ABSTRACT
Introducción: La baciloscopia es la herramienta primaria en el diagnóstico de la tuberculosis (TBC) pulmonar activa, siendo esta la técnica más utilizada internacionalmente en la búsqueda de casos infecciosos. El control de calidad consiste en la relectura de las láminas por un observador altamente calificado. Objetivo: Evaluar y destacar la importancia del control de la calidad de la baciloscopia en los laboratorios provinciales encargados del diagnóstico de TBC en Cuba. Material y Métodos: Este estudio fue realizado en el Laboratorio Nacional de Referencia e Investigaciones de Tuberculosis, Lepra y Micobacterias del Instituto de Medicina Tropical "Pedro Kourí", La Habana, Cuba. Fueron evaluadas 2.676 láminas recibidas en el período de enero de 2013-diciembre de 2014, procedentes de los diferentes Centros Provinciales de Higiene, Epidemiología y Microbiología de Cuba, incluido el Municipio Especial Isla de la Juventud. Resultados: Hubo 2.664 (99,5%) láminas concordantes, la concordancia obtenida para las láminas positivas fue 96,5% y las negativas 99,8%. Se identificaron 12 errores de lectura: 7 (3,5%) falsos positivos, 5 (0,2%) falsos negativos. Se calificaron láminas con calidad de la muestra adecuada en 2.039 (76,2%), presentaron deficiencias en la realización de la extensión 1.464 (54,7%), y la tinción fue adecuada en 2.343 (87,6%). El índice de kappa fue de 0.9674. Conclusión: Aunque hubo una adecuada concordancia entre las observaciones realizadas, se recomienda mejorar la calidad del extendido, mantener programa de entrenamiento al personal que realiza esta actividad, al igual que las supervisiones periódicas por parte de especialistas, para continuar mejorando la calidad del diagnóstico.
Background: Baciloscopy is the primary tool for pulmonary tuberculosis diagnosis, being this technique the most used internationally in the search for infectious cases. Quality control is the process of the rechecking smears by a highly qualified observer. Aim: To evaluate and highlight the importance of quality control of smear microscopy in the Provincial Laboratories diagnosticians of Tuberculosis in Cuba. Methods: This study was conducted at the National Reference Laboratory and Research in Tuberculosis, Leprosy and Mycobacteria in the Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba, Were evaluated 2676 smears received from January 2013 to December 2014, from Provincial Centers of Hygiene, Epidemiology and Microbiology of Cuba, including the special municipality Isla de la Juventud. Results: 2,664 (99.5%) were concordant smears, the correlation obtained for the positive smears were 96.5% and 99.8% for negative. Were identified12 reading errors: 7 (3.5%) false positive and 5 (0.2%) false negatives. Slides were classified with adequate quality of smears in 2039 (76.2%), showed difficulties in realizing the extension in 1464 (54.7%) and staining were adequate in 2343 (87.6%). The kappa index was 0.9674. Conclusion: Although there was good agreement between observations it is recommended to improve the quality of extended, maintain staff training program that performs this activity, like regular supervision by specialists, to further improve the quality of diagnosis.
Subject(s)
Humans , Quality Control , Sputum/microbiology , Tuberculosis/diagnosis , Microscopy/standards , Mycobacterium tuberculosis , Reference Standards , Staining and Labeling/methods , Predictive Value of Tests , Cuba , Diagnostic ErrorsABSTRACT
Background: Granulomatous lesions occur in tuberculosis (TB), other infections, toxic, allergic, and autoimmune diseases among others. In absence of a an acid-fast bacilli (AFB) confirmation of TB is necessary. Objective: To assess the efficacy of PCR for TB detection and to correlate with granuloma histology and AFB staining. Methods: We analyzed 380 fixed paraffin-embedded tissues (PETs) of granulomas with and without caseous necrosis; suppurative; sarcoidal; or of chronic nonspecific nature. Nested PCR-IS6110 for Mycobacterium tuberculosis complex (MTB) and a nested pan-Mycobacterium for the hsp65 gene were used for Mycobacterium spp detection. Results: PCR was more sensitive than AFB staining for all five catageories of granulomas: G1: PCR 71%, AFB staining 28%. G2: PCR 37%, AFB 8%. G3: PCR 17%, AFB staining 7%. G4: PCR 8%, AFB staining 4%. G5: PCR 6%, AFB staining 0%. Conclusions: Molecular diagnosis of TB using PCR-based testing is a fast, efficacious and sensitive method that increased the accuracy of PET histological diagnosis associated with granulomatous lesions.
Introducción: Lesiones granulomatosas ocurren en tuberculosis (TBC), otras infecciones, condiciones tóxicas, alérgicas y autoinmunes, entre otras. Con baciloscopia negativa, es necesario confirmar el diagnóstico de TBC. Objetivo: Evaluar la eficacia de la RPC para detectar TBC comparado con baciloscopia en relación a la histología del granuloma. Métodos: Analisis de 380 tejidos fijados en formalina e incluidos en parafina (TFFP) con diferentes tipos de granulomas: con necrosis caseosa; sin necrosis caseosa; supurativo; sarcoidal; a cuerpo extraño/inespecífico. Utilizamos RPC anidada-IS6110 para detección del complejo Mycobacterium tuberculosis (MTB) y una pan-RPC anidada-hsp65 para Mycobacterium spp. Resultados: La detección de TBC mediante RPC fue significativamente superior a baciloscopia en los cinco tipos de granuloma: G1: RPC 71%, baciloscopia 28%; G2: RPC 37%, baciloscopia 8%; G3: RPC 17%, baciloscopia 7%; G4: RPC 8%, baciloscopia 4%; G5: RPC 6%, baciloscopia 0%. Conclusión: El diagnóstico de TBC por RPC es un método rápido, eficaz y de gran sensibilidad, que aumenta la precisión del diagnóstico diferencial de lesiones granulomatosas de TFFP procesados rutinariamente en histopatología.
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Young Adult , DNA, Bacterial/genetics , Granuloma/microbiology , Mycobacterium tuberculosis/genetics , Tuberculosis/diagnosis , Diagnosis, Differential , Formaldehyde , Granuloma/diagnosis , Paraffin Embedding , Polymerase Chain Reaction , Predictive Value of Tests , Sensitivity and Specificity , Staining and Labeling , Tissue FixationABSTRACT
Tuberculosis control is a priority for the Ministry of Health policies in Brazil. In the present work, the detection of Mycobacterium tuberculosis by the Polymerase Chain Reaction (PCR) was standardized, and the laboratory diagnosis of pulmonary tuberculosis was evaluated comparing baciloscopy, culture and PCR tests. The study was carried out with 117 sputum samples from different patients suspected of having pulmonary tuberculosis, for whom physicians had ordered a baciloscopy test. Baciloscopy was performed using the Ziehl-Neelsen method, and culture was performed by incubation of treated samples in Lowenstein-Jensen's medium at 37ºC for eight weeks. For PCR, DNA was amplified with a specific pair of primers to the M. tuberculosis complex, with a resulting product of 123 bp from the insertion element IS6110. Three (2.56 percent) samples presented a positive baciloscopy result and a positive PCR result (100 percent agreement), and nine (7.69 percent) presented Mycobacterium sp. growth in culture (P= 0.1384). Among six samples with positive results in culture, one was identified by PCR-RFLP as belonging to the M. tuberculosis complex and one was identified as a non-tuberculosis mycobacteria. Sensitivity and specificity of PCR compared to culture were 33.3 percent and 100 percent, respectively.
A tuberculose é um dos agravos prioritários para as políticas do Ministério da Saúde. No presente trabalho, o método de detecção de Mycobacterium tuberculosis pela Reação em Cadeia da Polimerase (PCR) em amostras de escarro foi padronizado e o diagnóstico laboratorial da tuberculose pulmonar foi avaliado, comparando-se as metodologias de baciloscopia, cultura e PCR. Foram analisadas 117 amostras de escarro de diferentes pacientes com suspeita de tuberculose pulmonar, com solicitação de baciloscopia. A baciloscopia foi realizada com a coloração de Ziehl-Neelsen e a cultura pela semeadura das amostras em meio de Lowenstein-Jensen, incubadas a 37ºC por oito semanas. Para realização da PCR, o DNA foi amplificado com um par de oligonucleotídeos específicos para o complexo M. tuberculosis, resultando em um produto de 123 pb do elemento de inserção IS6110. Das 117 amostras analisadas, três (2,56 por cento) apresentaram baciloscopia positiva e PCR positiva para M. tuberculosis (concordância de 100 por cento), e nove (7,69 por cento) tiveram crescimento de Mycobacterium sp. na cultura (P= 0,1384). Das seis amostras que tiveram resultado positivo somente por cultura, uma foi identificada ainda como pertencente ao complexo M. tuberculosis por PCR-RFLP, e outra foi identificada como micobactéria não tuberculosa. A sensibilidade e a especificidade da baciloscopia e da PCR em relação à cultura foram 33,3 por cento e 100 por cento, respectivamente.
Subject(s)
Humans , Clinical Laboratory Techniques , In Vitro Techniques , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction , Tuberculosis, Pulmonary , Culture Media , Methods , Patients , MethodsABSTRACT
Agencias internacionales para el control de la tuberculosis (TBC) (WHO, IUATLD, KNCV) consideran fracasado el tratamiento anti-tuberculoso si finalizado el mismo, persiste la baciloscopia (BK) positiva. Describimos el caso de un paciente con TBC pulmonar que, tras completar correctamente el tratamiento por 6 meses, continuaba siendo bacilífero. Después de 9 meses con terapia específica, a pesar de una BK positiva persistente, su tratamiento fue suspendido, porque estaba asintomático, con el peso corporal recuperado y ninguna muestra de esputo arrojó un cultivo positivo. Una revisión de la literatura médica permitió concluir que una BK positiva después de completar el tratamiento ocurre hasta en 5 por ciento de los casos pero, no siempre indica un fracaso en la terapia como señalan definiciones internacionales. Para una mejor evaluación de este tipo de pacientes, en circunstancias en que no se dispone del cultivo, discutimos un algoritmo que toma en cuenta la gravedad de la enfermedad al ser diagnosticada, el cumplimiento del tratamiento, la condición clínica del paciente al finalizar el mismo y la tasa de resistencia in vitro de M. tuberculosis en la comunidad, para así definir mejor un fracaso de tratamiento.
International agencies for tuberculosis (TB) control (WHO, IUATLD, KNCV) consider the presence of acid-fast bacilli (AFB) in the sputum smear after completion of TB therapy as treatment failure. We describe a case of a pulmonary TB patient who received and correctly completed 6 months of antituberculous treatment with a positive AFB sputum smear. Treatment was continued and sputum cultures were performed for resistance determination. After 9 months, despite the persistence of AFB in the sputum smear, the treatment was suspended because none of the sputum samples yielded a positive culture, indicating no viable bacilli. In addition, the patient was asymptomatic and had recovered his body weight. An exhaustive review of medical literature allowed us to conclude that a positive sputum smear after therapy has been detected in up to 5 percent of cases and does not always represent a treatment failure as defined by international guidelines. For scenarios where culture methods are not available we propose a scheme to evaluate these patients. This includes compliance with the treatment, severity of the disease at the moment of diagnosis, clinical symptoms after specific therapy and rate of in vitro resistance of M. tuberculosis in the community.