Species identification of Enterococcus spp: Whole genome sequencing compared to three biochemical test-based systems and two Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) systems.

AIM: Here, we evaluated the performance of two commercial MALDI-TOF MS systems and three biochemical-based systems and compared them to WGS as the gold standard for identifying isolates of vancomycin-resistant enterococci (VRE). METHODS: A total of 87 VRE clinical isolates were included. The mass spectrometers were the Microflex system with Biotyper software 3.1 and the Vitek MS system. The biochemical-based systems included the Vitek 2, Phoenix, and MicroScan WalkAway systems. WGS was performed on an Illumina MiSeq instrument using the MiSeq v3 reagent kit. Vancomycin resistance was determined according to CLSI criteria. RESULTS: Among the 87 VRE, 71 and 16 were identified as Enterococcus faecium and Enterococcus faecalis by WGS. All 71 E faecium were correctly identified by both mass spectrometers, as well as the Vitek 2 and Phoenix instruments. However, only 51 E faecium isolates were correctly identified by the MicroScan system. The most frequent misidentification was Enterococcus casseliflavus (n = 20). For vancomycin-resistant E faecium, the Microflex Biotyper system had the highest sensitivity (85.54%), and all instruments (except for the Microscan) had a 100% specificity and PPV. Up to 87% of E faecalis isolates were misidentified by VITEK MS and VITEK2, 81% by Microscan and Phoenix, and 75% by Bruker biotyper. CONCLUSION: As the coverage of type strain-genome sequence database continues to grow and the cost of DNA sequencing continues to decrease, genome-based identification can be a useful tool for diagnostic laboratories, with its superior accuracy even over MALDI-TOF and database-driven operations.

Comparison of standard and on-plate extraction protocols for identification of mastitis-causing bacteria by MALDI-TOF MS.

The objective was to compare standard versus on-plate sample preparation protocols for identification of mastitis bacteria by MALDI-TOF MS. A total of 186 bacterial isolates from cows with subclinical mastitis were identified by MALDI-TOF MS after preparation using two extraction protocols. On-plate protocol was performed by applying the bacterial colony directly from the culture plate onto the plate spot. For the standard protocol, lysis of bacterial colonies using reagents was performed in a cryotube, and the resulting extract was applied onto the plate spot for analysis. The on-plate protocol showed a similar bacteria identification rate (91.4%, n = 170/186) in comparison to the standard (94.6%, n = 176/186). Identification was higher for both protocols when scores used for species-level identification (≥ 2.0) was reduced to genus-level (≥ 1.7); genus-level identification score rate increased from 94.6 to 100% when using the standard protocol, and from 91.4 to 94.6% when using the on-plate protocol. However, when compared standard (as gold standard) versus on-plate protocol, genus-level identification score rate ranged from 87.1 to 89.8%. Therefore, when the on-plate protocol fails to identify any specie, the standard extraction may be more suitable as a reference protocol for use. Strategy for increasing identification with the on-plate protocol may include upgrading the reference database library. Choice of protocol for preparation may be influenced by the bacterial type to be identified. Standard and on-plate extraction protocols of bacterial ribosomal proteins associated with MALDI-TOF MS might be alternatives to conventional microbiology methods for identification of subclinical mastitis pathogens.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences.

BACKGROUND: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. RESULTS: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. CONCLUSIONS: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species.

Comparison of methods for the identification of microorganisms isolated from blood cultures.

BACKGROUND: Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. METHODS: This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. RESULTS: The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). CONCLUSIONS: The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.

Discrepancies among three laboratory methods for Clostridium difficile detection and a proposal for their optimal use.

Clostridium difficile is the major cause of nosocomial diarrhoea. Several detection methods are available for the laboratory diagnosis of C. difficile, but these vary in terms of sensitivity and specificity. In this study, we compared the performance of three following laboratory tests to detect C. difficile: in-house real-time PCR aiming for toxin B gene (tcdB), EIA for detection of toxins A and B (Premier Toxins A & B) and C. difficile culture in selective medium (bioMerieux). Our results were grouped into three categories as follows: (1) C. difficile-associated diarrhoea (CDAD); (2) asymptomatic carriers; and (3) negative results. Among the 113 patients included in the study, 9 (8.0%) were classified as CDAD, 19 (16.8%) were asymptomatic carriers, 76 (67.2%) had negative results and 9 (8.0%) could not be categorized (positive test for C. difficile toxins only). PCR was found to be the most sensitive diagnostic test in our study, with the potential to be used as a screening method for C. difficile colonization/CDAD. Diagnosis of CDAD would be better performed by a combination of PCR and EIA tests.

Design of two molecular methodologies for the rapid identification of Colombian community-acquired methicillin-resistant Staphylococcus aureus isolates.

INTRODUCTION: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are found with increasing the frequency, both in healthy individuals in the community and in hospitalized patients. In Colombia and the Andean region, CA-MRSA isolates have a genetic background that is related to the pandemic USA300 clone. OBJECTIVE: Two molecular methods are designed and standardized for the rapid differentiation of Colombian community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates. MATERIALS AND METHODS: Two molecular methods were standardized for the identification of CA-MRSA isolates. The first method was based on the differential digestion of the carbamate kinase (arcC)and guanylate kinase (gmk) genes in the sequences type 5 (ST5) in the HA-MRSA isolates and 8 (ST8) in the CA-MRSA isolates. The second method was based on the PCR amplification of 5 specific virulence factors found in CA-MRSA and HA-MRSA isolates. The specificity and precision of each method were evaluated using 237 clinical MRSA isolates. RESULTS: The first method identified 100% and 93.2% of the CA-MRSA and HA-MRSA isolates, respectively. The second method also correctly identified the two isolates types (CA-MRSA and HA-MRSA). CONCLUSIONS: These two methods are a convenient alternative for the rapid identification of the CA-MRSA isolates, compared with other techniques such as pulsed field gel electrophoresis and multilocus sequence typing, which are time-consuming and more expensive.

Design of two molecular methodologies for the rapid identification of Colombian community-acquired methicillin-resistant Staphylococcus aureus isolates / Diseño de dos metodologías moleculares para la rápida identificación de aislamientos de Staphylococcus aureus resistente a meticilina asociados a la comunidad en Colombia

Introduction. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are found with increasing the frequency, both in healthy individuals in the community and in hospitalized patients. In Colombia and the Andean region, CA-MRSA isolates have a genetic background that is related to the pandemic USA300 clone. Objective. Two molecular methods are designed and standardized for the rapid differentiation of Colombian community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates. Materials and methods. Two molecular methods were standardized for the identification of CA-MRSA isolates. The first method was based on the differential digestion of the carbamate kinase (arcC)and guanylate kinase (gmk) genes in the sequences type 5 (ST5) in the HA-MRSA isolates and 8 (ST8) in the CA-MRSA isolates. The second method was based on the PCR amplification of 5 specific virulence factors found in CA-MRSA and HA-MRSA isolates. The specificity and precision of each method were evaluated using 237 clinical MRSA isolates. Results. The first method identified 100% and 93.2% of the CA-MRSA and HA-MRSA isolates, respectively. The second method also correctly identified the two isolates types (CA-MRSA and HA-MRSA). Conclusions. These two methods are a convenient alternative for the rapid identification of the CA-MRSA isolates, compared with other techniques such as pulsed field gel electrophoresis and multilocus sequence typing, which are time-consuming and more expensive.

Introducción. Los aislamientos de Staphylococcus aureus resistente a la meticilina asociado a la comunidad (SARM-AC), están aumentando la frecuencia de infecciones en personas sanas de la comunidad y en pacientes hospitalizados. En Colombia y en la región andina estos aislamientos tienen un componente genético relacionado con el clon pandémico USA300. Objetivo. Diseñar y estandarizar dos metodologías para la diferenciación rápida de aislamientos colombianos de S. aureus resistente a la meticilina asociado a la comunidad de los asociados al hospital (SARM-AH). Materiales y métodos. Se estandarizaron dos metodologías moleculares para la identificación de aislamientos de S. aureus resistente a la meticilina asociado a la comunidad. La primera se basa en la digestión diferencial con tres enzimas de restricción de los genes cinasa de carbamato (arcC)y cinasa de guanilato (gmk)para los tipos de secuencia 5 (ST5) y 8 (ST8), correspondientes a aislamientos de S. aureus resistente a la meticilina asociado al hospital y asociado a la comunidad, respectivamente. La segunda se basa en la amplificación por reacción en cadena de la polimerasa de cinco factores de virulencia que se encuentran de manera diferencial en estos aislamientos. Las dos metodologías fueron validadas en 237 aislamientos clínicos de S. aureus resistente a la meticilina. Resultados. Con la primera metodología se identificaron el 100 % y 93,2 % de los aislamientos de S. aureus resistente a la meticilina asociado a la comunidad y asociado al hospital, respectivamente. Con la segunda metodología se identificaron correctamente los dos tipos de aislamientos. Conclusiones. Estas dos metodologías son una buena alternativa en términos de ahorro en tiempo y dinero comparadas con otras técnicas, como la electroforesis en campo pulsado y la tipificación de secuencias multilocus para la rápida identificación de aislamientos de S. aureus resistente a la meticilina asociado a la comunidad en Colombia.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media / Detecção do fator corda e avaliação do aspecto macroscópico das colônias de micobactérias: um eficiente teste de triagem combinado para a identificação presuntiva do complexo Mycobacterium tuberculosis em meios só

OBJECTIVE: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. METHODS: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. RESULTS: The overall accuracy of cord factor detection alone was 95.4 percent (95 percent CI: 90.7-98.1 percent), and that of the combined screening test was 99.3 percent (95 percent CI: 96.4-100 percent). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. CONCLUSIONS: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

OBJETIVO: A diferenciação rápida entre Mycobacterium tuberculosis e micobactérias não-tuberculosas é fundamental para os pacientes coinfectados com tuberculose e HIV. Para tanto, utilizamos duas metodologias em nosso laboratório: detecção do fator corda e PCR-restriction enzyme analysis (PRA). O objetivo do estudo foi avaliar a acurácia desse teste de triagem em meio sólido como um método rápido para a identificação presuntiva do complexo M. tuberculosis, considerando custos e tempo de resultado. MÉTODOS: Foram processadas 152 cepas pelo teste de triagem combinado, que consistiu da detecção do fator corda por microscopia (esfregaço corado por Ziehl-Neelsen) e avaliação do aspecto macroscópico das colônias, e PRA (padrão ouro). Os custos foram estimados através da obtenção dos preços dos insumos necessários para a realização de cada teste. RESULTADOS: A acurácia da detecção do fator corda foi de 95,4 por cento (IC95 por cento: 90,7-98,1 por cento) e a do teste de triagem combinado foi de 99,3 por cento (IC95 por cento: 96,4-100 por cento). O custo da detecção do fator corda foi de R$ 0,60 e do PRA de R$ 16,00. Os resultados da detecção do fator corda estão prontos em 2 dias, ao passo que os de PRA necessitam de 4 dias. CONCLUSÕES: A identificação presuntiva de M. tuberculosis usando o aspecto macroscópico das colônias em conjunto com a detecção de fator corda por microscopia é um teste simples, rápido e de baixo custo. Recomendamos o teste de triagem combinado para rapidamente identificar M. tuberculosis em sítios com poucos recursos financeiros e em laboratórios menos equipados, enquanto se aguarda a identificação definitiva por métodos moleculares ou bioquímicos.

PCR-restriction fragment length polymorphism analysis as a tool for Mycobacterium species identification in lepromas for lepromin production.

OBJECTIVES: The aim of the present work was to standardise a PCR-Restriction Fragment Length Polymorphism analysis (PRA) as a tool to detect the mycobacteriologic composition of lepromas from leprosy patients used in the production of lepromin to improve the quality of the Mitsuda test. DESIGN: PCR-Restriction Fragment Length Polymorphism analysis using hsp65 and rpoB genes were applied to 11 reference strains of mycobacteria, including M. leprae, and the obtained PRA profiles were compared to mycobacteria in clinical specimens. RESULTS: Out of the biopsies studied, 522% had DNA fragment amplified for both genes (hsp65 and rpoB) for M. leprae. However, other Mycobacterium species were observed in samples of lepromatous leprosy patients. Here we discussed the importance of mycobacteria identification in the antigen of Mitsuda production to be used in the evaluation of leprosy. CONCLUSIONS: Our results suggest that the use of the molecular approach for sample selection can contribute to an improvement in the quality of produced lepromin.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media.

OBJECTIVE: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. METHODS: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. RESULTS: The overall accuracy of cord factor detection alone was 95.4% (95% CI: 90.7-98.1%), and that of the combined screening test was 99.3% (95% CI: 96.4-100%). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. CONCLUSIONS: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

[Rapid identification of non tuberculous mycobacteria by restriction pattern analysis]. / Identificación rápida de micobacterias no tuberculosas mediante análisis de patrones de restricción.

BACKGROUND: The frequency of diseases caused by non tuberculous mycobacteria has increased in the last years. Their clinical diagnosis is difficult, mainly in immunocompromised patients. The identification of these mycobacteria by traditional methods is based on phenotypic characteristics and the results are obtained two to four weeks after their isolation in primary cultures. AIM: To report a new identification method for non tuberculous mycobacteria. MATERIAL AND METHODS: The restriction pattern analysis method was implemented. It is based on the amplification, using polymerase chain reaction (PCR), of a polymorphic region of 440 base pairs that codifies Hsp65 protein, followed by a digestion with BstE II and Hae III restriction enzymes. The results were compared with patterns established for each strain. RESULTS: Sixty four strains of mycobacteria obtained from clinical samples and seven reference mycobacteria, were identified using the traditional methods and restriction pattern analysis. The latter method identified the same strain as the former in 87.5% of cases. In the remainder 12.5% of cases there was no agreement between both methods. In these, the sequencing of a fragment of a gene that codifies 16S ribosomal RNA, confirmed the correct identification by restriction patterns. CONCLUSIONS: Restriction pattern analysis is a rapid identification method for non tuberculous mycobacterial strains.

Identificación rápida de micobacterias no tuberculosas mediante análisis de patrones de restricción / Rapid identification of non tuberculous mycobacteria by restriction pattern analysis

Background: The frequency of diseases caused by non tuberculous mycobacteria has increased in the last years. Their clinical diagnosis is difficult, mainly in immunocompromised patients. The identification of these mycobacteria by traditional methods is based on phenotypic characteristics and the results are obtained two to four weeks after their isolation in primary cultures. Aim: To report a new identification method for non tuberculous mycobacteria. Material and methods: The restriction pattern analysis method was implemented. It is based on the amplification, using polymerase chain reaction (PCR), of a polymorphic region of 440 base pairs that codifies Hsp65 protein, followed by a digestion with BstE II and Hae III restriction enzymes. The results were compared with patterns established for each strain. Results: Sixty four strains of mycobacteria obtained from clinical samples and seven reference mycobacteria, were identified using the traditional methods and restriction pattern analysis. The latter method identified the same strain as the former in 87.5% of cases. In the remainder 12.5% of cases there was no agreement between both methods. In these, the sequencing of a fragment of a gene that codifies 16S ribosomal RNA, confirmed the correct identification by restriction patterns. Conclusions: Restriction pattern analysis is a rapid identification method for non tuberculous mycobaterial strains.

[PCR-RFLP/Hsp70 for identification and tipification of leishmania from the tropical region]. / PCR-RFLP/Hsp70 para identificar y tipificar Leishmania de la región neotropical.

The optimization of the PCR conditions for amplification of the gene coding for the 70 kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, Lamazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosornma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi.

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

Molecular techniques for MRSA typing: current issues and perspectives.

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially for therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Molecular techniques for MRSA typing: current issues and perspectives

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially as to therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose