Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses.
BACKGROUND
Severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD).


METHODS
Nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples.


RESULTS
Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 - 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more.


CONCLUSIONS
The custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.
Department of Microbiology & Immunology, Advanced Basic Sciences & Clinical Research Laboratory, (ICMR Grade - I Viral Diagnostics & Research Laboratory), Sawai Man Singh Medical College, Jawahar Lal Nehru Marg, Jaipur, 302 004, India. drbhartimalhotra@gmail.com.
Bharti
B
Malhotra
Department of Microbiology & Immunology, Advanced Basic Sciences & Clinical Research Laboratory, (ICMR Grade - I Viral Diagnostics & Research Laboratory), Sawai Man Singh Medical College, Jawahar Lal Nehru Marg, Jaipur, 302 004, India.
M Anjaneya
MA
Swamy
Department of Microbiology & Immunology, Advanced Basic Sciences & Clinical Research Laboratory, (ICMR Grade - I Viral Diagnostics & Research Laboratory), Sawai Man Singh Medical College, Jawahar Lal Nehru Marg, Jaipur, 302 004, India.
P V Janardhan
PV
Reddy
Department of Microbiology & Immunology, Advanced Basic Sciences & Clinical Research Laboratory, (ICMR Grade - I Viral Diagnostics & Research Laboratory), Sawai Man Singh Medical College, Jawahar Lal Nehru Marg, Jaipur, 302 004, India.
Neeraj
N
Kumar
Department of Microbiology & Immunology, Advanced Basic Sciences & Clinical Research Laboratory, (ICMR Grade - I Viral Diagnostics & Research Laboratory), Sawai Man Singh Medical College, Jawahar Lal Nehru Marg, Jaipur, 302 004, India.
Jitendra Kumar
JK
Tiwari
D002675:Child, Preschool; D003365:Costs and Cost Analysis; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D009305:Nasopharynx / Q000821:virology; D010614:Pharynx / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012141:Respiratory Tract Infections / Q000175:diagnosis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D013997:Time Factors; D014777:Virus Diseases / Q000175:diagnosis*
Concordance; Custom assay; FTD assay; Multiplex real time RT-PCR