and easy analytical method to the multi-component phenylurea herbicides detection.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity.

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Detection of Rickettsia spp. in ticks parasitizing toads (Rhinella marina) in the northern Brazilian Amazon.

This study evaluated rickettsial infection in ticks collected on toads from the northern Brazilian Amazon (Amapá state), where to our knowledge there are neither records of ticks from amphibians nor rickettsial infections in ticks. During 2016-2017, a total of 22 free-living toads were captured and identified as Rhinella marina. Overall, 12 (54.5%) toads were parasitized by a total of 97 ticks (6 males, 39 females, 31 nymphs, 21 larvae), giving a mean intensity of 8.1 ticks per infested toad. Two tick species were morphologically identified: Amblyomma rotundatum Koch (31 females, 14 nymphs), and Amblyomma dissimile Koch (6 males, 8 females, 17 nymphs). The 21 larvae were morphologically denoted as Amblyomma sp. Five toads were co-infested by A. rotundatum and A. dissimile. Morphological identifications were confirmed by nucleotide sequencing of fragments of the mitochondrial genes 16S rDNA, 12S rDNA and/or COX1. A total of 54 ticks were analyzed for the presence of rickettsial DNA. Eleven (9 females and 2 nymphs) out of 14 A. rotundatum ticks contained Rickettsia bellii. None of the 25 specimens of A. dissimile (6 males, 6 females, 13 nymphs) contained amplifiable rickettsial DNA. From 15 Amblyomma sp. larvae, a pool of 10 individuals contained Rickettsia sp. strain Colombianensi. Sequencing of the 16S rDNA amplicon derived from the positive pool yielded a sequence of A. dissimile. We detected Rickettsia sp. strain Colombianensi for the first time in Brazil. Prior records of this agent were restricted to Colombia and Honduras. In addition, we report the presence of A. rotundatum for the first time in the state of Amapá, where the only other record of A. dissimile was registered over 20 years ago.

Restoration Effects of the Riparian Forest on the Intertidal Fish Fauna in an Urban Area of the Amazon River.

Urbanization causes environmental impacts that threaten the health of aquatic communities and alter their recovery patterns. In this study, we evaluated the diversity of intertidal fish in six areas affected by urbanization (areas with native vegetation, deforested areas, and areas in process of restoration of vegetation) along an urban waterfront in the Amazon River. 20 species were identified, representing 17 genera, 14 families, and 8 orders. The different degrees of habitat degradation had a major effect on the composition of the fish fauna; the two least affected sectors were the only ones in that all 20 species were found. Eight species were recorded in the most degraded areas. The analysis revealed two well-defined groups, coinciding with the sectors in better ecological quality and degraded areas, respectively. The native vegetation has been identified as the crucial factor to the recovery and homeostasis of the studied ecosystem, justifying its legal protection and its use in the restoration and conservation of altered and threatened environments. These results reinforce the importance of maintaining the native vegetation as well as its restoration in order to benefit of the fish populations in intertidal zones impacted by alterations resulting from inadequate urbanization.

Crystallization of progesterone polymorphs using polymer-induced heteronucleation (PIHn) method.

Progesterone is a natural hormone steroid used in humans for several treatments and in livestock for artificial insemination, which exhibits two polymorphic forms at ambient conditions: form 1 and form 2. Form 2 is metastable and more soluble than form 1; however, it is not suitable to use as powder raw material because it transforms into form 1 by the effects of grinding. A polymorphic screening of progesterone based on polymer-induced heteronucleation method was performed as an alternative to prepare the metastable form. Polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC), dextran, gelatin, polyisoprene (PI) and acrylonitrile-butadiene (NBR) copolymer were used. Crystals were prepared from 0.5, 10 and 40 mg/mL solutions in acetone at room temperature by solvent evaporation. The samples were characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), scanning electron microcopy and attenuated total reflectance infrared Fourier transform spectroscopy. Form 1 was nucleated from 40 mg/mL solutions on the six polymers and from 10 mg/mL solutions on PI and NBR. The mixture of form 1 and form 2 was obtained from 10 mg/mL solution on HPMC, dextran and gelatin and from 0.5 mg/mL solution crystallizations. Therefore, the polymeric devices, which crystallized the metastable and more soluble polymorph (2) of progesterone, would be a promissory alternative for the pharmaceutical applications.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.

Dissolution properties, solid-state transformation and polymorphic crystallization: progesterone case study.

Progesterone is a natural steroid hormone and a poor soluble drug which presents two polymorphs (forms 1 and 2). Different methods to obtain form 2 were tested and a complete solid-state characterization of both polymorphs (forms 1 and 2) was conducted. X-ray powder diffraction, hot stage microscopy, Fourier transform infrared, dispersive Raman, (13)C solid-state nuclear magnetic resonance spectroscopy, thermal analysis, scanning electron microscopy techniques and intrinsic dissolution rates (IDR) were applied to investigate physical-chemical and dissolution properties of these two polymorphs. Form 2 was obtained from diluted solutions and from melting after cooling at room temperature. Form 1 was obtained from concentrated solutions and, a mixture of both polymorphs was crystallized from intermediate solutions. The crystal habit was not a distinctive characteristic of each polymorph. The effect of mechanical stress was evaluated in the metastable polymorph (form 2). We observed that grinding form 2 produced seeds of form 1 that induced the transformation of form 2 into form 1 at high temperature. The polymorphic quantification from XRD patterns of ground samples were carried out by the Rietveld method. After grinding and at room temperature conditions (∼25 °C), it was observed the transformation of 17% of form 2 into form 1 in 10 days.

Ticks parasitizing cold-blooded animals from three different Brazilian biomes; with note for males of Amblyomma rotundatum.

Ticks parasitize a wide variety of wild animals, including amphibians and reptiles. In addition to the possibility of microorganism transmission to these hosts, ticks can also cause severe bleeding, and high parasitism can lead to death. Therefore, knowing the diversity of ticks parasitizing amphibians and reptiles is important for conservation and preservation measures for these vertebrates. In the present study, we report parasitism by ticks in amphibians and reptiles from different Brazilian biomes (Amazon, Caatinga, Cerrado and Atlantic Forest). Ticks were collected from amphibians and reptiles deposited from the Herpetological Collection of the Federal University of Maranhão (UFMA), São Luís (Maranhão State), the State University Santa Cruz (UESC), Ilhéus (Bahia State), and the Federal University of São Francisco Valley (Univasf), Petrolina (Pernambuco State). Additionally, ticks were collected from amphibians and reptiles captured and road-killed in the Amazon biome, at Maranhão and Amapá States. Specimens of ticks were photographed under a Zeiss stereomicroscope (5.1 zoom). Map with the locations were made using the Qgis program. Overall, 1973 specimens of amphibians and reptiles were examined. A total of 927 ticks were collected: 98 larvae, 421 nymphs and 408 adults. Six species of ticks were identified: Amblyomma rotundatum and Amblyomma dissimile the most frequent, and Amblyomma cajennense sensu stricto, Amblyomma sculptum, Amblyomma nodosum and Amblyomma humerale, occasionally. Surprisingly, a total of twelve males of A. rotundatum were collected. Here we report new records of association between cold-blooded animals and ticks and reinforce the absence of A. dissimile in the Caatinga, Cerrado and Atlantic Forest biomes. Additionally, we report new records of A. rotundatum males on reptiles in the Amazon biome. This last record allows us to speculate about a possible association of A. rotundatum males with reptiles and the Amazon biome.

Ophiotaenia karipuna n. sp. (Eucestoda: Proteocephalidae), a parasite of Erythrolamprus miliaris (Linnaeus, 1758), with redescription of Ophiotaenia arandasi (Santos and Rolas, 1973) from the Brazilian Amazon.

Ophiotaenia is the most diverse genus among proteocephalids, composed of species commonly found parasitizing snakes worldwide. However, the diversity of Ophiotaenia in the Neotropical region is still underestimated. This study describes Ophiotaenia karipuna n. sp. found parasitizing Erythrolamprus miliaris in the State of Amapá, Brazil. Additionally, we redescribe Ophiotaenia arandasi based on a re-examination of type series and newly collected material from Erythrolamprus taeniogaster in the State of Pará, Brazil. The new species differs from its congeners in the following characteristics: scolex width, number of testes, relative length of the cirrus-sac, absence of a vaginal sphincter, presence of a vestigial apical organ resembling a sucker, and relative size of the ovary in relation to the surface of the proglottid. Furthermore, we provide taxonomic information for Ophiotaenia arandasi not reported in the original description, including morphology of embryophore, uterine development, and the absence of the vaginal sphincter in the species. Ophiotaenia karipuna n. sp. represents the 21st- species described in snakes -from the Neotropical region and the first formally described in the Brazilian Amazon. Additionally, we provide the first ultrastructural analysis, a new host, and locality records for O. arandasi.

Mycobacterium fragae sp. nov., a non-chromogenic species isolated from human respiratory specimens.

Three isolates of a slow-growing, non-chromogenic mycobacterium were grown from three sputum samples of a patient from the north-eastern Ceará state in Brazil. Identification at species level could not be obtained with PCR restriction analysis of the hsp65 gene. In order to characterize the isolates we carried out phenotypic and genotypic tests. We sequenced the nearly complete 16S rRNA gene and obtained partial sequences of the hsp65 (encoding the hypervariable region of the 65 kDa heat-shock protein) and rpoB (encoding the beta-subunit of RNA polymerase) genes. The three isolates turned out to be identical and most closely related to the species Mycobacterium celatum and Mycobacterium kyorinense. The results, however, showed significant differences between these species and the isolates studied, which led us to consider them members of a novel species for which we propose the name Mycobacterium fragae. The type strain is HF8705(T) ( = Fiocruz-INCQS/CMRVS P4051(T) = DSM 45731(T)).

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondônia, Brazil.

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

A new species of Aplectana Railliet & Henry, 1916 (Nematoda: Cosmocercidae) in the Brazilian Amazon and the taxonomic status of Aplectana longa.

Nematodes of the genus Aplectana Railliet & Henry, 1916 are common parasites of the digestive tract of amphibians and reptiles in the Neotropical region. During a parasite survey on Boana boans (Linnaeus, 1758), we found specimens of nematodes with Aplectana characteristics. We observed a set of characteristics that differs the species of our study from its congeners, and the present study describes a new species of Aplectana parasite of B. boans using light microscopy and scanning electron microscopy. Aplectana pella n. sp. has lateral alae and somatic papillae in males and females; males have equal short spicules, and the gubernaculum is absent. The arrangement of pairs of caudal papillae also differs from other species (2 precloacal,1 adcloacal, and 5 postcloacal+1 unpaired). In females, the vulva is simple, with non-prominent lips, and equatorial. This is the first record of the genus Aplectana parasitizing B. boans and the 58th species described for this genus. Additionally, we added the precloacal papillae pattern of A. delirae, and based on morphological and morphometric characteristics, we propose the reallocation of Aplectana longa to the genus Oxyascaris.

First isolation of Mycobacterium kyorinense from clinical specimens in Brazil.

In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.

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.