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 ~  Abstract
 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~  References
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  Table of Contents  
BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 2  |  Page : 219-221
 

Ribosomal RNA-based panbacterial polymerase chain reaction for rapid diagnosis of septicaemia in Intensive Care Unit patients


1 Department of Microbiology, Rajendra Institute of Medical Sciences, Ranchi, Jharkhand, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Anaesthesia, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission04-Dec-2014
Date of Acceptance24-Sep-2015
Date of Web Publication14-Apr-2016

Correspondence Address:
Vikas Gautam
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.180351

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 ~ Abstract 

Early diagnosis and treatment of sepsis by appropriate antibiotics is of utmost importance. Therefore, we evaluated 16S rRNA panbacterial polymerase chain reaction (PCR) for rapid diagnosis of sepsis in 49 adult patients in Intensive Care Units (ICUs) and compared it with an automated blood culture. 8 ml of 10 ml blood collected was inoculated into BACTEC® aerobic bottle and the remaining 2 ml was used for DNA extraction and PCR. 109 of 115 (93%) episodes of suspected sepsis showed concordant results between automated culture and PCR. Six episodes were positive by PCR only. Panbacterial PCR reduces turnaround time with rapid differentiation between systemic inflammatory response syndrome and sepsis.


Keywords: 16S rRNA, adult Intensive Care Unit, blood culture, polymerase chain reaction, sepsis


How to cite this article:
Gupta MD, Kaur H, Ray P, Gautam V, Puri G D. Ribosomal RNA-based panbacterial polymerase chain reaction for rapid diagnosis of septicaemia in Intensive Care Unit patients. Indian J Med Microbiol 2016;34:219-21

How to cite this URL:
Gupta MD, Kaur H, Ray P, Gautam V, Puri G D. Ribosomal RNA-based panbacterial polymerase chain reaction for rapid diagnosis of septicaemia in Intensive Care Unit patients. Indian J Med Microbiol [serial online] 2016 [cited 2019 Dec 5];34:219-21. Available from: http://www.ijmm.org/text.asp?2016/34/2/219/180351



 ~ Introduction Top


Intensive Care Unit (ICU) patients constitute a highly susceptible population with disproportionately high incidence of hospital-acquired infections (HAI) attributed to invasive procedures, drug resistance and inadequate asepsis. [1] For rational prescription of antimicrobials in ICUs, it is important to distinguish between systemic inflammatory response syndrome (SIRS) and sepsis where the latter is associated with proven or suspected infectious cause demanding antibiotic therapy. [2] Blood culture helps the recovery of microbes and determine drug susceptibility. However, isolation may be hampered by a low degree of bacteraemia, prior antibiotic use, the presence of intracellular or slow-growing organisms, the amount of blood sampled and timing and method of sample collection and conventional blood culture systems. [3],[4] Therefore, molecular methods including polymerase chain reaction (PCR), flow cytometry and mass spectrometry have gained importance for direct detection of pathogens in blood with a shorter turnaround time. [5],[6]

We studied 16S rRNA panbacterial PCR for rapid diagnosis of bacterial septicaemia in adult ICU patients and compared it with automated blood culture system.


 ~ Methods Top


From February to August 2010, a total of 115 episodes of SIRS in 49 subjects in the adult ICU qualified as cases and 20 healthy controls with no features of infection were also included to evaluate sensitivity and specificity of the test. The sample size was calculated using EpiInfo version 6 (Centers for Disease Control, Atlanta, GA). The study was approved by Institute Ethics Committee and informed consent was taken from all study subjects.

Sample collection

A volume of 10 ml of blood was collected from antecubital vein with aseptic precautions. 8 ml of blood was inoculated into BACTEC ® aerobic media bottle at the bedside of patient and 2 ml blood was collected in sterile EDTA Vacutainers ® . [7]

Blood culture

BACTEC ® aerobic media with patient's blood was incubated within half an hour into BACTEC ® 9240 system (Beckton Dickinson Diagnostics, USA). With aseptic precautions, around 2 ml of liquid was withdrawn from the positive bottle beeping within 5 days of incubation, using a sterile syringe and transferred into a sterile plugged glass tube. Two to three drops of this blood were used for Gram smear microscopy and subcultured on 5% sheep blood agar and MacConkey agar (HiMedia) and examined after overnight incubation at 37°C.

DNA extraction

DNA was extracted from blood samples using the QIAamp ® 96 DNA Blood Kit (Qiagen Pvt Ltd) according to manufacturer's instructions and stored at −20°C until use. [8]

Polymerase chain reaction amplification

The 16S rRNA primer sequences included RW01, 5'- AAC TGG AGG AAG GTG GGG AT-3' and DG74, 5'- AGG AGG TGA TCC AAC CGC A-3'. [8],[9] Each reaction contained 5 μl of the forward and reverse primers, 0.2 μl dNTPs, 0.5 μl Taq polymerase, 2.5 μl buffer and 4 μl of genomic DNA template in a reaction volume of 25 μl. PCR amplification conditions included initial denaturation of 10 min at 95°C, followed by 30 cycles each of 1 min at 94°C, 2 min at 60°C and 1 min at 72°C and a final extension of 7 min at 72°C.  Escherichia More Details coli ATCC 25923 DNA was used as positive control and sterile distilled water was used as negative control. Amplified products were electrophoresed on 2% agarose gel containing ethidium bromide and observed under UV illumination.

Statistical analysis

The Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, version 13.0 for Windows) software was used for analysing data. To see the concordance between PCR and blood culture, Kappa test of agreement was applied. A P ≤ 0.05 was considered significant.


 ~ Results Top


The demographic and clinical profiles of 49 cases are shown in [Table 1]. The outcome in ICU had a significant correlation with age showing increasing mortality with increasing age. No association was seen with gender of patient.
Table 1: Demographic profile of 49 patients admitted to the ICU


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Comparative analysis of blood culture and polymerase chain reaction

Of the 115 episodes of suspected sepsis in 49 patients, 109 (93%) episodes showed concordance between automated culture and PCR. Of these, 42 (39%) showed positive results while 67 (61%) gave negative results. The remaining six cases were only PCR positive [Table 2]. All control blood specimens gave negative results with PCR. Taking blood culture as the gold standard, PCR assay sensitivity, specificity, positive and negative predictive values were calculated as 100%, 91.8%, 87.5% and 100%, respectively. The diagnostic accuracy of PCR was 94.8%. The kappa value for agreement with blood culture results was 0.891. The average time to positivity for automated blood culture was 12-48 h whereas that for PCR was about 4 h.
Table 2: Comparative analysis of blood culture and PCR


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 ~ Discussion Top


Early diagnosis and management of sepsis is important, especially in ICUs. The study was planned to compare the usefulness of 16S rRNA panbacterial PCR assay with automated culture for early detection of bacteria in the blood of patients admitted to adult ICU. ICU surveillance performed in US and Europe in 1990s revealed the occurrence of severe sepsis in approximately 80% of adult patients hospitalised for some other diagnosis. [2] Such high incidence of infections in the ICU occurs due to increased immunocompromised patients as well as a breach in epithelium by use of numerous invasive devices. Previous studies carried out in neonatal sepsis have shown sensitivity and specificity of broad range PCR of 84-98% and 95-100%, respectively. [9],[10],[11],[12],[13] The present study conducted in adult ICU of a tertiary care hospital showed excellent correlation between the two diagnostic tests. A high negative predictive value of the assay helps to rule out infection in ICU patients. A slight difference in the sensitivity and specificity as compared to earlier studies in neonates could be explained by different sample size, an increased bacterial load in blood of neonates and requirement of a pre-amplification incubation step of 5 h in some studies.

Six episodes in the present study showing only PCR positivity highlight its higher sensitivity. These patients were on prior antibiotics, which could act as inhibitors during culture but did not affect the results of PCR. [14] Four of these PCR positive episodes were from two cases (two episodes each) on different occasions, and showed repeated positivity at an interval of 2-3 days which may be explained by presence of fastidious or dead organisms. Taking blood culture and/or PCR positivity for calculating sepsis, the incidence was 43% in the present study which is similar to an earlier study. [15]

The increased expenditure and mortality in ICUs is attributed to increased length of stay which further is due to delay in early diagnosis and antimicrobial treatment of sepsis. Rapid ruling out of sepsis, which strongly mimics SIRS in critically ill patients of ICU, by PCR will prevent unusually long stay in the ICU as well as the improper and inadvertent use of antibiotics. However, PCR has a limitation of differentiating Gram-positive and Gram-negative organisms, which is the advantage of automated blood culture system. Still, application of molecular methods for rapid diagnosis of sepsis in critically ill ICU patients can serve in more than one way for customised therapy, reduced health care burden and cost and better patient satisfaction.

The study supports the use of molecular method in diagnosis of sepsis as compared to conventional or automated culture methods. Therefore, molecular methods can be a useful adjunct to rule out sepsis in critical patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Vallés J, Ferrer R. Bloodstream infection in the ICU. Infect Dis Clin North Am 2009;23:557-69.  Back to cited text no. 1
    
2.
Munford RS, Suffredini AF. Sepsis. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 7 th ed. Philadelphia, PA: Churchill Livingstone Elsevier; 2010. p. 987-1010.  Back to cited text no. 2
    
3.
Tiwari DK, Golia S, Sangeetha KT, Vasudha CL. A study on the bacteriological profile and antibiogram of bacteremia in children below 10 years in a tertiary care hospital in Bangalore, India. J Clin Diagn Res 2013;7:2732-5.  Back to cited text no. 3
    
4.
He F, Zhang X, Zhou J, Liu Z. A new MSPQC system for rapid detection of pathogens in clinical samples. J Microbiol Methods 2006;66:56-62.  Back to cited text no. 4
    
5.
Hunfeld KP, Bingold T, Brade V, Wissing H. Molecular biological detection of pathogens in patients with sepsis. Potentials, limitations and perspectives. Anaesthesist 2008;57:326-37.  Back to cited text no. 5
    
6.
Klouche M, Schröder U. Rapid methods for diagnosis of bloodstream infections. Clin Chem Lab Med 2008;46:888-908.  Back to cited text no. 6
    
7.
Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott's Diagnostic Microbiology. 12 th ed. St. Louis, Missouri: Mosby; 2007.  Back to cited text no. 7
    
8.
Jordan JA, Durso MB. Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis. J Mol Diagn 2005;7:575-81.  Back to cited text no. 8
    
9.
Dutta S, Narang A, Chakraborty A, Ray P. Diagnosis of neonatal sepsis using universal primer polymerase chain reaction before and after starting antibiotic drug therapy. Arch Pediatr Adolesc Med 2009;163:6-11.  Back to cited text no. 9
    
10.
Pammi M, Flores A, Leeflang M, Versalovic J. Molecular assays in the diagnosis of neonatal sepsis: A systematic review and meta-analysis. Pediatrics 2011;128:e973-85.  Back to cited text no. 10
    
11.
Cursons RT, Jeyerajah E, Sleigh JW. The use of polymerase chain reaction to detect septicemia in critically ill patients. Crit Care Med 1999;27:937-40.  Back to cited text no. 11
    
12.
Reier-Nilsen T, Farstad T, Nakstad B, Lauvrak V, Steinbakk M. Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: A case control study. BMC Pediatr 2009;9:5.  Back to cited text no. 12
    
13.
Jordan JA, Durso MB. Comparison of 16S rRNA gene PCR and BACTEC 9240 for detection of neonatal bacteremia. J Clin Microbiol 2000;38:2574-8.  Back to cited text no. 13
    
14.
Handschur M, Karlic H, Hertel C, Pfeilstöcker M, Haslberger AG. Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood. Comp Immunol Microbiol Infect Dis 2009;32:207-19.  Back to cited text no. 14
    
15.
Vincent JL, Taccone F, Schmit X. Classification, incidence, and outcomes of sepsis and multiple organ failure. Contrib Nephrol 2007;156:64-74.  Back to cited text no. 15
    



 
 
    Tables

  [Table 1], [Table 2]



 

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