Wooden sticks for plaque streaking and microbiological inoculation might be more cost- effective, but is its large scale use feasible? Quality control methods and proof of concept / Métodos de controle de qualidade e prova de conceito para a utilização de palitos de madeira em microbiologia podem ser mais econômicas, mas seu uso em larga escala é viável? Método de controle de qualidade e prova de conceito

The loop is a material classically used in the laboratory for the purpose of plate streaking and handling biological materials. However, metal loops techniques might be time consuming, considering the amount of time spent to guarantee its cooling process through each inoculation. Furthermore, plastic loops may also represent environmental issues during its production and discard process and can also represent higher costs for the laboratory. Thus, in situations of limited resources, even the simplest materials can be restricted due to logistical and budgetary issues, especially in developing countries. Inspired by demands like these, facing an occasional shortage of supply of laboratory plastic handles, we hereby present a quality control for sterilization methods and cost-effectiveness studies towards the use of wooden sticks in a Latin American country and we discuss the possibility of the large-scale use of this technique.

A alça calibrada é um material usado classicamente em laboratório para fins de inoculação em placas e manuseio de materiais biológicos. No entanto, as técnicas de alças metálicas podem consumir muito tempo, considerando a quantidade de tempo gasto para garantir seu processo de resfriamento a cada inoculação. Além disso, alças de plástico também podem representar questões ambientais durante o processo de produção e descarte e também podem representar custos mais altos para o laboratório. Assim, em situações de recursos limitados, até os materiais mais simples podem ser restringidos devido a questões logísticas e orçamentárias, especialmente nos países em desenvolvimento. Inspirados por demandas como essas, diante de uma escassez ocasional de suprimentos de alças de plástico de laboratório, apresentamos um controle de qualidade para métodos de esterilização e estudos de custo-efetividade para o uso de varas de madeira em um país latino-americano e discutimos a possibilidade de grande uso em escala dessa técnica.

Biochemical identification techniques and antibiotic susceptibility profile of lipolytic ambiental bacteria from effluents / Técnicas de identificação bioquímica e perfil de suscetibilidade antibiótica de bactérias ambientais lipolíticas de efluentes

Abstract Different methodologies have been developed throughout the years to identify environmental microorganisms to improve bioremediation techniques, determine susceptibility profiles of bacteria in contaminated environments, and reduce the impact of microorganisms in ecosystems. Two methods of bacterial biochemical identification are compared and the susceptibility profile of bacteria, isolated from residential and industrial wastewater, is determined. Twenty-four bacteria were retrieved from the bacteria bank of the Environmental Microbiology Laboratory at the Institute of Biology (IB) of the Universidade Federal de Pelotas, Pelotas, Brazil. Bacteria were identified by conventional biochemical tests and by the VITEK ®2 automated system. Further, the susceptibility profile to antibiotics was also determined by the automated system. Six species of bacteria (Raoutella planticola, K. pneumoniae ssp. pneumoniae , Serratia marcescens, Raoutella sp., E. cloacae and Klebsiella oxytoca) were identified by conventional biochemical tests, while three species of bacteria (K. pneumoniae ssp. pneumoniae, S. marcescens and K. oxytoca ) were identified by VITEK®2 automated system. VITEK ®2 indicated agreement in 19 (79.17%) isolates and difference in five (20.83%) isolates when compared to results from conventional biochemical tests. Further, antibiotic susceptibility profile results showed that all isolates (100%) were resistant to at least one out of the 18 antibiotics tested by VITEK®2. Thus, no multi-resistant bacteria that may be used in effluent treatment systems or in bioremediation processes have been reported. Results indicate VITEK ® 2 automated system as a potential methodology in the determination of susceptibility profile and identification of environmental bacteria.

Resumo Diferentes metodologias foram desenvolvidas ao longo dos anos para identificar microrganismos ambientais para melhorar as técnicas de biorremediação, determinar perfis de suscetibilidade de bactérias em ambientes contaminados e reduzir o impacto de microrganismos nos ecossistemas. Dois métodos de identificação bioquímica bacteriana são comparados e o perfil de susceptibilidade de bactérias, isoladas de efluentes residenciais e industriais, é determinado. Vinte e quatro bactérias foram coletadas do banco de bactérias do Laboratório de Microbiologia Ambiental do Instituto de Biologia (IB) da Universidade Federal de Pelotas, Pelotas, Brasil. As bactérias foram identificadas por testes bioquímicos convencionais e pelo sistema automatizado VITEK®2. Além disso, o perfil de suscetibilidade aos antibióticos também foi determinado pelo sistema automatizado. Seis espécies de bactérias (Raoutella planticola , K. pneumoniae ssp. pneumoniae, Serratia marcescens, Raoutella sp., E. cloacae e Klebsiella oxytoca) foram identificadas por testes bioquímicos convencionais, enquanto três espécies de bactérias (K. pneumoniae ssp. pneumoniae, S. marcescens e K. oxytoca) foram identificados pelo sistema automatizado VITEK®2. VITEK®2 indicou concordância em 19 (79,17%) isolados e diferença em cinco (20,83%) isolados quando comparados aos resultados de testes bioquímicos convencionais. Além disso, os resultados do perfil de suscetibilidade aos antibióticos mostraram que todos os isolados (100%) foram resistentes a pelo menos um dos 18 antibióticos testados pelo VITEK®2. Assim, não foram relatadas bactérias multirresistentes que possam ser usadas em sistemas de tratamento de efluentes ou em processos de biorremediação. Os resultados indicam que o sistema automatizado VITEK ® 2 é uma metodologia potencial na determinação do perfil de suscetibilidade e identificação de bactérias ambientais.

Evaluation of a selective chromogenic medium for detecting vancomycin-resistant enterococci

ABSTRACT Rapid identification of vancomycin-resistant enterococci (VRE) can assist in choosing the appropriate treatment and preventing VRE spread. The performance of chromIDTM VRE agar was evaluated using 184 clinical isolates of Enterococcus spp. and reference strains. The test had a sensitivity of 95.52% but a low specificity of 30%.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

Abstract A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

Growth Dynamics of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa as a Function of Time to Detection in BacT/Alert 3D Blood Culture Bottles with Various Preincubation Conditions

BACKGROUND: Delayed entry of blood culture bottles is inevitable when microbiological laboratories do not operate for 24 hr. There are few studies reported for prestorage of these bottles. The growth dynamics of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were investigated with respect to various preincubation conditions. METHODS: Fifteen or 150 colony-forming units (CFU) of bacteria were inoculated into standard aerobic or anaerobic blood culture bottles. Bottles were preincubated at 25degrees C or 37degrees C for 0, 2, 4, 8, 12, 24, or 48 hr. The time to detection (TTD) then was monitored using the BacT/Alert 3D system (bioMerieux Inc., USA). RESULTS: Significant difference in TTD was observed following preincubation for 8 hr at 25degrees C vs. 4 hr at 37degrees C for S. aureus, 4 hr at 25degrees C vs. 4 hr at 37degrees C for E. coli, 12 hr at 25degrees C vs. 4 hr at 37degrees C for P. aeruginosa, compared to no preincubation (P<0.005). TTD values did not vary significantly with bacterial CFU or with aerobic or anaerobic bottle type. The BacT/Alert 3D system returned false negatives following preincubation of P. aeruginosa for 48 hr at 25degrees C or 24 hr at 37degrees C. CONCLUSIONS: TTD was mainly affected by preincubation temperature and duration rather than by input CFU quantity or bottle type for the 3 experimental bacteria.

Development and Evaluation of the Quick Anaero-system-A New Disposable Anaerobic Culture System / 대한진단검사의학회지

BACKGROUND: We developed a new disposable anaerobic culture system, namely, the Quick anaero-system, for easy culturing of obligate anaerobes. METHODS: Our system consists of 3 components: 1) new disposable anaerobic gas pack, 2) disposable culture-envelope and sealer, and 3) reusable stainless plate rack with mesh containing 10 g of palladium catalyst pellets. To evaluate the efficiency of our system, we used 12 anaerobic bacteria. We prepared 2 sets of ten-fold serial dilutions of the 12 anaerobes, and inoculated these samples on Luria-Bertani (LB) broth and LB blood agar plate (LB-BAP) (BD Diagnostic Systems, USA). Each set was incubated in the Quick anaero-system (DAS Tech, Korea) and BBL GasPak jar with BD GasPak EZ Anaerobe Container System (BD Diagnostic Systems) at 35-37degrees C for 48 hr. The minimal inoculum size showing visible growth of 12 anaerobes when incubated in both the systems was compared. RESULTS: The minimal inoculum size showing visible growth for 2 out of the 12 anaerobes in the LB broth and 9 out of the 12 anaerobes on LB-BAP was lower for the Quick anaero-system than in the BD GasPak EZ Anaerobe Container System. The mean time (+/-SD) required to achieve absolute anaerobic conditions of the Quick anaero-system was 17 min and 56 sec (+/-3 min and 25 sec). CONCLUSIONS: The Quick anaero-system is a simple and effective method of culturing obligate anaerobes, and its performance is superior to that of the BD GasPak EZ Anaerobe Container System.

Clinical Relevance of Time-to-positivity in BACTEC9240 Blood Culture System / 대한진단검사의학회지

BACKGROUND: Continuous monitoring systems have allowed determination of the time-to-positivity (TTP). We evaluated the clinical relevance of TTP in the BACTEC9240 system (Becton-Dickinson, USA). METHODS: A total of 2,354 vials of positive blood cultures were evaluated over 2 months. TTP was monitored from each of BACTEC Plus Aerobic/F (BD) or Pediatric Plus/F and Lytic Anaerobic/F bottles, and the differential time-to-positivity (DTP) for blood samples drawn simultaneously via catheter and a peripheral site was determined. RESULTS: The average TTP of the positive vials was 17.4 hr, and 79.9% and 95.2% of the vials showed positivity within 24 and 48 hr, respectively. While the average TTP values for Aeromonas hydrophila, Bacillus cereus, Acinetobacter baumannii, and Streptococcus pneumoniae were less than 10 hr, those for Candida spp., anaerobes, Propionibacterium acnes, Corynebacterium spp, Bacillus spp. other than cereus, and coagulase-negative staphylococci were 35.3, 27.0, 56.8, 45.8, 23.0, and 26.3 hr, respectively. The negative predictive values of TTP over 24 hr to predict Staphylococcus aureus among staphylococci and S. pneumoniae among alpha-hemolytic streptococci were 76.7% and 100%, respectively. Enterobacteriaceae and Enterococcus faecalis showed shorter TTP in anaerobic vials than in aerobic vials. DTP of more than 2 hr was observed for 27.8%, 72.2%, and 45.5% of S. aureus, S. epidermidis, and Candida spp. CONCLUSIONS: TTP can be used to discriminate pathogens and contaminants. The shorter TTP in anaerobic vials of certain Enterobacteriaceae and Enterococcus spp. would facilitate further identification. DTP is useful for diagnosing catheter-related bloodstream infection by S. aureus, S. epidermidis, and Candida spp.

Comparação entre dois meios de coleta e transporte para estudo da microbiota conjuntival de indivíduos normais / Comparison of two transportation media for study of normal individual conjunctival microbiota

OBJETIVO: Comparar o perfil microbiológico da microbiota de pessoas sadias, obtidas do esfregaço conjuntival, utilizando zaragatoa seca transportada no meio de Stuart e zaragatoa úmida transportada no tubo de ensaio vedado com algodão. MÉTODOS: Trata-se de estudo prospectivo, com amostras selecionadas aleatoriamente, realizado no Departamento de Oftalmologia e Patologia da Santa Casa de Misericórdia de São Paulo, no mês de agosto de 2006. Foram estudados 80 olhos normais de 40 indivíduos. No olho direito de cada paciente, foi realizada a coleta de material com a zaragatoa seca, armazenando-a no meio de transporte de Stuart, no qual todo o material microbiológico obtido fica imerso no meio e o tubo hermeticamente fechado. No olho esquerdo, o material conjuntival foi colhido com a extremidade de algodão da zaragatoa umedecida em solução salina a 0,9 por cento, e armazenando-a no tubo de ensaio seco e estéril vedado com algodão. As amostras foram analisadas no prazo máximo de 2 horas após a coleta do material. RESULTADOS: Das 40 amostras coletadas com a zaragatoa úmida transportadas em tubo seco, foram identificadas bactérias em 10 (25 por cento). Das 40 amostras coletadas com zaragatoa seca transportada em meio de Stuart, foram identificadas bactérias em 12 (30 por cento). CONCLUSÃO: Os resultados do perfil microbiológico da microbiota normal conjuntival utilizando o meio de transporte da zaragatoa seca em meio de Stuart mostraram-se estatisticamente semelhantes (p= 0,85) ao comparar com o meio utilizando a zaragatoa úmida em tubo seco para semeaduras realizadas em até 2 horas após a coleta de material conjuntival.

PURPOSE: To compare the microbiological profile of normal microbiota of healthy people obtained from conjunctival smear using dry swab in Stuart's transport medium and wet swab transported in test tube sealed with cotton. METHODS: A prospective study with random samples, performed at the Departments of Ophthalmology and Pathology of Santa Casa Misericórdia de São Paulo, in August of 2006. Eighty normal eyes of 40 healthy individuals were analyzed. Samples were collected in the right eye with a dry swab and stored in Stuart's medium, where all microbiological material is kept immersed in the medium and the tube is hermetically sealed. In the left eye, the conjunctival material was collected using a swab embedded in saline solution 0.9 percent, and stored in dry and sterile test tubes sealed with cotton. The samples were analyzed within 2 hours at most after collection. RESULTS: Out of 40 samples collected with wet swab and transported in dry tube, bacteria were observed in 10 (25 percent), whereas of 40 samples collected with dry swab and transported in Stuart's medium, 12 (30 percent) had bacteria. CONCLUSION: The results of the microbiological profile of normal conjunctival microbiota using dry swab in Stuart's medium were statistically similar (p=0.85) to those obtained in wet swab in dry tube for spreading performed within 2 hours after collection of conjunctival specimen.

Use of bactec 460 TB system in the diagnosis of tuberculosis.

PURPOSE: To evaluate, the efficacy of BACTEC 460 TB system for the diagnosis of tuberculosis in a tertiary care hospital in Mumbai, India. METHODS: We compared 12,726 clinical specimens using BACTEC 460 TB system and conventional method for detection of Mycobacterium tuberculosis over a period of six years. Result: The overall recovery rate was 39% by BACTEC technique and 29% using Lowenstein-Jensen (LJ) medium. An average detection time for B actec0 460 TB system was found to be 13.3 days and 15.3 days as against 31.2 days and 35.3 days by LJ method for respiratory and nonrespiratory specimens respectively. The average reporting time for drug susceptibility results ranged from 6-10 days for the BACTEC 460 TB system. CONCLUSIONS: The BACTEC system is a good system for level II laboratories, especially in the diagnosis of extrapulmonary and smear negative tuberculosis.

Detección precoz de infección de herida operatoria en pacientes apendicectomizados / Early detection of wound infection after appendectomy

Introducción: La infección de la herida operatoria es una de las complicaciones más importantes en los pacientes apendicectomizados, y en especial los de apendicitis aguda perforada. Se diseñó un método simple para la detección precoz de esta complicación: Cultivo de punto centinela. El objetivo de este estudio es la validación de este procedimiento como método de diagnóstico precoz de los pacientes que evolucionarán con infección de herida operatoria. Materiales y Métodos: El punto centinela consiste en el cultivo de un material de sutura trenzado instalado en el tejido subcutáneo de la herida operatoria, que se retira a las 24-48 h postoperatorias. Se obtuvo una muestra de pacientes operados por apendicitis aguda perforada en el Hospital Herminda Martin de Chillán desde Octubre del 2004 hasta Marzo del 2005, según criterios de selección establecidos. Los cultivos ( ± ) se compararon con una evaluación clínica que evidenciara una infección de herida operatoria ( Gold Standard) y se analizó su coeficiente de probabilidad (CP). Resultados: Durante el estudio se operaron 129 pacientes de apendicitis aguda perforada (30,4 por ciento de todas las apendicitis). Se incluyó en el análisis a 46 y se les aplicó el método. Se obtuvo una sensibilidad de 91,1 por ciento, una especificidad de 97,1 porcentaje, un CP de cultivos (+) de 31,2, y un CP de cultivos (–) de 0,09. Discusión: El método descrito es simple y efectivo en la detección precoz de infección de herida operatoria.Se requiere mayor estudio para determinar cuán anticipado es sobre otras metodologías, así como su costo-beneficio y su utilización en otras patologías que evolucionan con infección de herida operatoria.

Isolation of mycobacteria by BACTEC 460 TB system from clinical specimens.

This article reports our experience with the BACTEC 460 TB system in the past five years and its performance characteristics and its advantages over the conventional LJ medium for mycobacterial culture. Clinical specimens (3597) from patients suspected to have tuberculosis were submitted for mycobacterial culture between May 2000 and August 2005 and were processed using the BACTEC 460 TB system. Pulmonary samples were 1568 while the extra pulmonary samples were 2029. BACTEC achieved detection of 681 (18.93%) M. tuberculosis cases (499- pulmonary, 182- extrapulmonary) with a recovery time shorter by 13.2 days compared to conventional method, while 577 (84.7%) were non-tuberculosis mycobacteria. Automated systems can have a great impact and thrust on an early diagnosis of tuberculosis allowing an early and appropriate management of the patient and thereby a better disease outcome.

Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods.

PURPOSE: The purpose of this study was to evaluate three methods for detection of biofilm formation in staphylococci. METHODS: For detection of biofilm formation, 152 clinical isolates of Staphylococcus spp. were screened by tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) method. RESULTS: Of the 152 Staphylococcus spp. 88(57.8%) displayed a biofilm-positive phenotype under the optimized conditions in the TCP method and strains were further classified as high 22 (14.47 %) and moderate 60 (39.4 %) while in 70 (46.0 %) isolates weak or no biofilm was detected. Though TM correlated well with the TCP test for 18 (11.8 %) strongly biofilm producing strains, weak producers were difficult to discriminate from biofilm negative isolates. Screening on CRA does not correlate well with either of the two methods for detecting biofilm formation in staphylococci. CONCLUSION: The TCP method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices.

Comparison of the automated cobas amplicor mycobacterium tuberculosis assay with the conventional methods for direct detection of mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens

To evaluate the recently available Cobas Amplicor polymerase chain reaction [PCR] system for the detection of Mycobacterium tuberculosis complex [MTBC] in well-characterized clinical specimens and to compare the results with clinical classification, conventional culture and staining techniques. Three hundred and forty-four clinical specimens consecutively received for culture of acid-fast bacilli by the laboratory of Zayed Military Hospital, Abu Dhabi, United Arab Emirates, from 2004 to 2005, were used in this study. Acid-fast bacilli and culture for tuberculosis were compared with Cobas Amplicor PCR. The final diagnostic evaluation of the Cobas Amplicor MTB showed the followings: sensitivity 73.3%, specificity 99.4%, positive predictive value 84.6%, negative predictive value 98.8% and with accuracy of 98.2% for the Cobas Amplicor MTB assay. The automated Cobas Amplicor MTB assay is suitable for routine use in clinical laboratories

Evaluation of Petrifilm™ method for enumerating aerobic bacteria in Crottin goat's cheese / Evaluación de PetrifilmTM para la enumeración de bacterias aerobias en queso de cabra Crottin

The PetrifilmTM Aerobic Count Plate (ACP) developed by 3M laboratories, is a ready-to-use culture medium system, useful for the enumeration of aerobic bacteria in food. PetrifilmTMwas compared with a standard method in several different food products with satisfactory results. However, many studies showed that bacterial counts in PetrifilmTM were significantly lower than those obtained with conventional methods in fermented food. The purpose of this study was to compare the PetrifilmTM method for enumerating aerobic bacteria with a conventional method (PCA) in Crottin goat's cheese. Thirty samples were used for the colony count. The mean count and standard deviation were 7.18 ± 1.17 log CFU g-1 on PCA and 7.11 ± 1.05 log CFU g-1 on PetrifilmTM. Analysis of variance revealed no significant differences between both methods (t = 1.33, P = 0.193). The Pearson correlation coefficient (0.971, P=0.0001) indicated a strong linear relationship between the PetrifilmTM and the standard method. The results showed that PetrifilmTM is suitable and a convenient alternative to this standard method for the enumeration of aerobic flora in goat soft cheese.

PetrifilmTM Aerobic Count Plate (ACP) desarrollado por 3M es un sistema listo para usar, empleado para el recuento de bacterias aerobias en alimentos. PetrifilmTMfue comparado con los métodos estándar en diferentes productos alimenticios con resultados satisfactorios. Sin embargo, en alimentos fermentados, algunos estudios mostraron que el recuento de bacterias aerobias en PetrifilmTM fue significativamente menor que aquellos obtenidos con los métodos convencionales (PCA). El propósito de este estudio fue comparar el método PetrifilmTM para el recuento de bacterias aerobias con un método convencional en queso de cabra Crottin. Se usaron 30 muestras para el recuento de colonias. Las medias y desviaciones estándar fueron 7,18 ± 1,17 log UFC g-1 en PCA y 7,11 ± 1,05 log UFC g-1 en PetrifilmTM. El análisis de varianza mostró que no había diferencia significativa entre ambos métodos (t = 1,33, P = 0,193). El coeficiente de correlación fue 0,971 ( P = 0,0001) indicando una fuerte correlación lineal. Los resultados muestran a PetrifilmTM como un método apropiado y una alternativa conveniente a los métodos estándar para la cuantificación de flora aeróbica en queso blando de cabra.

Blood culture in India: a proposal for a national programme for early detection of sepsis.

Septicaemia is a major contributor of mortality. Blood culture is the essential investigation for the management of sepsis. Due to lack of resources blood culture is an irregularly used investigation in India. A three-tier level of development is being proposed to develop the blood culture based national programme for early detection of sepsis. The plan envisages the establishment of manual blood culture based elementary system in the health centre and district hospital level (Level 1), direct Gram stain and direct antibiotic sensitivity testing from the "positive" blood culture broths at the medical college hospital level (Level 2) and development of automated methods, enhancement of quality control and safety measures, clinical liaison and re-orientation of microbiology training at the tertiary care centre level (Level 3).

Falsos cultivos positivos por contaminación cruzada en laboratorios de tuberculosis / False-positive cultures due to cross contamination in tuberculosis laboratories

Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.

Detection of Mycobacterium tuberculosis from sputum collected on filter paper and stored at room temperature for 5 days by PCR assay and culture.

The efficacy of PCR assay and culture for direct detection of M. tuberculosis (MTB) from sputum specimens collected on filter paper and stored at room temperature for 5 days was evaluated in comparison with conventional culture of fresh sputum specimen. A total of 231 sputum specimens were examined. MTB was recovered from 124 samples by culture from fresh sputum samples before storage. The culture positivity rate was significantly decreased to 70 per cent after 5 day's storage on filter paper. For PCR assay, a fragment of 377-bp of the IS6110 sequence was amplified and detected using nested PCR. Compared with culture results performed on fresh sputum samples, the sensitivity, specificity, and efficiency for the nested PCR were 96.0, 97.2 and 96.5 per cent, respectively. The nested PCR showed sensitivity and specificity with no significant difference (p>0.05) from culture of fresh sputum specimens. CONCLUSION: The collection and storage of sputum on filter paper at room temperature for 5 days had no apparent effect on the performance of nested PCR. Sputum samples collected by this method could be sent by post in a minimum of space and inexpensive way and will enable a large number of samples collected in the field or from peripheral health centers to be sent to central laboratories for analysis by trained technicians and under a well-equipped and well-established quality control system. The rapid and reliable detection by PCR-based assay will be helpful for optimal patient management of therapy and effective control of tuberculosis.