PKO: alternative method for isolating mycobacteria from sputum

Abstract

We elaborated an alternative culture method, which we denominated PKO (initials in tribute of respect to Petroff, Kudoh and Ogawa), for isolating Mycobacterium tuberculosis from sputum for diagnosis of pulmonary tuberculosis (TB), and to compare its performance with the Swab and Petroff methods. For the technique validation, sputum samples from patients suspected of pulmonary TB cases were examined by acid-fast microscopy (direct and concentrated smear), PKO, Swab and Petroff methods. We found that Petroff and PKO methods have parity in the effectiveness of M. tuberculosis isolation. However, by the PKO method, 65% of isolated strains were detected in a period of ≤15 days, while by the Petroff method the best detection was in an interval of 16-29 days (71%). In positive smear samples, the average time of PKO isolation is only superior to the one related for Bactec 460TB. In conclusion, the exclusion of the neutralization stage of pH in the PKO reduces the manipulation of the samples, diminishes the execution time of the culture according to the Petroff method and facilitates the qualification of professionals involved in the laboratorial diagnosis of Tuberculosis. KEYWORDS Diagnostic method, Mycobacterium tuberculosis, PKO, Tuberculosis PKO -Um método alternativo para o isolamento de micobactérias em amostras de escarro RESUMOFoi elaborado um método de cultivo alternativo, denominado por nós PKO (iniciais referentes à Petroff, Kudoh e Ogawa), para o isolamento do Mycobacterium tuberculosis em amostras de escarro para o diagnóstico da tuberculose pulmonar (TB). Para validação da técnica, amostras de escarro de pacientes suspeitos de TB foram submetidas aos métodos de baciloscopia (direta e pós-concentração), PKO, Swab e Petroff. A análise comparativa entre o método de Petroff e o PKO mostrou paridade de resultados em relação ao isolamento e número de colônias de M. tuberculosis. Porém, pelo método PKO, 65% das cepas isoladas foi detectada em um período ≤ 15 dias, enquanto que pelo método de Petroff a melhor detecção ocorreu em um intervalo de 16-29 dias (71%). O tempo médio de isolamento pelo PKO é somente superior ao sistema comercial Bactec 460TB em amostras positivas na baciloscopia. A exclusão da etapa de neutralização de pH no método PKO reduz a manipulação das amostras, diminui o tempo de execução do cultivo em relação ao de Petroff e facilita o treinamento de profissionais que realizam o diagnóstico laboratorial da TB.

Single-Nucleotide Variants in the AIM2 – Absent in Melanoma 2 Gene (rs1103577) Associated With Protection for Tuberculosis

“…The study population was recruited from Policlínica Cardoso Fontes, a sanitary pneumology center (Manaus, AM, Brazil). The patients with PTB were positive for M. tuberculosis , which was determined through a molecular rapid test (TRM, GeneXpert MTB/RIF) ( 47 ) or through sputum smears and PKO culture method ( 47 , 48 ). The diagnosis of EPTB was based on recommendations of Brazilian National Guidelines for the Control of Tuberculosis ( 49 ).…”

Toll-Like Receptor-1 Single-Nucleotide Polymorphism 1805T/G Is Associated With Predisposition to Multibacillary Tuberculosis

“…Sputum samples were submitted to microscopy ( 12 ) and solid culture in according to PKO method (initials in tribute to Petroff, Kudoh, and Ogawa) ( 13 ). A smear of culture, stained by Kinyoun method, was carried out to confirm if the culture was composed of acid-fast bacilli.…”

Alleles of HLA-DRB1*04 Associated with Pulmonary Tuberculosis in Amazon Brazilian Population

“…Bacteriological tests were performed at the Micobacteriology Laboratory, of Instituto Nacional de Pesquisas da Amazônia (INPA). The sputum samples were processed for the realization of direct or concentrated sputum smear and culture by PKO method [ 37 , 38 ]. Patients were classified as multibacillary (individuals who had positive direct sputum smear or concentrated) and paucibacillary (individuals with negative sputum smear, but with positive results for the culture method).…”