IMC Journal of Medical Science https://www.imcjms.com/ Ibrahim Medical College Journal of Medical Science <![CDATA[Molecular detection of atypical microorganisms in patients with ventilator associated pneumonia]]> Shahida AkterRehana KhatunS.M Shamsuzzaman https://www.imcjms.com/registration/journal_full_text/252 2017-07-12 08:36:18 Original Article Ibrahim Med. Coll. J. 2015; 9(1): 22-25 48-h) or progressive radiographic infiltrate plus two of the following: temperature of >38°C or <36°C, blood leukocyte count of >10,000 cells/ml or <5,000 cells/ml, purulent tracheal secretions, and gas exchange degradation.2 Endotracheal tube aspirates (ETA) and blood samples were collected from clinically suspected VAP cases.  ETAwas collected using a 50 cm and 14Fr suction catheter, which was gently introduced through the endotracheal tube for a distance of approximately 25-26 cm. The ETA was obtained by suction, without instilling saline and the catheter was withdrawn from the endotracheal tube. Two milliliter of phosphate buffered saline (PBS) was injected into the lumen of the catheter with a sterile syringe to flush the exudates.The exudates were collected into a sterile 50 ml Falcon tube and transported immediately to the laboratory for further processing.7 Only one ETA sample was collected from each patient.8 Sample processing for culture and PCR: ETA was mechanically liquefied and homogenized by vortexing for one minute with glass bead (1-2 glass bead). After vortexing sample was centrifuged at 2000 rpm for 10 minutes. Supernatant was discarded using a sterile pipette and the deposit was further mixed by vortexing. The processed specimen was used for culture in recommended media, Gram staining and PCR. Extraction of DNA: One hundred µl lytic buffer (composition-tris–HCL, proteinase-K and Tween 20 solution) was added to the pellet and vortexed thoroughly. The mixture was incubated at 60°C for 2 hours. After incubation the tube was placed in a block heater (DAIHA Scientific, Seoul, Korea) at 100°C for 10 minutes. Then it was-immediately transferred to the ice and kept for 5 minutes. The solution was then centrifuged at 13000 rpm at 4°C for 10 minutes. The supernatant was used as template DNA.      PCR was performed in a final reaction volume of 25µl in a PCR tube, containing 10 µl of master mix (mixture of dNTP, taq polymerase, MgCl2 and PCR buffer), 2 µl forward primer and 2 µl reverse primer (Promega corporation, USA) 3 µl extracted DNA and 8 µl of nuclease free water. After a brief vortex, the PCR tubes were centrifuged in a micro centrifuge for few seconds.   A total of 65 suspected VAP cases were enrolled. Out of 65 VAP cases, M. pneumoniae and L. pneumophila were detected in 5 (7.69%) and 4 (6.15%) cases respectively by multiplex PCR (Table -2 and Fig -1). No C. pneumoniae was detected. Out of 9 positve cases which showed presence of M. pneumoniae and L. pneumophila, only 2 cases did not have any other pathogen by culture. Seven cases had mixed infection (Table-3) along with the presence of atypical bacteria. Table-2: Distribution of atypical bacteria identified by PCR from ETA of VAP patients (n=65) Table-3: Distribution of other organisms isolated from VAP cases positive for atypical bacteria   Fig-1: Multiplex PCR showing amplified DNA of L. pneumophila and M. pneumonia. Lane 1: negative control (DNA of Ps. Aerufinosa). Lane-2: positive control of Legionella pneumophila. Lane 3: ETA test sample. Lane 4: 100bp DNA ladder. Lane 5: ETA test sample. Lane 6: Positive control of M. pneumonia Lane 7: Negative control (DNA of K. pneumonia) The present study has revealed that atypical bacteria are important causes of VAP, besides typical bacteria which are routinely detected by culture of ETA or bronchoalveolar lavage. In the present study, 13.84% VAP cases had infection with atypical bacteria like M. pneumoniae and L. pneumophila. But it is to be noted that except 2 cases, majority of the cases had mixed infection with other bacteria. Studies in other countries also reported the presence of such atypical bacteria in VAP cases. The reported rate of infection ranged from 6.6% to 15%.12,13   1.    Richards MJ, Edwards JR, Culver DH, Gaynes RP, et al. Nosocomial infections in Medical intensive care units in the United States. Crit Care Med 1999; 27: 887-892. 3.    Chastre J, Fagon JY. Ventilator-associated pneumonia. Am J Respir Crit Care Med 2002; 165(7): 867–903. 5.    Park DR. The Microbiology of Ventilator-Associated Pneumonia. Respiratory Care 2005; 50(6): 742-765. 7.    Park DR. Antimicrobial treatment ofVentilator-associated pneumonia. Respiratory Care 2005; 50(7): 932-955. 9.    Verkooyen R, P, Willemse D, Casteren S.C.A.M, Joulandan S.A.M, et al. Evaluation of PCR, Culture and Serology for Diagnosis of Chlamydia Pneumoniae Respiratory infections. J Clin Microbiol 1998; 2301-2307. 11.  Afrin S. Bacterial causes of ventilator associated respiratory tract infection. (M.Phil Thesis) Dhaka, Bangladesh: Department of Microbiology, DMC 2013. ]]> 2024 Ibrahim Medical College. All rights reserved.