IMC Journal of Medical Science
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Ibrahim Medical College Journal of Medical ScienceShahida AkterRehana KhatunS.M Shamsuzzaman https://www.imcjms.com/registration/journal_full_text/252
2017-07-12 08:36:18Original ArticleIbrahim 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instilling saline and the catheter was withdrawn from the endotracheal tube.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
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pneumonia. Am J Respir Crit Care Med 2002; 165(7): 867–903.
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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.
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