|Year : 2015 | Volume
| Issue : 1 | Page : 73-77
Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases
S Dhakad1, PC Mali2, S Kaushik3, AA Lal4, S Broor1
1 INCLEN Trust International , New Delhi, India
2 Department of Zoology , University of Rajasthan, Jaipur, India
3 Department of Biotechnology , MDU University Rohtak, Haryana, India
4 Intern from Emori University , Georgia, USA
|Date of Submission||07-Oct-2013|
|Date of Acceptance||16-Jan-2014|
|Date of Web Publication||5-Jan-2015|
INCLEN Trust International , New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: Influenza epidemics and periodic pandemics occur worldwide resulting in significant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type influenza viruses, as cell culture has a long turnaround time. Materials and Methods: Nasopharyngeal swabs were collected from patients presenting with influenza-like illness (ILI) at AIIMS OPD and Primary Health Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of 1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for influenza A using primers for matrix gene and for influenza B using non-structural gene (NS) primers. All influenza A positives were sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September 2009. Results: Of the 544 samples, 136 (25%) were positive for influenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as influenza A and 47 (34.5%) as influenza B viruses and 3 (2%) samples showed dual infection with influenza A and B. Of the 86 influenza A positive samples 48 (55.8%) were identified as seasonal influenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of influenza positivity using virus culture revealed that only 97/136 (71.3%) were influenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and influenza B (38/47; 80.8%); all influenza A/H1N1pdm09 viruses were detected by both methods. Conclusion: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing influenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for influenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.
Keywords: Influenza, Pandemic H1N1, Cell culture, Reverse transcription-polymerase chain reaction
|How to cite this article:|
Dhakad S, Mali P C, Kaushik S, Lal A A, Broor S. Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases. Indian J Med Microbiol 2015;33:73-7
|How to cite this URL:|
Dhakad S, Mali P C, Kaushik S, Lal A A, Broor S. Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 9];33:73-7. Available from: http://www.ijmm.org/text.asp?2015/33/1/73/148383
| ~ Introduction|| |
Influenza viruses cause frequent epidemics and periodic pandemics, and are a major public health problem. Globally, influenza is responsible for 250,000-500,000 deaths annually.  Presently, H1N1, H3N2 sub-type of influenza type A and type B influenza viruses are circulating and causing epidemics every year, globally.  In 2009, a novel influenza virus (H1N1) pdm09 emerged, with at least 18,449 deaths reported by August 2010.  While A (H1N1) pdm09 is no longer a pandemic virus, however, A (H1N1) pdm09 continues to circulate widely and has become part of seasonal H1N1 virus.  In India, the first confirmed case of pandemic influenza A (H1N1) was reported in Hyderabad on 16 May 2009  and by July 2010, a total of 34,669 laboratory-confirmed cases including 1692 deaths were reported.
As clinical presentation of many respiratory illnesses may resemble influenza, diagnosis can only be confirmed by laboratory tests. Diagnostic methods currently used for detection of influenza viruses in clinical laboratories include rapid antigen tests, viral culture, enzyme immunoassay and molecular tests such as real time reverse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR.  From the public health perspective, virus isolation and characterisation of influenza viruses is still the gold standard and remains the backbone of global surveillance for epidemic influenza and for the emergence of novel potentially pandemic strains.  However, for the purpose of diagnosis in an epidemic or pandemic situation, when a large number of potentially positive samples are received, the mandatory rapid virus identification cannot be done through culture procedures as it is time consuming. Influenza virus sub-typing is a critical tool in making therapeutic and infection control decisions. Virus isolation and characterisation by hemagglutination inhibition (HI) in these situations is time consuming. Molecular techniques such as conventional and real time RT-PCR provide the rapidity and enhanced sensitivity for detection and typing and sub-typing of influenza viruses. However, in resource limited countries real-time RT-PCR cannot be used by many peripheral laboratories because of the high cost. Further with the emergence of pdm09 viruses in 2009 the need for an early and accurate diagnosis for pandemic influenza became important for epidemiological and therapeutic purposes. Thus we have developed and evaluated an in-house multiplex RT-PCR and compared it with virus isolation for detection and characterisation of influenza viruses including pandemic A (H1N1) pdm09 in this study.
| ~ Materials and Methods|| |
The study was conducted from June 2007 to January 2009 and then from September to November 2009. During this period, 1567 nasal and throat swab samples were collected from patients of all ages with influenza-like illness (ILI) and their clinical details were recorded. Of these, 544 samples were selected randomly for the present study. ILI was defined as fever (≥100°F) or history of fever accompanied with cough and/or sore throat. Written consent was obtained from all patients at the time of sample collection and the study was approved by the Institutional Ethics Committee of All India Institute of Medical Sciences, New Delhi (letter no IEC/P-23/04.01.2010).
Samples were transported in 3 ml of viral transport medium (VTM) to the virology laboratory at 4°C. Specimens were processed in biosafety level II cabinet, and divided into three aliquots. The first aliquot was used for virus isolation in cell culture, the second was used for RT-PCR and the third was stored at −80°C deep freezer as a backup.
Influenza viruses were isolated in Madin-Darby Canine Kidney (MDCK) cells, provided by National Institute of Virology (NIV), Pune and maintained in Dulbecco's Modified Eagle medium supplemented with 10% foetal calf serum (Gibco), 1% L-glutamine (Sigma, USA), 100 IU/ml penicillin (Sigma, USA), 100 mg/ml streptomycin (Hi Media, India). The cells were seeded onto T-25 flask (Corning Incorporated, NY, USA) either 24 or 48 h prior to sample inoculation at 35°C in presence of 5% CO 2 . The processed samples were inoculated (400 μl) on to monolayers of MDCK cells. The inoculum was allowed to adsorb for 30 min at 37°C, followed by addition of 5 ml of serum-free virus growth medium with 2 μg/ml trypsin (Sigma, USA) to each flask. The flasks were again incubated at 35°C with 5% CO 2 . Cell culture was observed daily for cytopathic effect (CPE). Cells were harvested either when 3+ or 4+ CPE was observed or on 7 th day even if no CPE was observed. Presence of influenza virus was confirmed by hemagglutination assay (HA) using 0.75% guinea pig red blood cells and sub-typing was done by hemagglutination inhibition assay (HI) using reference antisera provided by World Health Organisation (WHO) collaborative centre for influenza viruses at Centre for Disease Control and Prevention (CDC) Atlanta, USA.
Reverse transcription polymerase chain reaction
RNA extraction was done using 350 μl of sample with the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Influenza virus cDNA was synthesised using random primer (Promega Corps., USA) and avian myeloblastic reverse transcriptase (AMV RT), (Promega Corp. USA). For 12.5 μl reaction volume, 5.0 μl of RNA and 500 ng of random primer along with 200 μM of each deoxynucleotise triphosphates (dNTPs) (Promega, Corps., USA) and 10 units of AMV RT were used. The reaction mix was incubated at 37°C for 90 min, followed by 65°C for 10 min to inactivate the enzyme.
Primers used for detection and typing of influenza virus targeted amplification of matrix and non-structural gene (NS) for influenza A and influenza B, respectively, and sub-typing of seasonal influenza A into A/H1N1, A/H3N2 used specific primers from hemagglutinin gene (HA) and neuraminidase gene (NA) [Table 1].  New set of the typing primer (type A/Mat-II) from matrix gene and sub-typing primer from HA gene for influenza A were used on samples collected after July 2009 as pandemic A (H1N1) pdm09 viruses were not detected by the earlier primers [Table 1]. 
|Table 1: Primer sequences used for typing and sub-typing of influenza virus by Multiplex RT-PCR|
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Typing PCR for influenza A and B was carried out as a monoplex assay with 5 μl of cDNA in a total volume of 25 μl containing 50 picomoles each of forward and reverse primers of influenza A or influenza B, 200 μM each of four dNTPs, and 1.5 units of the Taq polymerase (Bangalore Genei, India). DNA amplification was performed using an initial denaturation for 3 min at 94°C, followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 52°C and extension for 1 min at 72°C, with final extension for 10 min at 72°C in a thermo cycler (Gene AMP PCR system 9700, Applied Biosystems, USA). Amplicons of 311 for influenza A [[Figure 1]b and c] and 108 bp for influenza B [[Figure 1]a] were visualised under a digital gel documentation system (Bio-Rad, UK). PCR mix for multiplex RT-PCR for pandemic A (H1N1) pdm09 influenza viruses contained 20 picomoles each of primers from matrix gene and 10 picomoles each of primer targeting the HA gene. Rest of the conditions of PCR reaction were the same as sub-typing multiplex PCR.
|Figure 1: (Panel a) monoplex PCR. For Influenza B Lane 1, molecular size marker (100 bp); Lanes 2, 3, 4, 5 and 7, samples are negative for influenza B; Lane 6, sample is positive for influenza B (amplicon size 108 bp); Lane 8, positive control; Lane 9, negative control. (Panel b): Sub-typing of influenza A virus by multiplex PCR. Lane 1, molecular size marker (100 bp); Lane 2, 3 and 5, samples are positive for influenza A/H3N2 (amplicon size 311 bp for influenza A, 232 bp for A/H3 and 173 bp for N2 visualised); Lane 4, samples is positive for influenza A/H1N1 (amplicon size 311 bp for influenza A, 164 bp for A/H1 and 106 bp for N1 visualised); Lane 6, negative control. (Panel c): Typing and sub-typing of pandemic influenza A/H1N12009 by monoplex RT-PCR. Lane 1, molecular size marker (100 bp); Lane 2, 3 and 4 samples are positive for pdm2009 influenza A (amplicon size 348 bp); Lane 5, negative control; Lane 6 positive control; Lane 7, 12 blank; Lane 8, 9, 10 samples are positive for pdm2009 A/H1 (amplicon size 465 bp); Lane 11 negative control; Lane 13 positive control|
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Sub-typing - Multiplex PCR
A multiplex PCR was performed with 5 μl of cDNA in 25 μl reaction volume containing 200 μM of each dNTPs, 50 picomoles each of forward and reverse primers for seasonal H1, H3, N1 and N2, 1.5 U of Taq DNA polymerase. DNA amplification was performed using initial denaturation for 3 min at 94°C, followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C and extension for 30 s at 72°C, with final extension for 10 min at 72°C in a thermocycler. Amplicon of 164, 232, 106 and 173 bp, respectively, were visualised for H1, H3, N1 and N2 [[Figure 1]b]. In addition, a multiplex RT-PCR for pandemic A (H1N1) pdm09 influenza viruses was also carried out on 96 samples collected after September 2009 with primers for matrix gene and HA gene giving amplicons of 348 and 465 bp, respectively [[Figure 1]c].
| ~ Results|| |
Influenza detection by RT-PCR and virus isolation
During the study period, June 2007 to January 2009 and September to November 2009, 1567 clinical samples from cases with ILI were collected, of which 544 samples were randomly selected and evaluated for typing, and sub-typing of influenza virus by cell culture and RT-PCR [Table 2]. Influenza viruses were detected in 136/544 (25%) samples by RT-PCR; of these, 64 (47%) were typed as influenza A and 47 (34.5%) as influenza B and in 3 (2%) samples dual infection with influenza A and B viruses was identified. In 22/96 (22.9%) samples from September 2009 onwards pandemic A (H1N1) pdm09 influenza viruses were detected.
|Table 2: Comparison of RT - PCR with virus isolation for detection and sub-typing of influenza A viruses in samples from ILI cases|
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By virus isolation and HI influenza viruses were detected in 97 of 544 (17.8%) specimens tested; of these, 59 (43.3%) were typed as influenza A and 38 (27.9%) as influenza B and no co-infections were identified [Table 2]. Of the 86 influenza A viruses detected by RT-PCR, only 59/86 (68.6%) were positive by virus isolation, whereas 38/47 (80.8%) of influenza B positive specimens were detected by virus culture. Thus RT-PCR increased the detection rates of influenza by 28.6% as compared with virus isolation. Influenza viruses were detected in additional 7.1% specimens by RT-PCR.
Influenza A sub-typing by multiplex RT_PCR and virus isolation
Of the 64 influenza A positive samples, 48 (75%) were identified as seasonal influenza A/H1N1, and 16 (25%) as A/H3N2 by mRT-PCR. In addition, in 96 samples tested by additional pdm09 m-RT-PCR, 22 (22.9%) were found positive for pandemic A (H1N1) pdm09. Comparison of influenza A sub-type detection by mRT-PCR and virus isolation revealed a marked difference in sub-type detection by virus isolation; whereas all of A (H1N1) pdm09 were detected by both culture and RT-PCR, only 26/48 (54%) of seasonal A/H1N1 from 2007 and 11/16 (68%) of A/H3N2 were detected by virus isolation, suggesting a lower sensitivity for seasonal influenza A/H1N1 viruses from 2007 to 2008 [Table 2]. The sensitivity of RT-PCR as compared with virus isolation taken as gold standard was 100% but the specificity varied between 94% and 100% and positive predicitive value (PPV) was between 54% and 100% and negative predictive value (NPV) was 100% [Table 2]. Analysis of clinical features of influenza positive and negative indiviuals revealed that headache and body ache were associated with influenza positivity (P < 0.05) [Table 3].
|Table 3: Clinical features of influenza positive and negative indiviuals|
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| ~ Discussion|| |
Traditionally influenza virus surveillance is based on detection of influenza viruses by culture, although culture will remain the back bone for characterisation of circulating influenza viruses and vaccine strain selection, the clinical and virological surveillance can be improved by timely and accurate diagnosis. Published studies using culture methods may have under estimated the prevalence and rates of influenza globally. In recent years, real time RT-PCR has replaced virus isolation for influenza surveillance but the high cost (approximately 30 US dollars) and the non-availability of sophisticated equipment makes it less feasible for smaller laboratories in resource poor countries. Early detection of influenza is also important for timely detection of the first cases in a community, and adoption of control measures to avoid the spread of infection in institutions and nursing homes.  Antiviral medications can be used as effective chemoprophylaxis to reduce the severity and duration of illness, particularly if used within the first 48 h after illness onset, hence timing of viral confirmation is important for clinical management of influenza. 
In the current study, we report the comparison of conventional RT-PCR with virus isolation for detection of influenza viruses and found that conventional RT-PCR is a sensitive and rapid molecular technique for detection of seasonal influenza viruses as compared with virus isolation. In addition, we also describe an in-house mRT-PCR for the detection of pandemic A (H1N1) pdm09 influenza viruses. While 136/544 (25%) samples were positive for influenza viruses by RT-PCR, only 97/544 (17%) were positive by virus isolation; thus RT-PCR provided an increase in detection rate of approximately 28.6% as compared with virus isolation. This data is comparable to previously published studies from Scotland where 20-27% increase in detection rate using RT-PCR was reported. Further a recent study from Iran has shown 24.2% increase in detection rate by RT-PCR.  The lower sensitivity of detection by virus isolation may be due to the fact that culture assays need a minimum number of viable virus particles in the clinical samples, and viability is often lost between collection of specimens and execution of laboratory tests due to improper storage.  In these circumstances, assays that are able to detect viral nucleic acid, without requiring viable viruses, offers a great advantage.  An additional unique feature of the current study was the ability of mRT-PCR to rapidly sub-type influenza A viruses including pandemic A (H1N1) pdm09 viruses. Whereas for typing and sub-typing by HAI one has to depend upon availability of specific antisera usually prepared in ferrets. Further by RT-PCR co-infections with influenza A and B viruses could also be detected, which were missed by virus isolation. Similar findings have also been reported by other studies. 
The only few food and drug administration (FDA)-approved molecular assays for sub-typing influenza A viruses are the xTAG respiratory virus panel assay (Luminex Corp., Australia), which is a multiplex RT-PCR assay that uses flow cytometry for detection and the single plex real time RT-PCR assays developed by CDC. However, the mRT-PCR assay developed in this study can rapidly sub-type circulating human influenza A viruses. A Fluplex multiplex RT-PCR has also been developed by a laboratory in Milwaukee Wisconsin, USA but the confirmation of results in this assay are based on an enzyme hybridisation technique, which further makes it more expansive and technically difficult.  Further the mRT-PCR for pandemic A (H1N1) pdm09 viruses can also be combined as a single multiplex assay to the sub-typing mRT-PCR to make this assay more economical and easy to perform. Taken together, these studies suggest that influenza detection by RT-PCR is much more sensitive than traditional culture methods, it has a faster turnaround time and the advantages over real time RT-PCR are that it does require high technical expertise and specialised equipment, and is cost effective. This assay should be useful in diagnostic and epidemiological settings in resource poor countries.
| ~ Conclusion|| |
In summary, this study describes a comparison of an in-house RT-PCR with virus isolation for influenza viruses. RT-PCR was found to be rapid, sensitive and cost-effectiveness molecular method for diagnosis of seasonal influenza A and pandemic A (H1N1) pdm09 influenza viruses that circulate regionally and globally. Further the study demonstrated that sub-typing by multiplex RT-PCR of influenza viruses can be achieved rapidly, which is important for epidemiologic surveillance, and rapid identification of circulating strains of influenza viruses. Our study demonstrates that sensitivity of virus isolation for A/H1N1 pdm09 viruses was 100% as compared to RT-PCR but seasonal influenza viruses were detected by RT-PCR in additional 39 samples.
| ~ Acknowledgements|| |
The authors acknowledge the Indian Council of Medical Research (ICMR), New Delhi for providing funds through "multisite monitoring of human influenza viruses in India " to Microbiology department of AIIMS. The authors also want to thank Dr. Guresh Kumar from department of Biostatistics AIIMS for data analysis.
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[Table 1], [Table 2], [Table 3]