Review on Application of Nucleic Acid Amplification Techniques in Pharmaceutical Products Analysis.

The aim of this study was to describe application of molecular diagnostic tests based on nucleic acid amplification technologies (NAATs) in pharmaceutical product analysis. NAAT have become widely established in clinical microbiology laboratories in recent years, as well as in quality control (QC) laboratories for food testing, and lately introduced to the pharmaceutical QC laboratories. The number of available nucleic acid and gene amplification-based rapid microbiological methods has increased over the last few years, and for good reason. When compare to standard culture based methods, NAAT provide a rapid, an accurate and reliable means for detecting specific microorganisms of interest, especially in pharmaceutical dosage forms that are required to be free of objectionable or specified pharmacopeial organisms. This article discussed about the different types of nucleic acid amplification techniques and methods available to pharmaceutical microbiologists working on quality control of pharmaceutical products and develop some awareness among other pharmaceutical scientists. This review also highlights limitations of these methods applied in industrial setup.

Identificación rápida de microorganismos de frascos de hemocultivos por espectrometría de masas: Comparación de 2 procedimientos diagnósticos / Rapid identification of microorganisms by mass spectrometry in a blood culture system: Comparison of two procedures

La identificación rápida de microorganismos es crítica, en especial en pacientes sépticos hospitalizados. La espectrometría de masas conocida como matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) permite la identificación directa desde botellas de hemocultivos positivos en forma rápida y sencilla. Este estudio evaluó el desempeño del procedimiento basado en el sistema MALDI Biotyper que utiliza el kit comercial MALDI Sepsityper de Bruker Daltonics (en adelante, MS) frente a uno artesanal (en adelante, HF). Se procesaron 840 botellas de hemocultivos positivos con HF y 542 de estas fueron evaluadas también con MS. Se logró la identificación de los microorganismos en 670 (79,76 %) y 391 (72,14 %) botellas, respectivamente (p = 0,0013). Se demostró la efectividad de ambos procedimientos para la identificación de microorganismos desde frascos de hemocultivos positivos. Sin embargo, el procedimiento HF fue superior al MS, en especial frente a bacterias gram positivas.

Rapid identification of microorganisms is critical in hospitalized infected patients. Blood culture is currently the gold standard for detecting and identifying microorganisms causing bacteremia or sepsis. The introduction of mass spectrometry by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) in microbiology laboratories, especially in microorganisms growing in blood culture bottles, provides rapid identification. This study evaluates the performance of the Maldi Sepsityper Biotyper procedure (hereinafter, MS) compared to that of an in-home method (hereinafter, HF). Eight hundred and forty (840) positive blood culture bottles were processed using the HF procedure, 542 of which were also processed using MS. The organisms were identified in 670 (79. 76 %) and 391 (72. 14 %) bottles respectively (p = 0,0013). This study demonstrates the effectiveness of both procedures for identifying microorganisms directly from positive blood culture bottles. However, the HF procedure proved to be more effective than MS, especially in the presence of Gram positive organisms.

Meta-análisis: diagnóstico de tuberculosis resistente por técnicas no comerciales de amplificación / Meta-analysis: drug-resistant tuberculosis diagnosis by non commercial amplification techniques / Meta-análise: diagnóstico de tuberculose resistente por técnicas não comerciais de amplificação

La emergencia de tuberculosis (TB) multidrogo y extensivamente-resistente reactivó la necesidad de contar con métodos rápidos para detectar resistencia a isoniacida (INH) y rifampicina (RIF). Por tal motivo, los objetivos de este trabajo fueron evaluar, mediante meta-análisis, la exactitud global y la posible utilidad de métodos caseros basados en PCR para la detección rápida de resistencia a INH y RIF en aislamientos clínicos de Mycobacterium tuberculosis. La búsqueda bibliográfica incluyó Medline/PubMed, BioMedLib. Para estimar la variabilidad entre los resultados de los estudios y el grado de exactitud diagnóstica de los métodos utilizados se realizaron gráficos "forest plot" y curvas SROC (summary receiver operating characteristic) mediante el software Meta-DiSc. Fueron seleccionados 15 estudios, conteniendo 1311 aislamientos resistentes a INH y 953 a RIF. Para la detección de resistencia a INH la sensibilidad y especificidad globales fueron: 84,0% y 96,0% respectivamente, mientras que para la detección de resistencia a RIF esos valores fueron 92,0% y 97,0%. Además, estos métodos mostraron alta exactitud diagnóstica, con áreas bajo la curva SROC>0,9. La alta sensibilidad y especificidad obtenidas con métodos moleculares caseros sugieren que algunos de ellos podrían ser aplicados para el diagnóstico rápido de resistencia a partir del aislamiento de M. tuberculosis.

Due to the emergency of multidrug and extensively-drug resistant tuberculosis, molecular methods for a rapid detection of isoniazid (INH) and rifampicin (RIF) resistance are urgently needed. For that reason, the objectivesof this study were to asses through a meta-analysis the global accuracy and the utility of the home-made molecular methods based in PCR for INH and RIF resistance rapid detection from Mycobacterium tuberculosis clinical isolates. The articles were searched using Medline/PubMed, BioMedLib. The variability among different studies results and the diagnostic accuracy of the used methods were estimated by forest plot and summary receiver operating characteristic (SROC) curves performed with software Meta-DiSc. Fifteen studies were chosed: 1311 containing INH resistant and 953 RIF resistant isolates. The pooled sensitivity and specificity for INH resistance detection was 84.0% and 96.0% respectively, while 92.0% and 97.0% were the pooled values for RIF resistance detection. Besides, these methods showed a high diagnostic accuracy, with the area under the SROC curve >0.9. Due to the high sensitivity and specificity obtained with the home-made molecular methods, some of these tests could be applied for a rapid detection of M. tuberculosis drug resistance in clinical practice.

A emergência de tuberculose (TB) multidrogas e extensivamente-resistente reativou a necessidade de contar com métodos rápidos para detectar resistência à isoniazida (INH) e rifampicina (RIF). Por isso, o objetivo deste trabalho foi a avaliação através da meta-análise, da exatidão global e da possível utilidade de métodos caseiros baseados em PCR para detectar rapidamente a resistência a INH e RIF em isolamentos clínicos de Mycobacterium tuberculosis. A pesquisa bibliográfica incluiu Medline/PubMed, Bio MedLib. Para estimar a variabilidade entre os resultados dos estudos e o grau de exatidão diagnóstica dos métodos utilizados, foram realizados gráficos "forest plot" e curvas SROC (summary receiver operating characteristic) com o software Meta-Disc. Foram selecionados 15 estudos, contendo 1311 isolamentos resistentes a INH e 953 a RIF. Para a detecção de resistência a INH, a sensibilidade e especificidade globais foram 84,0% e 96,0% respectivamente, enquanto que para a detecção de resistência a RIF esses valores foram de 92,0% e 97,0%. Alem disso, os mesmos métodos mostraram elevada exatidão diagnóstica, com áreas inferiores à curva SROC>0,9. A elevada sensibilidade e especificidade obtida através de métodos moleculares caseiros sugere que alguns deles poderiam ser aplicados para o diagnóstico rápido de resistência a partir do isolamento de M. tuberculosis.

Rapid identification of Mycobacterium tuberculosis complex by a novel hybridization signal amplification method based on self-assembly of DNA-streptavidin nanoparticles

Rapid detection of Mycobacterium tuberculosis complex (MTBC) is a critical step in controlling tuberculosis (TB). In this study, we used IS6110 as the specific identification target to develop a novel hybridization signal amplification method (HSAM) for the rapid and direct detection of MTBC from clinical sputum specimens. This system consists of magnetic bead-linked capture probes for target isolation, dextranbased nanoparticles for amplifying the reporter molecule (biotinylated-FITC), and detection probes (2B-DNA) for binding the nanoparticles. Both the capture and detection probes were specific to the IS6110 target sequence. Our results determined that as few as 10 copies of the IS6110 sequence or 10 M. tuberculosis bacteria could be detected, indicating that the HSAM assay is as sensitive as conventional PCR, and the assay was specific enough to distinguish MTBC from nontuberculosis mycobacteria (NTM). A total of 176 clinical sputum specimens were collected for HSAM evaluation, and the results were compared to those from traditional culture and biochemical identification techniques. This assay had a sensitivity of 88.3 percent, a specificity of 91.8 percent, a positive predictive value of 93.8 percent and a negative predictive value of 84.8 percent for the detection of MTBC. This technique is highly sensitive and specific, is easy to perform, and does not require any sophisticated detection equipment; thus, this approach has great potential in clinical TB detection and diagnostic applications.

Detection of salmonella sp in chicken cuts using immunomagnetic separation

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100 percent sensitivity and 94 percent specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.

A separação imunomagnética (IMS) é uma técnica que tem sido associada a diferentes métodos de detecção de Salmonella em alimentos para aumentar a sensibilidade e a especificidade e diminuir o tempo de análise. Neste trabalho é comunicada a obtenção de microesferas magnéticas sensibilizadas com anticorpos anti-Salmonella e seu uso em associação com a metodologia de cultivo convencional para desenvolver um método de detecção de salmonelas em cortes de frango (IMS-plaqueamento). Inicialmente, microesferas cobertas com proteína A foram sensibilizadas com anticorpos policlonais contra lipopolissacarídeo e flagelo de salmonelas e usadas na padronização de um procedimento que captura Salmonella Enteritidis em cultivo puro e faz posterior detecção em ágar seletivo. A seguir, amostras de carne de frango experimentalmente contaminadas com S. Enteritidis foram analisadas imediatamente após a contaminação e após 24h de refrigeração utilizando três protocolos de enriquecimento. O limite de detecção foi cerca de 2x10 UFC/mL. O protocolo que incluiu enriquecimento não-seletivo por 6-8h, enriquecimento seletivo por 16-18h e pós-enriquecimento por 4h foi o que proporcionou melhor resultado na detecção de S. Enteritidis em carne de frango experimentalmente contaminada. Este protocolo foi comparado à metodologia convencional em estudo de detecção de salmonelas em cortes de frango naturalmente contaminados e obteve 100 por cento de sensibilidade e 94 por cento de especificidade. O método desenvolvido foi capaz de diminuir em pelo menos um dia o tempo de detecção de salmonelas em cortes de frango pela metodologia convencional.

Estudo comparativo entre os métodos simplate e convencional de contagem padrão em placas, para a determinação de bolores e leveduras em polpa de cajá / Study comparative between the approaches simplate and conventional of tally standard in plates, for the determination of moulds and yeasts in pulp of cajá

Os Métodos Rápidos de Análise Microbiológica, surgidos na década de 70, tem-se mostrado instrumentos eficazes na agilização de resultados de análises em indústrias de alimentos. O Método Simplate de enumeração de bolores e leveduras permite a obtenção dos resultados em 48 horas. No entanto, apresenta resultados falso-positivos quando utilizado em alimentos com atividade enzimática endógena. Diversos estudos foram realizados objetivando a avaliação e validação de diversos métodos rápidos por órgãos oficiais. Neste estudo, 25 amostras de polpa de cajá congeladas foram inoculadas com leveduras Saccharomyces cerevisiae, em diversas concentrações e plaqueadas através dos métodos Simplate e Convencional (técnica pour plate). A aplicação do teste t-pareado aos resultados das análises mostrou não existir diferença entre os dois métodos, a nível de 5 por cento de significância.

The Rapid Methods of Analysis Microbiological is appeared in 70 decade and it has been identified one effective instrument in the obtainment Of rapid results of analysis in food industries. The Simplate method for yeast and mold enumeration has been used for determination of fungus within 48 hours; however, it presents false-positive results when used in foods that suffer endogenous enzymatic activity. Several studies have been done to evaluate the performance of the rapid methods and validacion them by official organizations. In this study was proceeded the inoculation of 25 samples of cajá frozen pulps with Saccharomyces cerevisiae in different concentrations. The samples were analysied by Simplate method and convencional pour plate method. The T-pareado test was applied in evaluation equivalence among of two methods. Results of this test revealed that at 5% level significance there is not difference among of two methods.