Detección de Mycoplasma pneumoniae mediante PCR-hibridación in vitro en niños con infección respiratoria / Mycoplasma pneumoniae detection using PCR-in vitro hybridization in children with respiratory infection

Antecedentes: Las infecciones respiratorias agudas (IRA) son una de las causas más frecuentes de enfermedades en el mundo y pueden asociarse a complicaciones graves, entre las que la neumonía ocupa un lugar prioritario; diversos agentes virales y bacterianos se implican en su desarrollo. Recientemente Mycoplasma pneumoniae ha adquirido gran importancia ya que estudios epidemiológicos sugieren un aumento en la incidencia de enfermedades respiratorias causadas por este patógeno. Sin embargo, los laboratorios clínicos enfrentan varias dificultades para su detección haciendo necesario el estudio de nuevas técnicas moleculares. Objetivo: Detección de M. pneumoniae mediante PCR-hibridación in vitro en niños con infección respiratoria. Métodos: Utilizando la técnica de PCR-hibridación in vitro se analizaron 36 hisopados orofaríngeos de niños entre los 0 y 9 años de edad con infección respiratoria; 36% presentaron neumonía y el 30% bronconeumonía. Resultados: La implementacion y utilización de la técnica de PCR-hibridación in vitro para la detección de M. pneumoniae permitió detectar su ADN en el 67% de los pacientes estudiados. De éstos, el 33% tenía diagnóstico de neumonía. Conclusión: La técnica de PCR-hibridación in vitro demostró ser útil en la detección de M. pneumoniae en las muestras analizadas. Los resultados de este estudio evidencian que la presencia de ácidos nucleicos de este patógeno es frecuente en las muestras respiratorias analizadas y sugiere que el microorganismo puede ser un agente etiológico frecuente de neumonías atípicas y de otras patologías respiratorias.

Background: Acute respiratory infection is considered one of the main causes of morbidity worldwide; it can be associated to serious complications. A great number of viral and bacterial agents have been implicated in the development of the disease. Recently, importance has been given to Mycoplasma pneumoniae as a respiratory pathogen, since epidemiologic studies suggest a raise in the incidence of diseases due to this microorganism. Nevertheless, nowadays clinical laboratories deal with a variety of difficulties in the diagnosis of this agent, raising the need for other molecular methods for its detection. Objective: The detection of M. pneumoniae in children with respiratory infection by a PCR-in vitro hybridization technique. Methods: 36 oropharyngeal swabs from children between 0 and 9 years of age with respiratory infection were analyzed by PCR-in vitro hybridization; 36% had pneumonia and 30% bronchopneumonia. Results: The implementation of the PCR-in vitro hybridization for the detection of M. pneumoniae in patients with respiratory infection allowed the detection of the pathogen's DNA in 67% of the patients studied. Of these, 33% had pneumonia. Conclusion: PCR-in vitro hybridization is useful for the detection of M. pneumoniae. Our results show the frequent presence of nucleic acids of M. pneumoniae in the samples studied and suggest this microorganism can be a frequent causal agent of respiratory infection.

Comparison of Three Radiolabeled Probes for PCR-Hybridization to Detect Mycobacterium tuberculosis / 대한진단검사의학회지

BACKGROUND: Three homemade radiolabeled probes to detect DNA of Mycobacterium tuberculosis by PCR-hybridization (PCRH) assay were compared in order to select the most sensitive and economic probe with the longest lifespan. METHODS: One full length probe, probe 1, prepared by the random priming method and two oligonucleotide probes, probes 2 and 3, prepared by the 5' end-labeling method were designed and assessed for sensitivity, specificity, and life span. The detection limit of each probe was determined on sample membranes containing serially diluted M. tuberculosis DNA from 5 ng to 5 fg on weekly intervals. To assess the specificity of each probe, DNA samples from 4 species of nontuberculous mycobacteria (NTM) and 9 species of bacteria other than mycobacteria were also tested. RESULTS: Each probe with PCRH showed the same detection limits of 50 fg of M. tuberculosis DNA after a 48-hr film exposure time. There were no nonspecific reactions to bacteria when tested for specificity. When we defined the life span of each probe as the longest period for detecting the lowest detection limits of M. tuberculosis DNA, the life spans of probes 1, 2, and 3 after a 3-hour film exposure were 7, 0, and 0 weeks, respectively. For probes 2 and 3, no band was visible even on the day of preparation. The life spans after a 48-hour film exposure were 9, 3, and 2 weeks for probes 1, 2 and 3, respectively. CONCLUSIONS: Probe 1, a full length probe prepared by the random priming method, was more sensitive and was a cheaper probe with a longer life span compared to probes 2 and 3, oligoprobes prepared by the 5' end-labeling method.

Evaluation of Two PCR-Hybridization Methods for the Detection of Mycobacterium tuberculosis / 대한진단검사의학회지

BACKGROUND: We evaluated two homemade polymerase chain reaction-hybridization (PCRH) assays for the detection of M. tuberculosis. METHODS: Two PCRH assays, using a digoxigenin-luminescent probe (DL-PCRH) and using a radioactive probe (R-PCRH) were developed and compared. To determine the detection limit of each assay, 10-fold serial dilution samples of M. tuberculosis DNA were tested. To determine the specificity of each assay, 4 nontuberculous mycobacteria (NTM) and 13 bacteria other than mycobacteria were tested. Sputum samples from 50 patients with tuberculosis and 26 patients with nontuberculous diseases were tested by DL-PCRH, R-PCRH, culture, AFB stain and PCR assay methods, respectively. RESULTS: Both of the DL-PCRH and R-PCRH methods showed the same detection limit of 100 fg of M. tuberculosis DNA which was a log higher than that for the PCR. Sensitivity of both methods for the detection of M. tuberculosis in sputum specimens was 84%, which were slightly inferior to that of the culture (90%) and similar to that of the PCR (82%). However, both DL-PCRH and R-PCRH methods correctly identified M. tuberculosis for all six specimens that showed weakly-positive or negative PCR results. No false-positive results were obtained for patients with nontuberculous diseases (n=26), NTM (n=4) and other bacteria (n=13). CONCLUSIONS: Although the diagnostic efficiency of the two PCRH assays were similar to that of the PCR, both methods have greater objectivity in reading results than the PCR. This suggests that the PCRH is suitable for use in large number of clinical samples for the rapid detection of M. tuberculosis.

Comparison of Acid-Fast staining, PCR, LCR, PCR=Hybridization for dection of mycobacterum tuberculosis in clinical specimens / 결핵

BACKGROUND: Mycobacterial culture is a confirmatory test to detect M.tuberculosis, but it takes at least 6 weeks to diagnose. PCR is a rapid and sensitive method, but it is known that PCR has a high false positive rate due to contamination, and a high false negative rate due to inhibitors. It is also known that LCR and PCR-Hybridization, recently developed methods, are more specific methods than PCR in terms of detection M.tuberculosis. In this study, we estimated the clinical utility of in house PCR, LCR and PCR-Hybridization for the detection of M.tuberculosis. METHODS: We evaluated 75 specimens, upon which M.tuberculosis culture based testing was requested, by PCR LCR, and PCR-Hybridization and compared results. Mycobacterial culture was performed on 3% Ogawa media for 8 weeks, and an in house PCR, LCx Mycobacterium tuberculosis assay kit(Abbott Laboratories, North Chicago, III) and the AMPLICOR M.tuberculosis test kit(Roche Molecular Systems, Inc. Branchburg, NJ, USA). RESULTS: In the view of the culture results, the sensitivities of the three tests were 40%, 80%, and 100% and their specificities were 98.6%, 94.3%, and 94.3%. CONCLUSION: LCR and PCR-Hybridization and rapid and sensitive methods for detecting M.tuberculosis in clinical laboratories.

Quantification of Serum Hepatitis C Virus in Patients with Chronic C Viral Liver Disease / 대한임상병리학회지

BACKGROUND: The quantification of hepatitis C virus (HCV) is useful in diagnosis and monitoring of HCV infection. We evaluated clinical usefulness of HCV quantification and two quantification methods using different assay principles. METHODS: HCV RNA quantities and liver function were measured in patients with different disease severity using bDNA assay (QuantiplexTM, Chiron, USA). HCV RNA loads were quantified at the time of pre/post-interferon treatment in some of them using RT-PCR hybridization assay (AMPLICORTM, Roche, USA). These two quantification methods were also compared. RESULTS: HCV RNA loads showed no significant difference according to disease severity (group I, 3.8 5.3 MEq/mL; group II, 3.8 7.4 MEq/mL; group III, 5.9 13.0 MEq/mL; P=0.181) or interferon response (complete responders, 1.5 105/mL; partial or non responders, 2.2 105/mL; P=0.670). But HCV viral loads decreased at 6th month after interferon treatment (P=0.063) and correlated poorly with liver function tests. The bDNA assay correlated well with the RT-PCR hybridization method (r2=0.854). CONCLUSIONS: The quantificaion of HCV RNA is useful in following up treatment effect but not in predicting therapeutic failure or assessment of disease severity. HCV RNA quantities are independent of liver function. The bDNA assay showed good correlation with the RT-PCR hybridization method.