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Easy and Rapid Diagnosis of Mycoplasma Pneumonia: Is It Possible? Reham M

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Easy and Rapid Diagnosis of Mycoplasma Pneumonia: Is It Possible? Reham M Original article 394 Easy and rapid diagnosis of Mycoplasma pneumonia: is it possible? Reham M. Elkolalya, Maii A. Shams Eldeenb Background Atypical pneumonia (AP) with its different Results Using the PCR method; M. pneumonia was 42%, pathogens comprises a reasonable ratio of community- mostly by bronchoscopic lavage because of dry cough, with acquired pneumonia. Mycoplasma pneumoniae (M. significant correlation to arrhythmia, disturbed pneumoniae) constitutes a known pathogen causing AP with consciousness, and positive radiologic infiltrations (74, pulmonary and extrapulmonary symptoms that necessitate 65,76%, respectively). early diagnosis and treatment. Serology and culture give Conclusion PCR is considered a highly specific diagnostic diagnosis but after few days of infection onset. method for M. pneumonia. AP incidence is high in our region Aim Study the incidence of M. pneumonia using PCR and with special consideration to M. pneumonia as a causative relation to clinical symptoms. agent with high percentage. Egypt J Bronchol 2019 13:394–402 Settings and design Comprehensive, prospective study. © 2019 Egyptian Journal of Bronchology Materials and methods A total of 80 patients with suspected Egyptian Journal of Bronchology 2019 13:394–402 AP were examined for clinical symptoms and signs such as cough, crepitations, arrhythmia and conscious level, and Keywords: mycoplasma, PCR, pneumonia sputum was investigated using PCR for M. pneumoniae. Departments of, aChest, bPharmaceutical Microbiology, Faculty of Those with dry cough were subjected to fiberoptic- Medicine, Tanta University, Tanta, Egypt bronchoscopic bronchoalveolar lavage and the fluid was *Correspondence to Correspondence to Reham M. Elkolaly, MD, Chest examined by PCR. Department, Tanta University Hospitals, ElGharbyia, Tanta, 31511, Egypt. Tel: +20 122 765 0268; Statistical analysis Data were analyzed with the SPSS 22 e-mail: [email protected] software package. Received 25 June 2018 Accepted 6 February 2019 Introduction It is crucial to diagnose the causative pathogens early, Atypical pneumonia (AP) comprises a reasonable ratio rapidly, and accurately to avoid unnecessary antibiotic of pneumonia requiring hospital admission because of use and to treat and protect the patient from serious unusual presentation and complications [1,2]. It is complications [15]. ‘atypical’ because of unusual causative organisms and atypical clinical picture [3]. Real-time PCR is a recent technique that varies in reliability according to age, disease severity, and Itpreviouslyincludedrickettsia,viruses,andfungi[4],butby specimens’ type [16,17]. It is a rapid technique with time it became restricted to three pathogens: Mycoplasma high sensitivity [18]. pneumoniae (M. pneumoniae), Chlamydophila pneumoniae, and Legionella pneumophila [3]. Aim M. AP represents a rising cause of pneumonia either single The aim was to evaluate the incidence of pneumoniae [5,6] or mixed with other bacteria [7] and the highest in patients with AP using real-time percentage of AP is caused by M. pneumonia [6,8]. PCR in addition to its relation to clinical picture. Patients with M. pneumonia may complain of dry Patients and methods cough, dyspnea, exacerbation of chronic obstructive In this comprehensive prospective study, 80 pulmonary disease [9], and otitis media [10] in nonredundant clinical specimens were collected addition to nonspecific symptoms such as myalgia, between January 2017 and January 2018, from the heart affection [6] and leukopenia [11], all of which Chest Department, Tanta University Hospital of may aggravate disease severity. Egypt. Accurate diagnosis of M. pneumonia rarely depends on the clinical picture [12], but necessitates other diagnostic methods like culture and serological This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 confirmation. However, they are time consuming License, which allows others to remix, tweak, and build upon the work and require complicated techniques [13] that limit non-commercially, as long as appropriate credit is given and the new their commercial use [14]. creations are licensed under the identical terms. © 2019 Egyptian Journal of Bronchology | Published by Wolters Kluwer - Medknow DOI: 10.4103/ejb.ejb_46_18 Mycoplasma pneumonia diagnosis Elkolaly and Shams Eldeen 395 Inclusion criteria: in the form of suspicion of AP (2) Purification: DNA concentration was measured at infection and clinical and radiological data in the 260 nm and was greater than 10 ng/ml and the form of low-grade fever, cough with scanty sputum, A260/A280 ratio was greater than 1.8. cardiac arrhythmia, increased heart rate, wheeze, (3) Preparation of master-mix: for each sample, four crepitations, pleural rub, chest radiography patchy separate PCR reactions were prepared, including infiltrations with absent consolidation or pleural controls for positive PCR control, no template effusion [19–21]. control, microbial DNA positive control, and microbial DNA QPCR assay. Patients were excluded from the study if they were on mechanical ventilation, with lung tumor, tuberculosis, The kit was utilized to calculate the load of M. or hospital-acquired pneumonia (infection after 48 h of pneumoniae by targeting the cytadhesin P1 gene admission). (130bP) which is particular for M. pneumoniae. All patients were subjected to complete history taking, Real-time PCR primers for P1 cytadhesin gene of M. full clinical examination, general and local chest pneumoniae: examination, routine laboratory investigation, for example, complete blood picture, erythrocyte Forward primer: CCAACCAAACAACAACGT sedimentation rate, C-reactive protein for collected TCA. samples and chest radiology [chest radiography and computed tomography (CT) when needed]. Reverse PRIMER: TAACGGCAACACGTAATC AGGTC. Sample collection Total volume per sample was 25 ml. The PCR mixture consisted of 12.5 ml microbial qPCR 12.5 ml, 1 ml (1) Sputum samples: patients sit upright or at 45°, microbial DNA qPCR assay, 5 ng genomic DNA inspired deeply and hold and then coughed with sample, and variable amounts of microbial DNA-free expectoration into a clean container. water. (2) Patients with dry cough were subjected to fiberoptic bronchoscopy (PENTAX EB-1970UK Europe Plate preparation GmbH Julius Vosseler Strasse 104, Hamburg, After vortexing for 1–2 s (for mixing), the mix was put Germany) and bronchoalveolar lavage: under into the PCR wells. A plastic PCR plate was sealed complete aseptic technique; bronchoscope was with an adhesive optical cover and was inserted into the – introduced until peripheral bronchioles, 30 50 ml PCR instrument. of sterile saline was injected throughout. Saline was aspirated and collected in a sterile plastic container Performing real-time PCR with a firmly fitted cover. (1) Cycling condition: amplifications were done using All samples were located in tubes containing PBS, a Light-Cycler (step 1; Applied Biosystem, Foster vortexed and then stored at −20°C until processed [22]. City, California, USA). (2) The threshold (CT) was calculated for each well using the cycler’s software (Roche Diagnostics Ltd, Processing Forrenstrasse, CH-6343 Rotkreuz, Switzerland). Quantitativereal-timePCR was done forM.pneumoniae in different respiratory samples using Microbial DNA Standard curve generation QPCR assay kit (M. pneumoniae) (catalog no. 330033; With absolute standard curve, the copy number of the QIAGEN Sciences, Germantown, USA). Sensitivity target was known. The template was accurately and specificity of real-time PCR assays in the quantified and the sample is accurately determined to detection of M. pneumoniae is 100%. As reported by contain 1×1011 copies and diluted 10-fold eight times Templeton et al. [23], the sensitivity of the real-time down to 1×103 and the PCR was performed on each PCR assay reduceswith thedelay incollectionofsamples dilution for three replicates. from the onset of the disease. The standard curve correlated the copy number with a (1) Nucleic acid extraction from 200 μl of the particular CT. The copy number values for the specimen was done. The volume was reduced to unknown samples were derived by comparison to 100 μl, and 7 μl aliquots were stored at −70°C. this standard curve. 396 Egyptian Journal of Bronchology, Vol. 13 No. 3, July-September 2019 Informed consent was taken from patients or relatives PCR group (46 patients) with no significant difference for the procedures and for usage of their medical data in between the two groups as regards age, sex, bronchial the present study. asthma, and steroid intake as risk factors for M. pneumonia infection (Table 3) and also regarding Tanta university ethics committee approved the study erythrocyte sedimentation rate and leukocytic count protocol. in both groups (Table 4). Statistical analysis But there was statistically significant differences The data were analyzed with the SPSS 22 software between the two groups as regards pleural rub, chest package (SPSS Inc., Chicago, Illinois, USA). wheeze, crepitations, state of consciousness, heart rate, Quantitative variables presented as mean±SD and and arrhythmia (Table 3). In addition, there was a range. Qualitative variables presented as percentages. significant difference between the type of examined χ2 t Comparisons between groups were done with or sample for M. pneumonia (Table 4). independent test. A P value of less than 0.05 was considered statistically significant. The PCR positive group and the PCR negative
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