Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria.

BACKGROUND: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specific PCR commercial kits, paired serology, and urinary antigen. RESULTS: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1%, mPCR-IS = 57.1%, Seeplex®-IS = 52.4%, and Speed-oligo®-NPA/NPS = 11.1%, and the specificities were mPCR-NPS = 97.1%, mPCR-IS = 77.8%, Seeplex®-IS = 92.6%, and Speed-oligo®-NPA/NPS = 96.1%. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs. CONCLUSIONS: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60%; thus, better diagnostic techniques for these three bacteria are required.

Mycoplasma pneumoniae in Children With and Without Community-acquired Pneumonia. What do PCR and Serology Say?

BACKGROUND: IgM titers of Mycoplasma pneumoniae can remain high for months or years, and specific DNA can be detected in asymptomatic people. METHODS: We compared the performance of serology and PCR in children with and without community-acquired pneumonia (CAP) for the diagnosis of M. pneumoniae. RESULTS: In children with CAP, a positive test by M. pneumoniae (PCR and/or paired serology or both) were found in 13.9%. Of these, 10.3% were positive by multiplex PCR (Seeplex-Seegen), and 6.7% exhibited quadrupled titers (22 for IgG, 6 for IgM and 5 for both). Both tests were positive in 2.8% of cases. In the group without CAP, 3.3% were positive by PCR. Thirty-two percent of children with CAP and 38.3% of healthy children had IgM titers >11 in the acute phase. CONCLUSIONS: The detection of IgM is not useful for diagnosing acute M. pneumoniae infection, and a positive PCR result can be due to colonization and not infection. New and better diagnostic techniques are required.

[Molecular and immunological analyses suggest the absence of hydrophilic surface proteins in Leishmania (Viannia) panamensis]. / El análisis molecular y el inmunogénico sugieren la ausencia de las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis.

INTRODUCTION: The genus Leishmania is divided into two subgenera: Leishmania and Viannia. The two subgenera present several important differences such as the pathology they cause in susceptible hosts, their in vitro growth behavior, their genetic characteristics, and the expression pattern of several proteins, including those of the hydrophilic surface protein family. OBJECTIVE: To characterize the hydrophilic surface protein family in Leishmania (Viannia) panamensis. MATERIALS AND METHODS: The hasp genes were amplified in L. (V.) panamensis, using specific primers previously designed to amplify this gene in Leishmania (Leishmania) major. The PCR products were cloned, sequenced, and the sequences analyzed using common bioinformatics tools. Secondly, a serological screening was undertaken with an enzyme-linked immunosorbent assay and Western blot to detect specific antibodies against the hydrophilic surface recombinant protein from L. (L.) major. RESULTS: A copy of a pseudogene was amplified in L. (V.) panamensis which was 60% homologous with the L. (L.) major orthologous gene. Antibodies responded to the hydrophilic surface recombinant proteins only in sera from patients with visceral leishmaniasis [Leishmania (Leishmania) chagasi]. CONCLUSION: These results suggest the lack of a functional hasp gene in L. (V.) panamensis, suggesting probably the loss of the complete gene family in this species of the Viannia subgenus.

Genotyping and macrolide resistance of Mycoplasma pneumoniae identified in children with community-acquired pneumonia in Medellín, Colombia.

OBJECTIVES: The aim of this study was to describe the genotypes and the main characteristics of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae in hospitalized children in Medellín and neighboring municipalities during the period 2011-2012. METHODS: The M. pneumoniae genotype was determined by PCR and sequencing of the p1 and 23S rRNA genes from induced sputum samples and nasopharyngeal swabs (NPS). Samples were obtained from children with CAP who were hospitalized in 13 healthcare centers. In addition, a spatio-temporal analysis was performed to identify the potential risk areas and clustering of the cases over time. RESULTS: A variant of type 2 was the dominant genotype in the induced sputum (96.1%) and NPS (89.3%) samples; the type 1 variant was identified in 3.9% and 10.7% of these samples, respectively. No strains with mutations in the 23S rRNA gene associated with macrolide resistance were found. The cases in Medellín were mainly concentrated in the northeastern areas and western districts. However, no temporal relationship was found among these cases. CONCLUSIONS: A variant of type 2 of M. pneumoniae prevailed among children with CAP during the study period. No strains with mutations associated with macrolide resistance were found.

[Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia]. / Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumonía.

INTRODUCTION: The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests. OBJECTIVE: The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia. MATERIALS AND METHODS: Cross-sectional evaluation diagnostic tests were used in 166 immunosuppressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were stained with toluidine blue and fluorescent antibody. The proportion of positive results from each stain and concordance between them were determined. RESULTS: Twenty-four cases (14.5%) of P. jirovecii were detected in bronchoalveolar lavage samples. Of them, 21 were positive by both toluidine blue and fluorescent antibody stains, whereas 3 cases were detected by fluorescent antibody alone. None of the 166 oropharyngeal wash samples were positive by either of these techniques. No significant differences were found between proportions from positive results (p=0.63). Concordance (kappa coefficient) between both stains was 0.92 (95% CI: 0.84-1.00). CONCLUSIONS: Both techniques were useful to diagnose P. jirovecii in bronchoalveolar lavage samples. However, toluidine blue stain did not detect 12% of fluorescent antibody positive cases. Oropharyngeal wash samples do not provide sufficient material for the microscopic identification of this fungus.

Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumoníA / Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia

Introducción. El diagnóstico de neumonía por Pneumocystis jirovecii se fundamenta en la visualización microscópica del hongo en secreciones respiratorias. Técnicas moleculares recientes también lo han detectado en muestras de orofaringe, pero su utilidad diagnóstica es discutible. En Colombia, hay poca información al respecto. Objetivo. Comparar el rendimiento de dos coloraciones, azul de toluidina O e inmunofluorescencia directa, en muestras de lavado broncoalveolar y lavado orofaríngeo en pacientes inmunocomprometidos con neumonía. Materiales y métodos. Se llevó a cabo un estudio transversal de evaluación de pruebas diagnósticas en 166 pacientes inmunocomprometidos con sospecha de neumonía por P. jirovecii. Por protocolo, las muestras de lavado broncoalveolar y orofaríngeo se citocentrifugaron y se colorearon con azul de toluidina e inmunofluorescencia. Se determinó la proporción de resultados positivos con ambas tinciones en cada una de las muestras, y la concordancia entre ellas. Resultados. Se detectaron 24 casos de neumonía por P. jirovecii en las muestras de lavado broncoalveolar (14,5 %), 21 de los cuales fueron positivos por ambas pruebas, mientras que tres casos se detectaron sólo por inmunofluorescencia. Ninguna de las 166 muestras de lavado orofaríngeo fue positiva por cualquiera de estas técnicas. Al comparar las proporciones de resultados positivos, no se encontraron desacuerdos significativos (p=0,63). La concordancia (coeficiente kappa) entre ellas fue de 0,92 (IC95%: 0,84-1). Conclusiones. Ambas coloraciones son útiles para diagnosticar neumonía por P. jirovecii en muestras de lavado broncoalveolar. Sin embargo, el azul de toluidina no detecta, aproximadamente, el 12 % de los casos positivos por inmunofluorescencia. Las muestras de lavado orofaríngeo no son apropiadas para detectar microscópicamente P. jirovecii.

Introduction. The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests. Objective. The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia. Materials and methods. Cross-sectional evaluation diagnostic tests were used in 166 immunossupressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were stained with toluidine blue and fluorescent antibody. The proportion of positive results from each stain and concordance between them were determined. Results. Twenty-four cases (14.5%) of P. jirovecii were detected in bronchoalveolar lavage samples. Of them, 21 were positive by both toluidine blue and fluorescent antibody stains, whereas 3 cases were detected by fluorescent antibody alone. None of the 166 oropharyngeal wash samples were positive by either of these techniques. No significant differences were found between proportions from positive results (p=0.63). Concordance (kappa coefficient) between both stains was 0.92 (95% CI: 0.84-1.00). Conclusions. Both techniques were useful to diagnose P. jirovecii in bronchoalveolar lavage samples. However, toluidine blue stain did not detect 12% of fluorescent antibody positive cases. Oropharyngeal wash samples do not provide sufficient material for the microscopic identification of this fungus.

El análisis molecular y el inmunogénico sugieren la ausencia de las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis / Molecular and immunological analyses suggest the absence of hydrophilic surface proteins in Leishmania (Viannia) panamensis

Introducción. Los dos subgéneros en los cuales se divide el género Leishmania: Viannia y Leishmania, presentan diferencias significativas en las manifestaciones clínicas que causan, en su comportamiento de crecimiento en cultivos in vitro, en sus características genéticas y en la expresión de varias proteínas, entre ellas las de la familia hidrofílica de superficie superficie. Objetivo. Caracterizar las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis. Materiales y métodos. Se amplificaron los genes hasp en L. (V.) panamensis usando cebadores específicos para la especie Leishmania (Leishmania) major. Los productos de la amplificación fueron clonados, secuenciados y analizados con herramientas bioinformáticas. Posteriormente, se realizó un análisis serológico por medio de ensayo inmunoabsorbente ligado a enzimas y Western blot para detectar la presencia de anticuerpos específicos contra las proteínas hidrofílicas recombinantes de superficie de L. (L.) major en sueros de pacientes con leishmaniasis de zonas endémicas de Colombia. Resultados. Se encontró una copia de un pseudogen en L. (V.) panamensis, el cual presentó una identidad del 60 por ciento con el gen haspa de L. (L.) major. Sólo se encontraron anticuerpos contra las proteínas recombinantes de superficie hidrofílicas en sueros de pacientes con leishmaniasis visceral. Conclusión. Estos resultados sugieren que no existe ninguna copia de un gen funcional hasp en L. (V.) panamensis, lo que indica una pérdida de la familia de genes en esta especie de Leishmania perteneciente al subgénero Viannia.