Características microbiológicas de los pacientes con úlcera del pie diabético / Microbiologic characteristics of patients with diabetic foot ulceration

RESUMEN Introducción: Las infecciones de las úlceras del pie diabético son comunes, complejas, de alto costo y constituyen la principal causa de amputación no traumática de las extremidades inferiores. Objetivo: Identificar los microorganismos aislados para estimar tanto la sensibilidad a los antibióticos como la coincidencia entre el tratamiento empírico y los resultados microbiológicos en pacientes con úlceras del pie diabético. Métodos: Se realizó una investigación descriptiva-retrospectiva. La población de estudio estuvo constituida por 210 pacientes ingresados en el Hospital Universitario Clínico Quirúrgico "Comandante Faustino Pérez Hernández" de Matanzas entre junio de 2017 y junio de 2020. Las variables de salida fueron la frecuencia y el tipo de germen, la cantidad de gérmenes por úlcera, la sensibilidad para cada tipo de antibiótico, y el porcentaje de coincidencia entre el tratamiento empírico y el resultado microbiológico. Resultados: Se identificaron 259 gérmenes y se observaron 1,23 gérmenes por úlcera. El 62,5 por ciento de los gérmenes encontrados fueron Gram negativos, pero el germen más representado fue el Staphylococcus aureus. El 58,8 por ciento de los Staphylococcus aureus se mostraron resistentes a la meticillin. La vancomicina y el linezolid resultaron efectivos en el 100 por ciento de los Gram positivos. La amikacina fue el antibiótico más efectivo para los Gram negativos. Se observó coincidencia entre el tratamiento empírico y el resultado del antibiograma en el 27,6 por ciento de los pacientes. Conclusiones: Resulta necesario un apropiado diagnóstico microbiológico de las úlceras del pie diabético para identificar los gérmenes presentes en las lesiones y diseñar algoritmos de terapia antimicrobiana adecuados(AU)

ABSTRACT Introduction: Diabetic foot ulcer infections are common, complex, high cost and are the leading cause of non-traumatic lower extremity amputation. Objective: To identify the microorganisms isolated to estimate both the sensitivity to antibiotics and the coincidence between empirical treatment and microbiological results in patients with diabetic foot ulcers. Methods: A descriptive-retrospective investigation was performed. The study population consisted of 210 patients admitted to the University Hospital "Comandante Faustino Pérez Hernández" of Matanzas between June 2017 and June 2020. The output variables were the frequency and type of germ, the number of germs per ulcer, the sensitivity for each type of antibiotic, and the percentage of coincidence between the empirical treatment and the microbiological result. Results: A total of 259 germs were identified and 1.23 germs per ulcer were observed. The 62.5 percent of the germs found were Gram negative, but the most represented germ was Staphylococcus aureus. Of the Staphylococcus aureus, 58.8 percentwere resistant to methicillin. Vancomycin and linezolid were effective in 100 percent of Gram positives. Amikacin was the most effective antibiotic for Gram-negatives. Agreement between empirical treatment and antibiogram result was observed in 27.6 percent of patients. Conclusions: An appropriate microbiological diagnosis of diabetic foot ulcers is necessary to identify the germs present in the lesions and to design adequate antimicrobial therapy algorithms(AU)

Detection of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in Brazilian mastitic milk goats by multiplex-PCR / Detecção de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em leite caprino mastítico no Brasil, por multiplex-PCR

This study evalueted the prevalence of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in milk samples from 257 goats (513 half-udders) and ten bulk tanks, from ten dairy goat farms of São Paulo State, Brazil, by multiplex-PCR. The samples were screened by microbiological culture (gold-standard), and tested by different multiplex-PCR protocols for the detection of each bacterium. A total of 178 half-udders resulted positive by microbiological culture, with coagulase-negative staphylococci (70%), S. aureus (13.5%), S. intermedius (7.9%), and Enterobacteriaceae (4%) the prevalent pathogens. In other way, multiplex-PCR detected 173 pathogens in 151/523 (28.9%; CI95% 25.2-32.9%) milk samples 144/513 (28.1%) half-udders and 7/10 (70%) bulk tanks, with E. coli (86/162, 51.9%) and S. aureus (50/162, 30.9%) the prevalent ones in half-udders, and S. aureus (6/10, 60%) and E. coli (4/5, 36.4%) in bulk tanks. Multiplex-PCR showed a high performance for the detection of three bacteria at a time in mastitic goat milk direct from half-udders or bulk tanks. Thus, this multiplex-PCR protocol proved to be an adequate tool for the identification of the most common mastitis pathogens, independent of their phenotypic characteristics in the diagnosis of clinical mastitis in goats, allowing a continuous and better vigilance and monitoring the herd, being included in quality programs.(AU)

Este estudo avaliou por multiplex-PCR a prevalência de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em amostras de leite de 257 caprinos (513 tetos) e dez tanques de expansão, em dez fazendas leiteiras do estado de São Paulo, Brasil. As amostras foram triadas por cultura microbiológica (padrão-uro) e testadas por diferentes protocolos multiplex-PCR para a detecção de cada bactéria. Um total de 178 amostras de leite foram positivos na cultura microbiológica, com estafilococos coagulase-negativos (70%), S. aureus (13,5%), S. intermedius (7,9%) e Enterobacteriaceae (4%) como patógenos prevalentes. Por outro lado, a PCR multiplex detectou 173 patógenos em 151/523 (28,9%, IC95% 25,2-32,9%) amostras de leite, 144/513 (28,1%) amostras de tetos e 7/10 (70%) em tanques de expansão, E. coli (86/162, 51,9%) e S. aureus (50/162, 30,9%) foram identificados nas amostras de tetos e S. aureus (6/10, 60%) e E. coli (4/5, 36,4%) em tanques expansão. Multiplex-PCR mostrou um alto desempenho para a detecção das três bactérias em leite de cabra com mastite ou em tanques de expansão. Dessa forma, este protocolo multiplex-PCR provou ser uma ferramenta adequada para a identificação dos patógenos mais comuns da mastite, independentemente de suas características fenotípicas no diagnóstico de mastite clínica em caprinos, permitindo uma vigilância contínua e melhor acompanhamento do rebanho, sendo incluído em programas de qualidade.(AU)

Effect of somatic cell count on composition and hygiene indicators of bulk tank milk / Efeito da contagem de células somáticas sobre a composição e indicadores de higiene do leite de tanque

This study evaluated the effect of somatic cell count (SCC) on composition and hygienic quality of dairy herd bulk tank milk ­ specifically, the effect of SCC of bulk tank of dairy herds on composition (fat, protein, total solids, nonfat dry solids) and on total bacterial count (TBC), psychrotrophic count (PC) and coliform count (CC) were evaluated. A total of 230 dairy herds located south of Minas Gerais and west of São Paulo were selected based on SCC geometric mean obtained from five monthly analyses preceding the start of the sampling. The dairy farms were classified according to SCC in three groups: low (< 250,000 cells/mL, n = 84), medium (> 250,000 and < 750,000 cells/mL, n = 79) and high SCC (> 750,000 cells/mL, n = 67). After herd selection, bulk tank milk samples were collected every 14 days for three months totaling 1380 samples, which were subjected to analysis of composition, TBC, PC, and CC. A decrease of TBC and CC was observed in herds with low SCC; however, herds with medium and high SCC had an increase in fat, crude protein, and total solids contents. A medium correlation was observed between TBC and PC (r = 0.6215), and also between PC and CC (r = 0.3692). Based on hygiene indicators and milk composition, a low and negative correlation between TBC and fat (r = -0.0585), PC and fat (r = -0.0585), and PC and total solids (r = -0.0662) was observed. Dairy herds with SCC < 250,000 cells/mL had higher bulk tank milk hygienic quality; however, considering the composition, herds with higher SCC produced higher milk fat and protein concentration.(AU)

Este trabalho avaliou o efeito da contagem de células somáticas (CCS) na composição e na qualidade higiênica do leite de tanque de rebanhos leiteiros. Especificamente, foram avaliados o efeito da CCS do tanque de rebanhos leiteiros na composição (gordura, proteína, sólidos totais, extrato seco desengordurado) e nas contagens bacteriana total (CBT), de psicrotróficos (CP) e de coliformes (CC). Um total de 230 rebanhos leiteiros localizados no sul de Minas Gerais e oeste de São Paulo foram selecionados com base na média geométrica da CCS obtida de cinco análises mensais anteriores ao início das coletas das amostras. As fazendas foram classificadas de acordo com a CCS em três grupos: baixa (< 250.000 células/mL, n = 84), média (> 250.000 e < 750.000 células/mL, n = 79) e alta CCS (> 750.000 células/mL, n = 67). Após a seleção dos rebanhos, amostras de leite do tanque foram coletadas a cada catorze dias durante três meses, totalizando 1.380 amostras, as quais foram submetidas às análises de composição, CBT, CP e CC. Uma redução da CBT e da CC foi observada em rebanho com baixa CCS; entretanto, rebanhos com média e alta CCS tiveram aumento nos teores de gordura, proteína bruta e sólidos totais. Uma média correlação foi observada entre CBT e CP (r = 0,6215) e também entre CP e CC (r = 0,3692). Com base nos indicadores de higiene e na composição do leite, foi observada uma correlação baixa e negativa entre CBT e gordura (r = -0,0585), CP e gordura (r = -0,0585) e CP e sólidos totais (r = -0,0662). Os rebanhos leiteiros com CCS < 250.000 células/mL apresentaram maior qualidade higiênica do leite de tanque; entretanto, considerando a composição, rebanhos com maior CCS tiveram maiores concentrações de gordura e proteína.(AU)

Microorganismos aislados en cultivo bacteriológico de muestras de leche de vacas holstein clínicamente sanas / Microorganisms isolated in bacteriological culture from milk samples from clinically healthy holstein cows / Microrganismos isolados em cultivo bacteriológico de amostras do leite de vacas holandesas clinicamente saudáveis

Resumen El objetivo del presente estudio fue determinar la frecuencia de aislamiento de microorganismos de tipo infeccioso y ambiental en leche de un grupo vacas clínicamente sanas en dos diferentes tipos de ordeño. Para ello se utilizaron muestras de leche de 289 vacas ubicadas en hatos de cuatro municipios del Norte del departamento de Antioquia en diferentes sistemas de ordeño (manual y mecánico), a cada muestra de leche se le hizo cultivo bacteriológico para determinar la presencia de microorganismos. De 289 muestras de leche evaluadas, 193 (66,78%) fueron positivas para aislamiento de cualquier tipo de patógeno (ocho diferentes patógenosaislados), de las cuales 81 (28,02%) muestras provenían de ordeño manual y 112 (38,75%) pertenecían a ordeño mecánico, encontrando un mayor porcentaje de aislamiento de patógenos bacterianos en muestras de leche provenientes de sistema de ordeño mecánico (p=0,0236). El patógeno de mayor aislamiento fue el Arcanobacterium haemolyticum (antes clasificado como Corinebacterium sp), microorganismo que hace parte de la flora saprófita del animal, con presencia de manera individual en el 20,14% de las muestras analizadas y en confección con otros patógenos en 0,7% de las muestras. El microorganismo que siempre se reporta como mayor productor de mastitis subclínica en bovinos es el Streptococcus agalactiae, un patógeno infeccioso, que en el presente estudio se aisló del 12,50% de las muestras de leche. Se determinó el Odds ratio (OR), entre aislamiento de Streptococcus agalactiae y Score de Células somáticas (SCS) que fue estadísticamente significativo, indicando que cuando está presente este patógeno el SCS aumenta y los animales son más susceptibles a padecer mastitis.

Abstract A microbiological analysis was performed to determine the frequency of isolation of microorganisms of infectious and environmental type in milk from a group of clinically healthy cows in two different types of milking (manual and mechanical). To each sample of milk was made bacteriological culture to determine the presence of microorganisms. Of 289 milk samples evaluated, 193 (66.78%) were positive for isolation of any type of pathogen, of which 81 (28.02%) samples came from manual milking and 112 (38.75%) belonged to mechanical milking, finding a higher percentage of isolation of bacterial pathogens in milks coming from mechanical milking system (p = 0.0236). The most isolated pathogen was the Arcanobacterium haemolyticum, A microorganism that forms part of the saprophytic flora of the animal, with an individual presence in 20.14% and in coinfection with other pathogens in 0.7% of the samples. The most common microorganism of subclinical mastitis in cattle is Streptococcus agalactiae, which in the present study was isolated from 12.50% of milk samples. The odds ratio (OR) between the isolation of Streptococcus agalactiae and the Somatic Cell Score (SCS) was determined, which was statistically significant, indicating that when this pathogen is present the SCS increases and the animals are more susceptible to mastitis.

Resumo O objetivo deste estudo foi determinar a frequência de isoLamento de microorganismos de tipo infecciosos e ambientaL no Leite de um grupo de vacas cLinicamente saudáveL em dois diferentes tipos de ordenha. Para este fim utiLizaram-se amostras do Leite de 289 vacas LocaLizadas em rebanhos de quatro municipaLidades do Norte de Antioquia com dois tipos diferentes de ordenha (manuaL e mecânica), a cada amostra do Leite foi feito um cuLtivo bacterioLógico para determinar a presença de microorganismos. De 289 amostras do Leite avaLiadas, 193 (66,78%) foram positivas para o isoLamento de quaLquer tipo de agente patogênico (oito diferentes agentes patogênicos isoLados), das quais 81 (28,02%) amostras eram de ordenha manuaL e 112 (38, 75%) foram de ordenha mecânica, encontrando uma maior percentagem de isoLamento dos agentes patogênicos bacterianos nos Leites provenientes do sistema de ordenha mecânica (p = 0,0236). O agente patogênico mais isoLado foi Arcanobacterium haemolyticum (Corinebacterium sp), microrganismo que faz parte da flora saprófita do animaL, presentes individuaLmente em 20,14% das amostras anaLisadas, e em reLação com outros agentes patogênicos o 0,7% das amostras. O microrganismo que sempre se reporta como o maior produtor de mastite subcLínica em gado é o Streptococcus agalactiae, um agente patogênico infeccioso, que neste estudo foi isoLado no 12,50% das amostras do Leite. Foi determinado o Odds ratio (OR) entre o isoLamento de Streptococcus agalactiae e Pontuação das céLuLas somáticas (SCS), e foi estatisticamente significativo, indicando que quando o agente patogênico está presente, o SCS aumenta e os animais são mais susceptíveis à mastite.

Estudio comparativo de la sensibilidad entre el cultivo y la reacción en cadena de la polimerasa para la detección de bordetella pertussis a partir de muestras de hisopado nasofaríngeo / Comparative study of culture and sensitivity polymerase chain reaction in the diagnosis of bordetella pertussis from nasopharyngeal swab specimens

La tosferina es una enfermedad infecciosa causada por Bordetella pertussis, que se diagnostica mediante el cultivo como técnica de referencia, la cual tiene como limitante una baja sensibilidad. Es debido a esto, que el presente trabajo se centró en evaluar a la técnica de PCR a punto final amplificando las secuencias IS481 y PT, como una metodología alternativa diagnóstica a partir de 100 muestras pareadas de hisopado nasofaríngeo de pacientes provenientes de distintas regiones de Venezuela. Dichas muestras pareadas constaron de un hisopado para realizar el cultivo bacteriano y el otro para la detección de ADN específico. La identificación microbiológica de B. pertussis incluyó el aislamiento del microorganismo en agar Regan-Lowe, identificación bioquímica y la confirmación por coaglutinación. El análisis de los resultados fue realizado empleando el programa SPSS 21.0.0., usando como herramienta estadística el test de McNemar. La sensibilidad obtenida por el protocolo de PCR a punto final fue 50%, en concordancia con reportes previos. De acuerdo al valor de p=0,002 obtenido, la detección de B. pertussis mediante los dos métodos presentó una diferencia para el diagnóstico de tosferina estadísticamente significativa, por lo que no es indiferente emplear ambas técnicas diagnósticas. Sin embargo, se recomienda emplear en forma combinada ambas metodologías para incrementar la probabilidad de realizar el diagnóstico.

Whooping cough is an infectious disease caused by Bordetella pertussis, diagnosed by culture as a reference technique, which has low sensitivity as limiting. It is because of this that the present work focused on evaluating the PCR technique endpoint amplifying IS481 and PT sequences, as an alternative methodology diagnostic from 100 paired samples of nasopharyngeal swabs from patients from different regions of Venezuela. Such paired samples comprised a swab for bacterial culture and the other for detection of specific DNA. Microbiological identification of B. pertussis included the isolation of the microorganism in agar Regan-Lowe, biochemical identification and confirmation by Coagglutination. The analysis of the results was performed using the SPSS program 21.0.0., as a statistical tool using the McNemar test. The sensitivity obtained by the PCR protocol endpoint was 50%, consistent with previous reports. According to the value of p = 0.002 obtained, detection of B. pertussis by the two methods showed a difference for the diagnosis of pertussis statistically significant, so it is not indifferent both diagnostic techniques employed. However, it is recommended to use both methods in combination to increase the likelihood of diagnosis.

Diagnostic comparison of Corynebacterium pseudotuberculosis through microbiological culture and PCR in sheep samples / Comparação diagnóstica de Corynebacterium pseudotuberculosis pelo cultivo microbiológico e PCR em amostras ovinas

Caseous lymphadenitis is a chronic infectious disease caused by Corynebacterium pseudotuberculosis , which is a bacterium responsible for a great number of economic losses on goat and sheep production. It is characterized by the formation of abscesses in superficial lymph nodes and in internal organs and lymph nodes. This study aimed at determining the agreement between microbiological culture and PCR in the identification of C. pseudotuberculosis , in samples collected from animals in slaughterhouses and in animals that presented lymph node enlargement in field conditions. From the 202 samples analyzed through microbiological culture, 113 (56%) were positive for Corynebacterium sp.; from these positive samples, 38 (34%) were identified as C. pseudotuberculosis by microbiological culture. From the amount of samples, 110 (54%) were positive and 92 (46%) were negative in the PCR. Kappa index (0.193) presented a weak agreement between PCR and microbiological culture. We concluded that molecular diagnosis (PCR) in clinical samples proved to be more efficient, reproducible, and faster than the microbiological culture, both on clinical samples analyses and in the confirmation of C. pseudotuberculosis in colonies that were classified by Corynebacterium genus. Thus, the present study demonstrated the importance of PCR to confirm C. pseudotuberculosis diagnosis, and the best contribution for the epidemiological surveillance of the disease in sheep.(AU)

A linfadenite caseosa é uma doença infectocontagiosa crônica, causada pelo agente etiológico Corynebacterium pseudotuberculosis , que é uma bactéria responsável por grandes perdas econômicas na produção de ovinos e caprinos. Caracteriza-se pela formação de abscessos em nódulos linfáticos superficiais e em órgãos internos e linfonodos. O presente trabalho teve por objetivo verificar a concordância entre as metodologias de isolamento microbiológico com a PCR na identificação do C. pseudotuberculosis , em amostras clínicas colhidas em abatedouros e em animais que apresentavam aumento de linfonodo em condições de campo. Das 202 amostras analisadas no cultivo microbiológico, 113 (56%) foram positivas para o gênero Corynebacterium sp., e 38 (34%) colônias foram identificadas como C. pseudotuberculosis por meio de cultura microbiológica. Das amostras clínicas extraídas, 110 (54%) foram positivas e 92 (46%) foram negativas na PCR. A concordância estimada entre as técnicas de PCR e o cultivo microbiológico pelo indicador Kappa foi considerada fraca (0,193). Concluímos que o diagnóstico molecular (PCR) provou ser mais eficiente, rápido e com reprodutibilidade quando comparado ao cultivo microbiológico das amostras clínicas bem como da confirmação do C. pseudotuberculosis de colônias pertencentes ao gênero Corynebacterium . Dessa forma, o presente trabalho demonstrou a importância do uso da PCR na confirmação diagnóstica do C. pseudotuberculosis , visando contribuir com a melhoria da vigilância epidemiológica da doença em ovinos.(AU)

Nocardiose cutânea em felino: relato de caso* / Feline cutaneous nocardiosis: case report

A nocardiose cutânea é uma condição supurativa a piogranulomatosa, que ocorre após penetração do agente em ferimentos na pele que foram expostas a água ou ao solo contaminado. A doença se manifesta com formação de abscessos localizados, com fístulas e úlceras necróticas que drenam uma secreção serossanguinolenta, atingindo membros ou abdômen. É rara em gatos, e nestes, a maior parte se deve a infecção por Nocardia asteroides. Este relato objetivou descrever o diagnóstico clínico e laboratorial de um felino doméstico com diagnóstico microbiológico de Nocardia asteroides, descrevendo o tratamento preconizado com base no antibiograma. Uma gata castrada, sem raça definida (SRD), com cinco anos de idade, desenvolveu lesão cutânea em forma de tratos fistulosos drenando secreção piossanguinolenta sob a região frontal da face, membros torácicos e abscesso em membro pélvico, além de inapetência, emagrecimento e apatia. Havia sido tratada anteriormente de forma empírica para infecção bacteriana com enrofloxacina, seguida de cefalexina após identificação bacteriana de Staphylococcus spp. Realizou-se coleta do exsudato para fins de cultura e antibiograma. O diagnóstico foi de nocardiose e o agente identificado como Nocardia asteroides e os micro-organismos foram sensíveis apenas a amicacina, neomicina e canamicina. Foi instituído tratamento com amicacina (10 mg/kg, a cada 12 horas), passando após sete dias a ser administrado a cada 24 horas devido a cilindrúria constatada através de urinálise. A remissão dos sinais ocorreu com 20 dias de uso contínuo do antibiótico, o qual foi mantido por mais três semanas sem outras complicações com resolução da cilindrúria.

The cutaneous nocardiosis is a suppurative to pyogranulomatous condition that occurs after penetration of the agent on wounds in the skin that were exposed to contaminated water or soil. The disease is manifested with formation of localized abscess, with necrotic fistula and ulcers that draining a serosanguineous secretion, reaching limbs or abdomen. It is rare in cats, and in these, the most are due to infection by Nocardia asteroides. This report aimed to describe the clinical and laboratory diagnosis of a domestic feline with microbiological diagnosis of Nocardia asteroides, describing the recommended treatment based on antibiogram. A spayed female cat, mongrel, with five years of age, developed cutaneous lesions in the form of fistulous tracts draining piosanguinolenta secretion on the frontal region of the face, forelimb and abscess in hind limb, and loss of appetite, weight loss and apathy. It had previously been treated empirically for bacterial infections with enrofloxacin, followed by cephalexin after bacterial identification of Staphylococcus spp. Held collection of exudate for culture and antibiogram. It was diagnosed nocardiosis and the identified agent as Nocardia asteroides and microrganisms were sensitive only to amikacin, kanamycin and neomycin. Treatment with amikacin (10 mg / kg every 12 hours) was introduced, and seven days after being administered once every 24 hours due to cilindruria detected by urinalysis. The remission of the signs occurred 20 days of continuous use of the antibiotics, which was maintained for three weeks without further complications with resolution of the cilindruria.

Brucella canis infection in dogs attended in veterinary clinics from patos, Paraíba state, Brazil

To determine the frequency of anti-Brucella canis antibodies in dogs attended in veterinary clinics from Patos, Paraíba State, Brazil, as well as to identify risk factors and to isolate and identify the agent, 193 dogs were used. Agar gel immunodiffusion test (AGID) was used to detect B. canis antibodies in sera. Isolation of B. canis was carried out in blood and bone marrow from seropositive animals. Six animals tested seropositive in AGID, resulting in a frequency of 3.11 percent. B. canis was isolated from bone marrow of one seropositive animal, with confirmation by PCR. Lack of cleaning of the dog's environment was identified as risk factor (odds ratio = 7.91). This is the first report of isolation of B. canis in dogs from the Northeast region of Brazil.

Diagnosis of Mycobacterium tuberculosis using molecular biology technology

<p><b>OBJECTIVE</b>To present an integrated molecular biology dedicated system for tuberculosis diagnosis.</p><p><b>METHODS</b>One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining, by cultivation on solid medium and by a balanced heminested fluorometric PCR system (Orange G3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorometer. Produced double stranded DNA was flurometrically detected. The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.</p><p><b>RESULTS</b>The assay was able to detect 30 bacillus per sample mL with 99.8% interassay variation coefficient. PCR was positive in 23 (21.9%) tested samples (21 of them were smear negative). In our study it showed a preliminary sensitivity of 94.5% for sputum and an overall specificity of 98.7%.</p><p><b>CONCLUSIONS</b>Total run time of the test is 4 h with 2.5 real working time. All PCR positive samples are also positive by microbiological culture and clinical criteria. Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples. Furthermore, its low cost and friendly using make it feasible to run in poor regions.</p>

Diagnosis of Mycobacterium tuberculosis using molecular biology technology

Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining, by cultivation on solid medium and by a balanced heminested fluorometricPCR system (OrangeG3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorometer. Produced double stranded DNA was flurometrically detected. The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.Results: The assay was able to detect30 bacillus per sample mL with99.8% interassay variation coefficient.PCR was positive in23 (21.9%) tested samples (21 of them were smear negative). In our study it showed a preliminary sensitivity of 94.5% for sputum and an overall specificity of98.7%.Conclusions:Total run time of the test is4 h with2.5 real working time. AllPCR positive samples are also positive by microbiological culture and clinical criteria. Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples. Furthermore, its low cost and friendly using make it feasible to run in poor regions.