Evidencia molecular de la infección por Chlamydophila pneumoniae en reptiles de Argentina / Molecular evidence of Chlamydophila pneumoniae infection in reptiles in Argentina

En la región central de Argentina, las características epidemiológicas y moleculares de las infecciones por Chlamydophila pneumoniae en reptiles son desconocidas. Para detectar C. pneumoniae, se usó la reacción en cadena de la polimerasa anidada que amplifica el gen rpoB en muestras de hisopado cloacal de 19 reptiles. Once (57,89 %) reptiles resultaron positivos. La secuenciación y el análisis filogenético corroboraron la presencia de esta bacteria. No se detectó ADN de C. pneumoniae en la faringe ni IgM anti-C. pneumoniae en el suero de los cuidadores; sin embargo, ellos presentaron títulos muy elevados de IgG anti-C. pneumoniae. La detección de ADN de C. pneumoniae en los reptiles demostró la circulación de este agente en el centro recreativo donde se realizó este estudio, lo que podría explicar la exacerbada respuesta inmunitaria en los cuidadores; este hallazgo sugiere la presencia de un potencial ciclo zoonótico. Se reporta aquí por primera vez la detección de C. pneumoniae en reptiles en Argentina

In the central area of Argentina, the epidemiological and molecular characteristics of Chlamydophila pneumoniae infections in reptiles are still unknown. A nested polymerase chain reaction of the rpoB gene was used to detect C. pneumoniae in cloacal swab samples from 19 reptiles at a recreational area. Eleven (57.89%) reptiles were positive; the sequencing and phylogenetic analysis confirmed the presence of this bacterium. Neither C. pneumoniae DNA in the caregivers'pharynges nor IgM antibodies anti-C. pneumoniae in their serum samples were detected; however, caregivers presented very high titers of IgG anti-C. pneumoniae. The detection of C. pneumoniae DNA in reptiles demonstrated the circulation of this agent in the recreational area and could be responsible for the exacerbated immune response of the personnel handling the reptiles, which suggests a potential zoonotic cycle. This is the first report of the detection of C. pneumoniae in reptiles in Argentina

Application of three uniplex polymerase chain reaction assays for the detection of atypical bacteria in asthmatic patients in Kuwait

Respiratory infections are known to exacerbate wheezing in many asthmatic patients. We aimed to use molecular methods for the fast detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in respiratory specimens from asthmatic patients in Kuwait. We used uniplex PCR assays to detect the three atypical bacteria in clinical specimens from 235 asthmatic and non-asthmatic patients in Kuwait. A regression analysis was used to identify the risk factors related to the bacterial type. Group comparisons for similarity were conducted and correlation coefficients were calculated using SPSS statistical software. The detection limits using uniplex PCR for C. pneumoniae, L. pneumophila and M. pneumoniae were approximately 1 pg, 2.4 fg and 12 pg of DNA, respectively. M. pneumoniae PCR positivity was more common in asthmatic patients [15%] than in non-asthmatic subjects [9%] [P < 0.05]. A marked difference was observed between patients with acute asthma exacerbation [11%] and patients with chronic [stable] asthma [7%] among Kuwaiti patients; these percentages were 16% for non-Kuwaiti acute asthma patients and 14% for non-Kuwaiti chronic asthma patients [P < 0.201]. There was a weak positive correlation between asthma severity and PCR positivity for M. pneumoniae. The PCR results for C. pneumoniae and L. pneumoniae were found to be statistically insignificant. The results of this study suggest that infection with M. pneumoniae may be related to the exacerbation of asthma symptoms and could possibly be a factor that induces wheezing

Comparison of Sputum and Nasopharyngeal Swab Specimens for Molecular Diagnosis of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila

BACKGROUND: Differentiation of atypical pathogens is important for community-acquired pneumonia (CAP). In this study, we compared sputum and nasopharyngeal swabs (NPS) for use in detection of Mycoplasma pneumoniae (MP), Chlamydophila pneumoniae (CP), and Legionella pneumophila (LP), using Seeplex PneumoBacter ACE Detection Assay (PneumoBacter; Seegene). METHODS: Sputum and NPS specimens were collected from patients in 15 hospitals. DNA was extracted from sputum using QIAamp DNA Stool Mini Kit (Qiagen) and from NPS using easyMAG (bioMerieux). Both types of specimens were evaluated by multiplex PCR using PneumoBacter. To determine the diagnostic performance of this assay, sputum samples were also tested using BD ProbeTec ET Atypical Pneumonia Assay (APA; Becton Dickinson). RESULTS: Among 217 sputum and NPS, 20 (9.2%), 2 (0.9%), and 0 sputum were positive for MP, LP, and CP, respectively, whereas 8 (3.7%) NPS were positive for MP. The sputum APA test yielded 186, 206, and 204 interpretable results for MP, LP, and CP, respectively. Of these, 21 (11.3%) were positive for MP, 2 (1.0%) were positive for LP, and 0 samples were positive for CP. Compared to APA, the sensitivity and specificity of the sputum assay for MP were 95.2% and 100.0%, respectively, whereas for the NPS assay, these were 38.1% and 93.9%. Sputum testing was more sensitive than NPS testing (P=0.002). For LP and CP diagnosis, PneumoBacter and APA tests agreed 100%. CONCLUSIONS: Specimen type is crucial and sputum is preferred over NPS for simultaneous detection of MP, LP, and CP using multiplex PCR in CAP.

Chlamydia pneumoniae and stroke: is there a direct relationship? / Chlamydia pneumoniae e acidente vascular cerebral aterotrombótico: existe relação direta?

OBJECTIVE: To investigate the possible relationship between atherothrombotic stroke and Chlamydia pneumoniae. METHOD: 150 patients with carotid atherothrombosis were enrolled. The casuistic was divided in three groups: ischemic stroke (IS): 65 patients; transient ischemic attack (TIA): 26 patients; and control: 59. The IS or TIA onset was up to 30 days from the beginning of the study. Carotid atheromatoses was diagnosed by Doppler-ultrasonography. Patients with cardioembolic risk or non-atherothrombotic origin were excluded. Comparisons were done between the three groups, and within each group according to the different age sub-groups, to the main arteries affected, and to the atherogenic risk factors. Bacteria detection was done using polimerase chain reaction. RESULTS: Only one patient tested positive for C. pneumoniae belonging to the control group. CONCLUSION: These results do not suggest that C. pneumoniae participated in the onset of IS or TIA or that it has a role in carotid plaque destabilization.

OBJETIVO: Investigar a possível relação entre Chlamydia pneumoniae e acidente vascular cerebral aterotrombótico (AVC). MÉTODO: 150 pacientes com aterotrombose carotídea foram estudados. A casuística foi dividida em 3 grupos: AVC: 65 pacientes; ataque isquêmico transitório (AIT): 26 pacientes e controles: 59. O início do AVC ou AIT era até 30 dias da inclusão no estudo. A ateromatose carotídea foi diagnosticada por ultrassonografia com Doppler. Os pacientes com risco cárdio-embólico ou sem evidência de aterotrombose foram excluídos. Foram estabelecidas comparações entre os 3 grupos e dentro de cada grupo, formado sub-grupos de acordo com diferentes idades, território arterial comprometido e fatores de risco. A detecção da bactéria foi feita por reação de polimerização em cadeia. RESULTADOS: Somente um paciente, pertencente ao grupo controle, teve resultado positivo. CONCLUSÃO: Estes achados não sugerem que a C. pneumoniae participe no desencadeamento do AVC ou AIT ou que tenha papel na desestabilização da placa.

Optimization of a polymerase chain reaction (PCR) for increasing its sensitivity to detect Chlamydia pneumoniae specific genome.

Being an intracellular parasite, Chlamydia pneumoniae disseminates to organs outside the respiratory tract and causes chronic diseases in human. Nucleic acid-based method such as polymerase chain reaction (PCR) as diagnostic test has greater sensitivity and specificity than conventional microbiological techniques. The PCR protocol consisting of touchdown technique to detect C. pneumoniae DNA using major outer membrane protein gene (MOMP) was carried out in our laboratory as described in reference paper, but analytical sensitivity reported in it was not reproducible. Hence, the PCR was optimized after modifications in annealing temperature and magnesium ion concentrations. First round PCR profile with annealing at 56 degrees C for 8 cycles followed by 32 cycles with annealing temperature maintained at 54 degrees C and second round profile modified with annealing temperature maintained at 49 degrees C had resulted in 3-fold increase in clinical sensitivity. The present work highlights the importance of optimization of PCR in laboratory settings.