Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Resistencia a la meticilina y producción de biopelícula en aislamientos clínicos de Staphylococcus aureus y Staphylococcus coagulasa negativa en México / Methicillin resistance and biofilm production in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus in México

Resumen Introducción. Las infecciones por Staphylococcus aureus y Staphylococcus coagulasa negativa multirresistentes a los antibióticos y asociadas con la atención en salud tienen un gran impacto epidemiológico por su alta morbimortalidad; además, se han relacionado con la formación de biopelículas, lo cual también se asocia con la resistencia a los antimicrobianos. Objetivo. Determinar la resistencia a la meticilina y cuantificar la producción de biopelículas para establecer su posible relación con los aislamientos clínicos de S. aureus y Staphylococcus coagulasa negativa. Materiales y métodos. Se estudiaron 11 cepas de S. aureus y 12 de Staphylococcus coagulasa negativa. La resistencia a la meticilina se determinó con discos de cefoxitina tomando como valores de referencia los estándares del Clinical Laboratory Standards Institute (CLSI) de 2018. La producción de biopelícula se cuantificó con cristal violeta. Los genes mecA e icaADBC se identificaron mediante reacción en cadena de la polimerasa (PCR), y se hizo un análisis bivariado con la prueba de ji al cuadrado y el coeficiente V de Cramér, utilizando el programa SPSS™, versión 20.0. Resultados. Nueve cepas de S. aureus fueron resistentes a la meticilina (SARM) y dos fueron sensibles. Ocho cepas de Staphylococcus coagulasa negativa fueron resistentes y cuatro fueron sensibles. El genotipo mecA se encontró en ocho de las nueve cepas de S. aureus y en seis de las ocho de Staphylococcus coagulasa negativa resistentes a meticilina. Todas las cepas formaron biopelícula. Diez cepas de S. aureus y 11 de Staphylococcus coagulasa negativa presentaron el genotipo icaADCB. No se encontró asociación entre la resistencia a meticilina y la formación de biopelícula. Conclusiones. La cefoxitina es suficiente para determinar el fenotipo resistente a meticilina y se asoció con el genotipo mecA. Las cepas resistentes a la meticilina y poseedoras del gen mecA pueden presentar un mecanismo de resistencia alterno. Los dos grupos de cepas formadoras de biopelícula se relacionaron con la presencia del operón icaADCB. La formación de biopelícula y la resistencia a la meticilina se expresaron como características independientes en los dos grupos de cepas.

Abstract Introduction: Infections associated with health care caused by S. aureus and coagulase- negative Staphylococci multi-resistant to antibiotics cause a high epidemiological impact due to their high morbidity and mortality. Biofilm formation, which has been associated with antimicrobial resistance, can also occur. Objectives: To determine methicillin resistance and to quantify the biofilm production to establish if there is a relationship in clinical isolates of S. aureus and coagulase-negative Staphylococci. Material and methods: A total of 11 strains of S. aureus and 12 of coagulase-negative Staphylococci were studied. Methicillin resistance was determined with cefoxitin discs and the Clinical Laboratory Standards Institute (CSLI), 2018 reference values. Biofilm production was quantified by the crystal violet method. The mecA and icaADBC genes were identified by PCR. A bivariate analysis was performed with chi-square (c2) and Cramér's V statistical tests, using SPSS™, version 20.0 software. Results: Nine S. aureus strains were methicillin-resistant and two were sensitive. Eight coagulase-negative Staphylococci strains were resistant and four were sensitive. The mecA genotype was found in eight of the nine S. aureus resistant strains and six of eight resistant coagulase-negative Staphylococci. All strains formed biofilms. Ten strains of S. aureus and 11 of coagulase-negative Staphylococci presented the icaADCB genotype. No association was found between methicillin-resistance and biofilm formation. Conclusions: Cefoxitin is enough to define the resistance phenotype and is associated with the mecA genotype. All strains formed biofilms and were related to the presence of the icaADCB operon. Biofilm formation and methicillin resistance were independent features in both groups of strains.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Comparison of five methods of extraction of Staphylococcus aureus DNA for molecular detection by PCR

Abstract INTRODUCTION Molecular techniques for the detection of pathogens have been shown to be effective diagnostic tools with high sensitivity and short turnaround times. METHODS This study compared five Staphylococcus aureus DNA extraction methods for detection by the polymerase chain reaction. RESULTS: The concentration and purity of the extracted DNA showed that the methods did not yield DNA of significant quality. However, most protocols yielded 100% positivity, even with low DNA concentrations. CONCLUSIONS: Although one protocol seemed more efficient than the others, PCR was sensitive enough to allow for detection of S. aureus with all the protocols.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Preanalytical conditions can interfere with M. tuberculosis detection by PCR in respiratory samples

OBJECTIVES: Tuberculosis is one of the most prevalent infections in humans. Although culture is the reference for diagnosis, its sensitivity is compromised, especially in paucibacillary samples. Because polymerase chain reaction (PCR) amplifies mycobacterial DNA, it is more sensitive than culture for the diagnosis of Mycobacterium tuberculosis (Mtb). However, its performance can be affected by intrinsic sample inhibitors and by the extraction/detection techniques used. METHODS: We evaluated the influence of preanalytical conditions on Mtb detection in samples of sputum (SPU), bronchoalveolar lavage (BAL), and pleural fluid (PF) using combinations of extraction/detection methods. Respiratory samples were prepared to contain different concentrations of red blood cells and nucleated cells to which increasing amounts of Mtb colonies were inoculated and submitted to PCR. RESULTS: Up to 102 CFU/ml of Mtb were detected in the SPU in all methods, except for the Roche extraction/detection method, regardless of the preanalytical sample condition. In BAL samples, medium and high concentrations of cells and high concentrations of red blood cells contributed to a lower Mtb detection, regardless of the extraction method used. In PF, red blood cells were the variable that most interfered with Mtb detection, with better recovery (102 CFU/ml) observed with the Qiagen/Nanogen combination. CONCLUSION: The choice of Mtb extraction and detection method is of fundamental importance for PCR analytical sensitivity, especially when paucibacillary samples and/or samples containing potential PCR inhibitors are analyzed.

Diagnóstico molecular de Porphyromonas gingivalis: optimización del método / Molecular diagnosis of Porphyromonas gingivalis: optimization of the method

El objetivo del presente trabajo fue estandarizar y optimizar la técnica de PCR convencional para detección de Porphyromonas gingivalis ATCC 33277. Materiales y métodos: la cepa de P. gingivalis ATCC332227 se sembró en agar Bruella enriquecido con sangre de cordero, suplementado con hemina y vitamina K. El ADN se extrajo empleando el protocolo que usa bromuro de cetil trimetilamonio (CTAB). Se evaluó la cantidad y calidad del material genético obtenido con el fotómetro UV Ampli-Quat, AQ-07 Nucleic Acid. Se realizó la PCR convencional con diferentes concentraciones de MgCl2 1 mM, 1,5 mM y 2.0 mM y a dos temperaturas de alineamiento: 60ºC y 55ºC. Los productos PCR se separaron por electroforesis en un gel de agarosa 1 por ciento. Las bandas se visualizaron en un fotodocumentador. La sensibilidad se calculó teniendo en cuenta el número de bacterias en diferentes diluciones. Resultados: se obtuvo una concentración 1,55x10(6) ng/ul de ADN genómico a partir de una suspensión bacteriana de 10a células bacterianas/ml, con índice de pureza 1,648 (relación de OD260/OD280). Los mejores resultados se obtuvieron con una concentración de 2 mM de MgCl2 y una temperatura de alineación de 55ºC. En cuanto a la sensibilidad, se obtuvo un límite de detección de 5 x 10/5 uL células bacterianas en suspensión. Conclusión: en la prueba de PCR convencional para Prophyromonas gingivalis ATCC 33277, las condiciones óptimas de estandarización son la concentración de 2 mM de MgCl y 55ºC y es necesaria una carga bacteriana mínima de 5 x 10 células/5 ul como límite de detección.

A duplex endpoint PCR assay for rapid detection and differentiation of leptospira strains

Abstract INTRODUCTION: This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. METHODS: Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. RESULTS: The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. CONCLUSIONS: Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Influence of storage time on DNA of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae for accurate detection by quantitative real-time polymerase chain reaction

The shipment and storage conditions of clinical samples pose a major challenge to the detection accuracy of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) when using quantitative real-time polymerase chain reaction (qRT-PCR). The aim of the present study was to explore the influence of storage time at 4°C on the DNA of these pathogens and its effect on their detection by qRT-PCR. CT, NG, and UU positive genital swabs from 70 patients were collected, and DNA of all samples were extracted and divided into eight aliquots. One aliquot was immediately analyzed with qRT-PCR to assess the initial pathogen load, whereas the remaining samples were stored at 4°C and analyzed after 1, 2, 3, 7, 14, 21, and 28 days. No significant differences in CT, NG, and UU DNA loads were observed between baseline (day 0) and the subsequent time points (days 1, 2, 3, 7, 14, 21, and 28) in any of the 70 samples. Although a slight increase in DNA levels was observed at day 28 compared to day 0, paired sample t-test results revealed no significant differences between the mean DNA levels at different time points following storage at 4°C (all P>0.05). Overall, the CT, UU, and NG DNA loads from all genital swab samples were stable at 4°C over a 28-day period.

Bacterial diversity of symptomatic primary endodontic infection by clonal analysis

Abstract The aim of this study was to explore the bacterial diversity of 10 root canals with acute apical abscess using clonal analysis. Samples were collected from 10 patients and submitted to bacterial DNA isolation, 16S rRNA gene amplification, cloning, and sequencing. A bacterial genomic library was constructed and bacterial diversity was estimated. The mean number of taxa per canal was 15, ranging from 11 to 21. A total of 689 clones were analyzed and 76 phylotypes identified, of which 47 (61.84%) were different species and 29 (38.15%) were taxa reported as yet-uncultivable or as yet-uncharacterized species. Prevotella spp., Fusobacterium nucleatum, Filifactor alocis, and Peptostreptococcus stomatis were the most frequently detected species, followed by Dialister invisus, Phocaeicola abscessus, the uncharacterized Lachnospiraceae oral clone, Porphyromonas spp., and Parvimonas micra. Eight phyla were detected and the most frequently identified taxa belonged to the phylum Firmicutes (43.5%), followed by Bacteroidetes (22.5%) and Proteobacteria (13.2%). No species was detected in all studied samples and some species were identified in only one case. It was concluded that acute primary endodontic infection is characterized by wide bacterial diversity and a high intersubject variability was observed. Anaerobic Gram-negative bacteria belonging to the phylum Firmicutes, followed by Bacteroidetes, were the most frequently detected microorganisms.

Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India

Abstract Yellow pigmented, filamentous, Gram-negative bacteria belonging to genus Flavobacterium are commonly associated with infections in stressed fish. In this study, inter-species diversity of Flavobacterium was studied in apparently healthy freshwater farmed fishes. For this, ninety one yellow pigmented bacteria were isolated from skin and gill samples (n = 38) of three farmed fish species i.e. Labeo rohita, Catla catla and Cyprinus carpio. Among them, only twelve bacterial isolates (13.18%) were identified as Flavobacterium spp. on the basis of morphological, biochemical tests, partial 16S rDNA gene sequencing and phylogenetic analysis. On the basis of 16S rDNA gene sequencing, all the 12 isolates were 97.6-100% similar to six different formally described species of genus Flavobacterium. The 16S rDNA based phylogenetic analysis grouped these strains into six different clades. Of the 12 isolates, six strains (Fl9S1-6) grouped with F. suncheonense, two strains (Fl6I2, Fl6I3) with F. indicum and the rest four strains (Fl1A1, Fl2G1, Fl3H1 and Fl10T1) clustered with F. aquaticum, F. granuli, F. hercynium and F. terrae, respectively. None of these species except, F. hercynium were previously reported from fish. All the isolated Flavobacterium species possessed the ability of adhesion and biofilm formation to colonize the external surface of healthy fish. The present study is the first record of tropical freshwater farmed fishes as hosts to five environmentally associated species of the Flavobacterium.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current enotyping procedures: a population-based study in northeast Mexico

The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman’s rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

Prevalence of Pseudomonas aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with chronic periodontal infection

P. aeruginosa and Acinetobacter spp. are important pathogens associated with late nosocomial pneumonia in hospitalized and institutionalized individuals. The oral cavity may be a major source of these respiratory pathogens, particularly in the presence of poor oral hygiene and periodontal infection. This study investigated the prevalence of P. aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with periodontal disease or health. Samples were obtained from 55 periodontally healthy (PH) and 169 chronic periodontitis (CP) patients. DNA was obtained from the samples and detection of P. aeruginosa and Acinetobacter spp. was carried out by multiplex and nested PCR. P. aeruginosa and Acinetobacter spp. were detected in 40% and 45% of all samples, respectively. No significant differences in the distribution of these microorganisms between men and women, subgingival biofilm and saliva samples, patients < 35 and > 35 years of age, and smokers and non-smokers were observed regardless periodontal status (p > 0.05). In contrast, the frequencies of P. aeruginosa and Acinetobacter spp. in saliva and biofilm samples were significantly greater in CP than PH patients (p < 0.01). Smokers presenting P. aeruginosa and high frequencies of supragingival plaque were more likely to present CP than PH. P. aeruginosa and Acinetobacter spp. are frequently detected in the oral microbiota of CP. Poor oral hygiene, smoking and the presence of P. aeruginosa are strongly associated with periodontitis.

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province.