Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses

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). 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. 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. 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.


Background
Severe acute respiratory infections (SARI) are one of the major causes of illness and death worldwide and are the third most common cause of death among children [1]. Acute respiratory infections (ARI) cause more deaths in children < 5 years with most cases reported from India (43 million), China (21 million), Pakistan (10 million), Bangladesh, Indonesia and Nigeria (56 million) [2]. Respiratory infections can be caused by many viruses, both DNA and RNA. These include the Respiratory Syncytial Virus (RSV), human Parainfluenza Virus (HPIV), Influenza A Virus (Flu A), Influenza B Virus (Flu B), human Adenovirus (HAdV), human Coronavirus (HCoV), human Rhinovirus (HRV), human Metapneumovirus (HMPV) and human Bocavirus (HBoV) [3]. A new wave of viral diagnosis was established with the development of Polymerase Chain Reaction (PCR) techniques in the 1990s [4]. PCR is more sensitive and rapid than conventional methods for detection of respiratory viruses. Different respiratory viruses present with similar signs and symptoms and can't be differentiated symptomatically or clinically. Tests capable of rapid simultaneous identification of various viruses at the same time can help expedite initiation of appropriate therapy. Uniplex RT-PCR requires individual amplification of each virus under study which is expensive, time consuming and laborious [5]. To overcome this, multiplex real-time PCRs targeting the detection of multiple pathogens simultaneously have been developed commercially but they are very expensive. There is a need to develop cheaper systems for rapid simultaneous identification of various viruses. The present study compares custom real-time multiplex PCR primers and probes for the simultaneous detection of 18 respiratory viruses with an in-vitro diagnostics (IVD) approved fast track diagnostics (FTD) kit.

Patient inclusion criteria
Children with SARI, admitted in J. K. Lone Hospital, a pediatric hospital attached to Sawai Man Singh (SMS) Medical College Jaipur were enrolled in the study and tested for respiratory viruses with prior consent of the parent/guardian. Duration of the study was 27 months i.e. between September, 2012 to December, 2014. Children enrolled were ≤ 5 years of age, presenting with fever, cough, sore throat, nasal catarrh, shortness of breath, bronchiolitis, pneumonia, and wheezing.

Patient exclusion criteria
Samples were not collected from patients with chronic respiratory ailments; non-consenting caregivers, with history of hospitalization in the preceding 14 days, not admitted in hospital and children aged > 5 years.

Sample collection and transportation
A total of 356 nasopharyngeal aspirate and throat swab samples were collected from patients with SARI by a trained technician using a sterile nylon flocked swab and placed in viral transport medium (VTM), labelled and transported on ice at the earliest to Advanced research lab (ICMR Grade-1 Virology Lab) of SMS Medical college Jaipur for further processing and storage of the samples. The study was approved by the institutional ethics committee.

Nucleic acid extraction
Viral nucleic acid from samples was extracted using an EasyMAG (Biomeurex) automated extractor according to the manufacturer's instructions. Briefly, the extraction was done from 400 μl homogenised sample which was added to 1500 μl lysis buffer and was incubated for 10 min off board. The samples were loaded into the EasyMAG and 100 μl of magnetic silica was added to each sample and mixed well. Finally, the nucleic acid was eluted in a volume of 110 μl of which 50 μl was used for the FTD assay and 54 μl for the custom assay.  (Table 1), 5 μl of extracted nucleic acid of the positive control/sample and made to a final volume of 20 μl with nuclease free water. The thermal cycling profile for the uniplex RT-PCR was 50°C for 30 min (1 cycle), 95°C for 10 min (1 cycle) followed by 90°C for 15 s and 55°C for 30 s (45 cycles). Specimens were considered positive when the Ct value was < 35.

Standardisation of multiplex real-time RT-PCR (Custom assay)
Uniplex PCR was followed by multiplex real-time RT-PCR standardisation. In multiplex PCR, each reaction . FTD positive samples were considered as standard. AgPAth (Ambion) one step RT-PCR master mix was used for the detection of respective viral nucleic acids as mentioned above in a reaction volume of 25 μl but with 9 μl of nucleic acid, 4 picomoles of each primer and 2 picomoles of probe. The thermal profile for the multiplex real-time PCR was as described above. Specimens were considered positive when the Ct value was < 35. After standardisation of multiplex PCR the same protocol was used for screening patient samples. Rnase P was used as an internal control in a separate uniplex RT PCR assay. Samples negative for Rnase P were not included in the study.

Results
A total of 356 samples were tested by both assays. Custom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively (Table 2). No significant differences were seen in the number of samples positive for each virus by the custom assay as compared to the FTD assay except with RSV A/B which was over detected in 18 samples and one sample being under detected by the custom assay as compared to the FTD assay. Further, to completely assess the results of these 18 discordant RSVA/B samples, testing was repeated using RSV A and RSV B specific primer and probe mix in uniplex real time RT-PCR as published previously [6]. All 18 samples were found to be positive for RSV B (Table 3).
Low concordance was observed between the two assays for RSV A/B (94.66 %) and EV (98.31 %).
The discordant results of the custom assay were seen in 19 co-infection samples, 13 single infection samples and four negative samples as compared to the FTD assay, and the discordance was predominant in the co-infected samples as compared to single infection samples (Table 5).
Comparisons between the custom assay and the FTD assay were made based on the different parameters listed in Table 6. Most of the findings between the custom assay and the FTD assay were similar except for the cost incurred for screening 18 respiratory viruses. In this regard, the custom assay was found to be more economical than the commercial FTD assay.

Discussion
The present study was performed to compare a custom multiplex assay and an FTD multiplex assay by testing of 356 respiratory samples obtained from children with SARI admitted in J K lone paediatric hospital Jaipur.
In the present study, the concordance between the custom assay and the FTD assay was found to be 100 % for Flu A, Influenza A(H1N1) pdm09, HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, and HPeV. Similarly Chen et al., [7] reported a concordance of 99.60 % for Flu A and Influenza A(H1N1) pdm09 when comparing a multiplex PCR assay with a uniplex assay.
The concordance between the two assays varied from 94.66 to 99.71 % for the remaining ten viruses;  [8,9]. Concordance for EV in the present study was different from an earlier study (93.00 %) [8]. The difference in concordance obtained in different studies may be due to the different primer binding regions or may be due to different methodologies employed by various studies. The number of samples positive for HCoV  229E, HPIV-4, HPIV-2, HCoV NL63, HPeV, HCoV HKU1, Flu A, were ≤ 5 in the present study. Studies based on larger numbers of samples are required to assess the concordance of these viruses more thoroughly. The limit of detection for some of the viruses in the custom assay (Table 7) ranged from 1 DNA copy/ml to 2×10 4 copies/ml [7,[10][11][12][13][14]. The detection limit of the FTD assay for different viruses was 10 2 copies/ml for FluA, HPIV-2, HMPV and HCoV OC43; 10 3 copies/ml for FluB, HCoV HKU1, HPIV-1, HBoV, HPIV-3, HCoV NL63, RSV, HAdV, EV, and HPeV; and 10 4 copies/ml for HRV, HCoV 229E and HPIV-4 [15].
In the present study RSV A/B was the most predominant virus detected by both the custom and FTD assays with positivity in 84 (23.60 %) and 67 (18.82 %) samples respectively and concordance of 94.66 %. This finding is different when compared with other studies [8,16] where comparisons were made between multiplex PCRs in which RSV was the second most predominant virus detected [16].
The custom primer and probes used for Influenza A (H1N1) [20], and 4.70 % [6] respectively in earlier studies where the same primer and probes were used. HBoV was mostly associated with co-infections in the present study in both assays. This is consistent with an earlier study [16] The major discrepancy in the present study was found with RSV A/B. The discrepancy in 18 samples which were over detected by the custom assay was resolved by RSV A and RSV B typing. The RSV typing results for the discrepant samples showed that all 18 samples were RSV B. Further all samples positive for RSV A/B by the FTD assay were also subjected to RSV typing which indicated RSV A in 13 (19. During the process of standardisation of the custom assay 3 μl of viral nucleic acid (positive control) was used for each virus including 4 picomoles of primers and 2 picomoles of probes. Each panel consisted of 3 viruses. In total 9 μl of viral nucleic acid was used for each panel. While the FTD assay used 10ul of nucleic acid in each tube with primers and probes for 4 viruses, the concentration of primer and probe was not disclosed by FTD. In total 4 μl more of viral nucleic acid was used in the custom assay compared to the FTD assay which may have increased the sensitivity/detection of different viruses in the custom assay.
Initially during the process of standardisation of the custom assay, different primer and probe concentrations were tried and the PCR was run for 45 cycles as per the protocol followed by various authors. Although data was analysed using PCRs run for 35 and 40 cycles, best results were achieved using a Ct value of 35 for both the FTD assay and the custom assay. Accordingly, a Ct value of <35 was considered as positive for both assays as per the FTD kit. With the custom assay being run for 40 cycles this reduces the custom assay run time by 8 min, thereby making it only 21 min longer than the FTD assay.
Comparisons were made between various aspects of the custom and the FTD assays (Table 6). No major differences were observed between the two assays except in the cost incurred for both assays. Similar comparisons were also done in an earlier study [21] where three multiplex PCRs were compared. The turn-around time of the custom assay was 29 min more as compared to the FTD assay. But both the assays reported the results on the same day. The excess time of 29 min taken by the custom assay as compared to the FTD assay may not greatly interfere with the treatment process. However, the custom assay was much more economical costing INR 1500/-per sample for screening 18 respiratory viruses compared to the commercial FTD assay which was expensive costing INR 4300/-per sample. This assay may prove to be highly cost effective in resource limited settings like ours. However the limitation of our study was that some of the viruses showed low positivity as a result it is difficult to assess the concordance accurately. Larger numbers of positive samples need to be tested to evaluate the concordance of these less prevalent viruses.

Conclusion
This study reported a high prevalence of respiratory viruses in children ≤ 5 years using a custom assay and an FTD assay. Good concordance was observed for all the viruses between both assays except for RSVA/B. However larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses.