Among the 117 episodes of single respiratory virus infections, arthralgia was more frequently observed in influenza A infections than in non-influenza infections (66.1% [39/59] vs. 46.6% [27/58], P = 0.033); for these 2 types of infections, the other examined symptoms, including sore throat, rhinorrhea, cough, purulent sputum, wheezing, dyspnea, and headache, were detected at similar frequencies.

Of 55 cases of LRTIs, coinfection with bacterial pathogens by sputum culture or blood culture was found in 3 (8.8%) of 34 patients who tested positive for respiratory viruses and in 2 (9.5%) of 21 patients who tested negative for respiratory viruses. Four of 6 cases of influenza A LRTI had received oseltamivir. Two patients died of pneumonia and the worsening of an underlying malignancy; 1 of these patients tested positive for hMPV, and the other patient tested negative for a respiratory virus. Four (16.7%) out of 24 patients vaccinated for influenza vaccine in 2012–2013 (A/California/7/2009 (H1N1)-like virus A/Victoria/361/2011 (H3N2)-like virus B/Wisconsin/1/2010-like virus) and 61 (25.1%) out of 243 unvaccinated patients had influenza A infections (P = 0.358).

All 267 respiratory samples from each RTI case were examined for viruses by both virus isolation and PCR/ESI-MS, and the results are presented in Table 2. For virus isolation, respiratory viruses were detected in 63 (23.6%) cases, including influenza A (48 cases, 18.0%), enterovirus (13, 4.9%), and parainfluenza virus (2, 0.7%), and no coinfection was detected. Virus isolation identified additional parainfluenza type 3 and enterovirus infections that were not found by PCR/ESI-MS in 2 samples.

Direct fluorescent antibody (DFA) staining of viral antigens in patient specimens is generally considered to be more sensitive than rapid immunoassays. The specificity of DFA is high but depends on experienced technologists. Food and Drug Administration- (FDA-) cleared commercial DFA reagents have long been available for detection and identification of influenza A and B viruses, RSV, parainfluenza viruses 1–3, and adenovirus. Recent advances in DFA tests include the availability of FDA-cleared commercial DFA reagents for detection of human metapneumovirus. A study showed excellent sensitivity (95% versus RT-PCR) of a human metapneumovirus DFA performed on nasopharyngeal aspirates from children. DFA was substantially more sensitive than rapid immunoassays for detection of the novel 2009 influenza A (H1N1) virus, though less sensitive than virus isolation and/or RT-PCR [31, 34].

Long considered the gold standard for detection of respiratory viruses, the turn-around time of conventional cell culture in tubes for respiratory viruses is generally too long to be clinically relevant. When performed on fresh specimens maintained at refrigerated temperature, virus isolation has excellent sensitivity for most respiratory viruses. The use of centrifugation-enhanced (shell vial) culture and mixed cell lines in the same vial has decreased turn-around time to 24–48 hours and streamlined workflow. A study showed that shell vials containing mixed cell lines had similar sensitivity as conventional tube culture for detection of influenza and parainfluenza viruses. For RSV detection, the shell vial system was more sensitive than conventional culture, but less sensitive than a direct antigen test. Virus isolation in shell vials was highly sensitive for detection of 2009 influenza A (H1N1).

Student’s t-test and chi-square test were used to analyze and compare the categorical demographic characteristics including clinical manifestations and laboratory tests. Kappa statistic was used to evaluate the consistency between PCR and original tests (categorical variables) and Cohen’s kappa coefficient (κ) was regarded as poor to fair consistency if κ ≤ 0.4; moderate consistency if 0.41 ≤ κ ≤ 0.60; and good consistency if 0.61 < κ. A two-sided p < 0.05 was considered statistically significant. Statistical analyses were performed using the SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA).

Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.

For the purpose of this study, nasopharyngeal swabs of all inpatients and outpatients who were screened for the respiratory viruses influenza, parainfluenza and RSV during the winter season 2012/2013 were retrospectively analysed. For some of the LRTI patients, additional bronchoalveolar lavage (BAL) samples were available. Readily available medical records were retrospectively reviewed from all patients to obtain basic characteristics, clinical and laboratory data.

Statistical analyses were performed with Stata® and Excel®. Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year.

The viral nucleic acids were extracted from 200-µL of each sample using the High Pure Viral Nucleic Acid Kit (Roche Applied Science, Castle Hill, Germany) following the manufacturer’s instructions. Extracted nucleic acids were eluted in 100 μL elution buffer and stored at −70 °C. Reverse transcription (RT) was carried out using High-Capacity Complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems Part Number: 4375575 Rev.C). The total volume of RT mix was 40 μL per reaction, containing 4 μL RT buffer (10×), 1.6 μL dNTP mixture (25 mM of each dNTP), 4 μL random primers (10×), 2 μL RNase inhibitor (20 U/μL), 2 μL MultiScribe Reverse Transcriptase (50 U/μL), and 26.4 μL template, whereby the RT reagent mix was prepared on ice. The thermal profile of the RT program consisted of 10 min incubation at 25 °C, 120 min RT at 37 °C, 5 min RT inactivation at 85 °C, and cooling down to 4 °C and was performed in a 96-well GeneAmp PCR System 9700. The resulting cDNA was stored at −20 °C.

The following multiplex PCR assays were performed for each sample to detect RNA/DNA of 15 respiratory viruses, including RSV A or B, FluA, FluB, human enterovirus (EV), MPV, human parainfluenza virus types 1–4, human rhinovirus (RV), coronavirus OC43/NL63/229E, human adenovirus (ADV), and human bocavirus (Boca). In the present study, previously published primers and PCR assays were used for multiplex RT-PCR and the details of primers are summarized in Table S1. Briefly, the PCR reaction was performed by adding 3 µL RT product to 22 µL PCR mix. The conditions of amplification were as follows: initial denaturation at 95 °C for 10 min; followed by 40 cycles of 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min; a final extension at 72 °C for 10 min. Amplification products were visualized by 1% agarose gel electrophoresis with ethidium bromide staining and observed under ultraviolet light. For each PCR assay, a positive and negative control for each parameter was performed. Internal control was also performed to detect sample inhibition and avoid false-negative results. External and internal amplification controls were designed for quality control and validation. The detection limits of the multiplex PCR assays were 10 to 100 copies of the individual virus.

A total of 400 Iranian military trainees with clinical diagnostic criteria for respiratory infection were enrolled in the survey. All participants were male, with a mean age of 21.69 ± 4.9 years (range from 18 to 57 years). Most prevalent complaints of patients referred to the military medical clinic center were sore throat (n=302; 75.5%), rhinorrhea (n=253; 63.2%), cough (n=237; 59.2%), fever (n=237; 59.2%), and nasal congestion (n=202; 50.5%) (Fig. 1). Of the 400 samples, 124 (31%) were positive for respiratory viruses. Human rhinovirus (n=29; 7.2%), human respiratory syncytial virus A (n=29; 7.2%), and influenza B virus (n=24; 6%) were the most frequently detected respiratory viruses in our study, followed by bocavirus (n=12; 3%), influenza A H1N1 (n=9; 2.2%), influenza A H3N2 (n=6; 1.5%), human respiratory syncytial virus B (n=6; 1.5%), adenovirus (n=6; 1.5%), and human coronavirus 229E (n=3; 0.7%). Other viruses including influenza C virus, human parainfluenza viruses, metapneumovirus, and echovirus have not been detected in any of the samples (Table 1, Fig. 2). It's worth noting that no co-infections were detected in our study. The most cases of dyspnea (n=5; 62.5%) were in the group of patients with respiratory syncytial viruses A and B, followed by influenza B (n=2; 25%) and coronavirus 229E (n=1; 12.5%). Sore throat and rhinorrhea were the most frequent symptoms in rhinovirus infection. The most cases of myalgia were seen in influenza B (n=5; 62.5%) and influenza A H1N1 (n=3; 37.5%) infections, respectively (Table 1).

Out of 369 swab specimens collected and analysed, 172 (46.6%) were positive for one or more respiratory agents. The most frequently-detected respiratory virus amongst all study participants was influenza A (n = 71, 19.2%). Others were adenovirus (n = 32, 8.7%), rhinovirus A (n = 29, 7.9%) and coronavirus OC43 (n = 16, 4.3%). The least-detected respiratory viruses were parainfluenza virus 2 (n = 2, 0.5%) and coronavirus 229E (n = 2, 0.5%) (Table 2). Approximately 63.0% of all viral detections, including all the parainfluenza viruses (1, 2 and 3) and 78.1% of adenovirus and 75.0% of coronavirus OC43, were detected from study participants who were aged 10 years or less. Only Rhinovirus A and all influenza viruses were detected across all ages.

Demographic data of patients were compared using Pearson's χ2 test. Weekly proportions positive for each virus were calculated to allow comparability and assess differences in virus epidemics between seasons. We compared our data to influenza notification rates in Victoria obtained from the National Notifiable Diseases Surveillance System (NNDSS) to assess the representativeness of inter-seasonal peaks we observed. To assess timing and magnitude of epidemics, the proportion of positive specimens and the peak week of the epidemic were considered: those in the lowest quartile were considered early or small and those in the highest quartile were considered late or large. Seasonality of viruses was assessed visually by time series analysis and for further investigation each virus was compared with influenza A and RSV using cross-correlations that estimated the association between peaks in epidemic curves at a lag or lead of up to 15 weeks.

Fisher's exact test was used to investigate any negative association between virus pairs among specimens with co-detections. Multivariate logistic regression, adjusted for age category (<5, 5–19, 20–64 and ⩾65 years), sex and season, was used to produce odds ratios (OR) and 95% confidence intervals for these associations and the chi-square test used to assess trend. Adjustment for multiple comparisons was not performed [23, 26]. The significance level for all tests was set at P < 0.05.

All data extraction, exclusion and analyses were performed in Stata (version 14.2, StataCorp, College Station, Texas).

RNA was extracted from respiratory specimens using the QIAamp® viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Reverse transcription, amplification and detection of viral RNA was performed with the RealStar® Influenza, Parainfluenza and RSV real-time RT-PCR kits (altona Diagnostics, Hamburg, Germany) on a LightCycler® 480 instrument II (Roche, Mannheim, Germany) according to the manufacturer's instructions. These assays distinguished influenza A, B, H1N1, RSV A, and RSV B. The RealStar Parainfluenza RT-PCR kit cannot differentiate between parainfluenza virus types 1 and 3 and between types 2 and 4. Therefore, the FTD Respiratory pathogens 21 kit (Fast Track Diagnostics, Luxembourg) was used for typing of samples positive for parainfluenza virus RNA.

This study only collected anonymous information that cannot be associated with individual patients. Patient samples were collected during the course of medical care provided at the participating facilities, and all examinations and testing for pathogens occurred at the request of the medical facilities for the purposes of diagnosis and treatment. This study used only existing medical records and documents, and oral informed consent was obtained from all patients.

The nucleic acid was subjected to multiplex amplification for all specimens using SureX 13 Respiratory Pathogen Multiplex Detection Kit (Cat. No. 1 060 144, Ningbo Health Gene Technology) on ABI GeneAmp PCR System 9700 (Thermo Fisher Scientific). The 13 respiratory pathogens were as following: influenza A virus, influenza A virus H1N1 (2009), seasonal H3N2 influenza virus, influenza B virus, adenovirus, boca virus, rhinovirus, parainfluenza virus, chlamydia, human metapneumovirus, Mycoplasma pneumoniae, coronavirus, and respiratory syncytial virus. The PCR product was subjected to capillary electrophoresis using GenomeLab™ GeXP Genetic Analysis System (Beckman Coulter) according to the instructions. Each pathogen, if detectable, produced a distinctive fragment size after PCR amplification. The results of fragment analysis were used to determine the outcomes of testing. In brief, if the peak height of a targeted fragment size is lower than the lower peak of the signal standard, the targeted pathogen is determined negative; if the peak height of a targeted fragment size is higher than the higher peak of the signal standard, the targeted pathogen is determined positive; if the peak height of a targeted fragment size is between the higher and the lower peaks of the signal standard, the targeted pathogen is determined uncertain and the test should be repeated.

As currently recommended, these tests have been used during the French RSV epidemic periods in children below 36 months old. Overall, 1529 RSV-IC samples were performed from 1508 children, 477 (31.3%) were positive with a slight decrease in the 6–36 months strata (31.6% vs 25.0% for 0–6 and 6–36 months strata, respectively, p = 0.24) (Fig 1). Interestingly, the reverse pattern is observed with the use of mPCR with a slight increase between the 0–6 and the 6–36 months groups (64.7% and 81.0% of positive viruses detection, respectively, p = 0.06).

Among children presenting a negative RSV-IC test, 43 were also tested by mPCR in the same 2 days period. Sixteen (33.3%) were positive by mPCR and 4 displayed viral co-infections. The mPCR test has been done specifically on physician request, reflecting a probable more symptomatic population. Among them, 36 (83.7%), 6 (14.0%) and 1 (2.3%) were belonging to the 0–6, 6–36 and >36 months old, respectively. Interestingly, mPCR were more frequently positive among children in paediatric units (7/11, 63.6%) rather than neonatology (4/25, 16.0%, p = 0.007). Among the 7 positive mPCR in paediatric units, 4 corresponded to viral co-infections. The two most frequently identified viruses were picornavirus (n = 12) and parainfluenzae (n = 3). Metapneumovirus, adenovirus, bocavirus and coronavirus were also identified (n = 1 in all cases). Underlying the good sensitivity of RSV-IC in children, only one RSV was identified by mPCR among the 43 RSV-IC negative samples. In this particular case, the mPCR test was performed two days after the RSV-IC test. Thus, we cannot conclude if this discrepancy is explained by a lower sensitivity of RSV-IC test or by the apparition of RSV in this child in these two days interval.

Statistical methods

Statistical analysis was performed using IBM SPSS version 18 (Inc., Chicago, USA) and Microsoft Office 2013 (Excel). Descriptive analysis was carried out using percentage and simple frequencies. Data was reported as count and percentage. Clinical signs and symptoms were counted, and the corresponding empirical proportions were calculated with 95% confidence intervals (CIs) to measure the overall symptom load.

For influenza virus‐negative samples, more PCR tests were performed to detect the following pathogens: adenovirus (ADV), bocavirus (BOV), human rhinovirus (HRV), parainfluenza virus (PIV), human metapneumovirus (HMPV), Mycoplasma pneumoniae (MP), and respiratory syncytial virus (RSV), using corresponding NAAT kits from Daan Gene. All tests were carried out on ABI Quant Studio 7 System (Thermo Fisher Scientific) according to the instructions. A typical S amplification curve and Cq value ≤38.0 were determined positive.

Between August and December 2008, a total of 369 study participants presenting with ILI at the two study centres were recruited. Of these, 286 (77.5%) participants were from Entebbe Hospital and 83 (22.5%) participants were from Kiswa Health Centre (Table 1). Both genders were represented in almost equal proportions (52.3% females and 47.7% males) and the median age was 6 years (range: 1–70). Over half of the study participants (61.5%) were aged 10 years or less, and had low or no form of education (74.5%).

All patients were seen at the outpatient departments of the two study sites and none required hospital admission at the time of enrolment. Apart from fever, the most common clinical symptoms were cough (98.4%), shortness of breath (43.1%) and headache (29.0%). Only 3.8% (n = 14) of the study participants reported the presence of a chronic condition or illness such as active tuberculosis, chronic cough and chest pain. Almost all of the participants (94.5%) reported to the clinic within three days (range: 1–31) of the onset of symptoms.

Data analysis was performed by using SPSS (version 22.0; IBM). Differences in the distribution of categorical variables were compared using chi-square or Fisher's exact tests. A P value of ≤0.05 was considered significant.

Between February 15, 2011 and January 18, 2012, 371 pediatric CAP patients from 1 year to 17 years old were enrolled in this study. CAP patients were stratified into three groups: 242 (65.2%) patients were in the pre-kindergarten group (≤3 years), 36 (9.7%) were in the kindergarten group (3–7 years) and 93 (25.1%) were in the school-age group (≥7 years). Of the patients enrolled, a fever was documented in 52.3% (194/371), 76% presented with sputum and chest pain (282/371), and 18.6% presented with lung consolidation (69/371). The clinical characteristics for each age group are shown in Table 1.

After testing the specimens, we provided the results to a medical institution within 4 days including the conveyance period. The 50 patients tested in this study included 2 infants (1 male and 1 female, aged <1 year), 25 children (12 males and 13 females, aged 1–6 years), 10 elementary school pupils (6 males and 4 females, aged 7–12 years), 4 minors (2 males and 2 females, aged 13–18 years), 8 adults (3 males and 5 females, aged >18 years), and 1 patient (age unavailable).

Table 2 lists the pathogens detected by the PCR analysis stratified by age in the 27 patients. In children, enterovirus, rhinoviruses, RSV, and parainfluenza viruses were detected, whereas M. pneumoniae was detected only in elementary school pupils and minors. In the remaining 23 patients, no pathogens were detected. These 23 patients were also found to be negative for coronavirus.

PCR was used to obtain definitive viral diagnoses via rapid RSV and adenovirus diagnosis kits, and the sensitivity and specificity were calculated for these test kits. For the rapid RSV diagnosis kit, sensitivity was 80% and specificity was 85%. For the rapid adenovirus diagnosis kit, no positive results were obtained; therefore, sensitivity could not be calculated and specificity was 100%.

RSV infections were detected using the rapid diagnosis kit, but rhinovirus, enterovirus, and parainfluenza virus infections were not. The causative pathogens were unknown in many patients, although they were nevertheless treated for upper respiratory tract infections.

Evaluation of the incidence of various symptoms in patients infected with different pathogens showed that rhinoviruses were detected in nasal swab specimens more often than other viruses and patients with rhinovirus infections were less likely to present with fever (Table 3).

All RSV-positive patients were children, 80% of whom presented with coughing. All patients who were tested using the rapid adenovirus detection kit showed negative results. However, all these patients also tested negative for adenovirus using sensitive PCR tests. Thus, adenovirus was not considered to be the causative organism of this suspected outbreak.

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. All 18 samples were found to be positive for RSV B (Table 3).

One hundred percent concordance was observed between the custom assay and the FTD assay for eight viruses; HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 while it varied from 94.66 to 99.71 % for the remaining ten viruses; Flu B, HRV, HPIV-3, HPIV-4, HCoV NL63, HMPV A/B, RSV A/B, EV, HCoV HKU1, HAdV. (Table 4).

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.

Data are expressed as median with interquartile range (IQR) for continuous variables and count with percent for categorical variables. Fisher’s exact test was used to analyze categorical variables. Statistical analyses were carried out using SPSS 12.0 (SPSS Inc., Chicago, IL, USA) and a P value of <0.05 was considered to be statistically significant.

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., 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; Flu B (99.71 %), HPIV-3 (99.71 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), HCoV HKU1 (99.71 %), HAdV (99.71 %), HRV (99.71 %), EV (98.31 %). Similar findings have been observed in earlier studies for Flu B (98.25 to 99.42 %), HPIV-3 (96.53 to 99.30 %), HPIV-4 (97.10 %), HCoV NL63 (95.95 to 100.0 %), HMPV A/B (99.65 to 100.0 %), RSV A/B (93.06 to 98.60 %), HCoV HKU1 (98.84 to 100.0 %), HAdV (97.20 to 100.0 %) [8, 9]. Concordance for EV in the present study was different from an earlier study (93.00 %). 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 2×104 copies/ml [7, 10–14]. The detection limit of the FTD assay for different viruses was 102 copies/ml for FluA, HPIV-2, HMPV and HCoV OC43; 103 copies/ml for FluB, HCoV HKU1, HPIV-1, HBoV, HPIV-3, HCoV NL63, RSV, HAdV, EV, and HPeV; and 104 copies/ml for HRV, HCoV 229E and HPIV-4.

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.

The custom primer and probes used for Influenza A(H1N1) pdm09, RSV A/B, Flu B, HMPV A/B, HBoV, HRV, HPIV-1-4, HAdV and HCoVs showed a positivity of 7.58, 23.60, 3.65, 11.80, 4.49, 18.54, 11.79, 7.58 and 3.93 % respectively for each virus in the present study in comparison to a positivity of 18.39 %, 14.1 %, 13.3 %, 2.9 %, 0.5–4.5 % [13, 18, 19], 20.78 %, 8.62 %, 3.5 %, and 4.70 % 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

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 (19.40 %) samples, RSV B in 50 (74.63 %) samples and RSV A & RSV B dual infections in 4 (5.97 %) samples

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 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.