Abraham, Asha Mary

Dhanapal, Pavithra

Giri, Sidhartha

Iturriza-Gomara, Miren

John, Jacob

Kang, Gagandeep

Kumar, Nirmal

Rajan, Anand K

Taniuchi, Mami

Venkatesan, Jayalakshmi

Sidhartha Giri

Anand K. Rajan

Nirmal Kumar

Pavithra Dhanapal

Jayalakshmi Venkatesan

Miren Iturriza-Gomara

Mami Taniuchi

Jacob John

Asha Mary Abraham

Gagandeep Kang

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Journal of Medical Virology

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant

University of Liverpool Repository

Received: 8 July 2016 | Accepted: 30 January 2017DOI 10.1002/jmv.24793SHORT COMMUNICATIONComparison of culture, single and multiplex real-time PCR fordetection of Sabin poliovirus shedding in recently vaccinatedIndian childrenSidhartha Giri1 | Anand K. Rajan2 | Nirmal Kumar1 | Pavithra Dhanapal1 |Jayalakshmi Venkatesan1 | Miren Iturriza-Gomara3 | Mami Taniuchi4 |Jacob John5 | Asha Mary Abraham2 | Gagandeep Kang11Department of Gastrointestinal Sciences,Christian Medical College, Vellore, India2Department of Clinical Virology, ChristianMedical College, Vellore, India3 Institute of Infection and Global Health,University of Liverpool, Liverpool, UnitedKingdom4Division of Infectious Diseases andInternational Health, University of VirginiaSchool of Medicine, Charlottesville, Virginia5Department of Community Medicine, ChristianMedical College, Vellore, IndiaCorrespondenceSidhartha Giri, Department of GastrointestinalSciences, Christian Medical College, Vellore,India.Email: sidharthgiri@cmcvellore.ac.inAlthough, culture is considered the gold standard for poliovirus detection from stool samples,real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirusfrom the stool of recently vaccinated children by culture, single andmultiplex real-timePCRwascompared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61(76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assaysrespectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture forpoliovirus detection in stool, although the difference was not statistically significant.K E YWORD Scell culture, poliovirus, shedding1 | INTRODUCTIONIdentification of poliovirus (Family Picornaviridae, genusEnterovirus, species Poliovirus) in clinical and environmentalsamples is a crucial component of the global polio eradicationprogram led by the World Health Organization (WHO).1 Earlierstudies aimed at detection of poliovirus in stool used culture inseveral cell lines including Vero, Hep-2, and MK cells.2 Since2004, the WHO has recommended L20B and RD cell lines forpoliovirus isolation,3 with samples cultured for 14 days beforebeing reported as negative. Identification of the three poliovirusserotypes in culture positive samples needs intratypic differenti-ation (ITD) by neutralization, enzyme-linked immunosorbentassay (ELISA), or polymerase chain reaction (PCR)4 extendingthe time taken to generate a report. Because of these limitations,conventional and real-time PCR have emerged as alternatives toculture for the early and more sensitive detection of poliovirusfrom clinical samples.2,5In this study, we evaluated the performance of culture in the L20Bcell line and two real-time PCR assays (for single poliovirus types and aone step multiplex real-time PCR) in the detection of Sabin poliovirusfrom stool samples of recently vaccinated Indian children.2 | MATERIALS AND METHODS2.1 | Study design and sample collectionA random set of 80 stool samples from infants vaccinated withtrivalent oral poliovirus vaccine (tOPV) at 6 weeks of age was selectedfor the study. The samples were collected as part of a clinical trial(CTRI/2012/05/002677) evaluating supplementation with zinc and/or probiotics to enhance the immune response of oral rotavirusvaccine and tOPV in Indian infants which was conducted atChinnallapuram, Vellore between July 2012, and February 2013.Institution where work was performed: Christian Medical College, Vellore,India.J Med Virol. 2017;9999:1–4 wileyonlinelibrary.com/journal/jmv © 2017 Wiley Periodicals, Inc. | 1A total of 3275 stool samples from 570 infants were collected on days0, 4, and 7 after administration of tOPV and oral rotavirus vaccine(Rotarix) at 6and 10 weeks of age. The study was approved by theInstitutional Review Board (IRB) of Christian Medical College (CMC),Vellore and informed consent was obtained from the parents/legalguardians of all the study participants.2.2 | CultureStool culture was performed in the L20B cell line according to WHOguidelines.3 A 20% stool suspension was filtered through a 0.22micron filter and the filtrate used for culture. The inoculated andcontrol flasks were incubated at 37°C (5% CO2). All observations wererecorded for 1 week or till culture positive. For negative samples, ablind passage was performed and the flasks observed for another7 days. If the cultures showed no cytopathic effect (CPE) by 14 days,the sample was reported as negative for poliovirus.2.3 | RNA extraction and cDNA conversionBefore extraction, a 20% stool suspension of each sample was spikedwith 1 μL of MS2 phage as an extraction and amplification control,which was calibrated to yield a cycle threshold (Ct) value of 25-26cycles by the multiplex real-time PCR. Positive and negative controls(nuclease free water) were included with each set of extraction. RNAwas extracted using Vx reagents on a QIAxtractor (Qiagen, GmbH,Hilden, Germany). Complementary DNA (cDNA) was generated fromthe eluted RNA by reverse transcription using random primeroligonucleotides (Invitrogen, Carlsbad, CA) and 200 U/μL of Moloneymurine leukemia virus reverse transcriptase (Invitrogen).2.4 | Real-time PCR for Sabin viruses 1, 2, and 3The serotype specific primers and probes targeting the VP1 regionof the Sabin poliovirus genome were adopted from Kilpatricket al.5 Plasmids constructed by ligation of poliovirus serotypes 1,2, and 3 region of the VP1 PCR fragment in TOPO-TA 2.1 vectorpropagated in Escherichia coli DH5α cells were used as plasmidDNA standards for calibration of assay interpretation (supple-mentary information). The detection limit was determined bystandard curves using 10-fold serial dilutions of Sabin plasmidsranging from 3 × 107 to 3 copies/μL. The detection limit wasthree copies corresponding to a Ct value of <40 for all threeSabin poliovirus types. The TaqMan based real-time PCRassays used Sabin 1 (S1), Sabin 2 (S2), and Sabin 3 (S3) specificforward and reverse primers (Sigma Aldrich, Bengaluru, India) andprobes labeled with FAM and black hole quencher 1 (BHQ-1)(Sigma Aldrich), according to Kilpatrick et al.5The serotype specific real-time PCR assays included the PlatinumQuantitative PCR UDG Super Mix (Invitrogen) in a 25 μL total volumecontaining 12.5 μL of the PCR mix, 0.5 μL each of 20 μM primers,0.25 μL of 10 μM probe, 0.025 μL of ROX dye, 6.225 μL of nucleasefree water (NFW), and 5 μL of the template cDNA. The cyclingconditions were 2min at 50°C, 2 min at 95°C, and 40 cycles of 15 sat 95°C, and 1min at 45°C on the ABI 7500 Fast real-time PCRdetection system (Applied Biosystems, Foster city, CA). Serotypespecific positive control, negative control, and a no template control(NTC) with only the master mix were included in each run.2.5 | One step multiplex real-time PCR for detectionof Sabin serotypesThe multiplex one step reverse transcription real-time PCR used todetect tOPV shedding for all three Sabin types simultaneously wereperformed according to Taniuchi et al.6 The S1 and S2 probeswere minor-groove binding TaqMan probes (Applied Biosystems)with the VIC and FAM fluorophores, respectively. The S3 probe waslabeled with Texas Red and BHQ2 (Integrated DNA technologies,Coralville, IA) while the MS2 probe was labeled with Quasar 670and BHQ2 (Biosearch Technologies, Novato, CA). Initially, a primer-probe mix was prepared by adding 4 μL each of 100μM forward andreverse primers, and 2 μL of each 100 μM probes. The PCR includedthe2XqScript XLTOne-StepRT-qPCRToughmix (QuantaBiosciences,Gaithersburg, MD) in a 20 μL total volume containing 10 μL of thePCR buffer, 0.4 μL of the primer-probe mix, 5.6 μL NFW, and 4 μL oftemplate RNA. The cycling conditions were 15min at 50°C, 1min at95°C, and40cyclesof10 s at95°C, and45 sat60°ContheQuantstudio12K Flex real-time PCR detection system (Applied Biosystems).A cutoff Ct value of 36 for S1 and 37 for S2 and S3 were applied.6Prior to testing the samples, the positive controls for Sabin 1, 2,and 3, were run on both the real-time PCR machines (ABI 7500 Fastand the Quantstudio 12 K Flex real-time PCR detection system), andthe results were reproducible (Ct value range of 28-30 for all threeSabin types). The Sabin plasmid dilutions ranging from 3 × 107 to3 copies/μL were also run on both the real-time PCR machine in threeseparate runs and the results were found to be reproducible betweenthe two equipment (Supplementary Information).2.6 | Statistical analysisAll the statistical analyses were performed by using SPSS StatisticalSoftware 17.0 (SPSS Inc, Chicago, IL), Microsoft Excel, and GraphPadPrism Version 5.0 for Windows (San Diego, CA). A P-value of <0.05was considered statistically significant. All P-values were two tailed.Inter assay agreement statistics (kappa values) were used to comparethe detection of Sabin polioviruses by the single and multiplexreal-time PCR assays.3 | RESULTSOf the 80 stool samples tested, 55 (68.75%) were positive by forpolioviruses by culture. In contrast, 61 (76.25%) and 60 (75%) sampleswere positive by the single and one step multiplex real-time PCRassays respectively (Table 1). The increased sensitivity for detection bythe two real-time PCR assays, compared to culture, was, however, notstatistically significant (P = 0.28 for singleplex, and P = 0.38 formultiplex real-time PCR). Overall, 51 samples (63.75%) were positiveand 14 samples (17.5%) were negative by all three methods. All the2 | GIRI ET AL.stool samples were positive for MS2 by multiplex real-time PCR, thus,ruling out any PCR inhibitors in the samples.If culture in L20B cell line is considered the gold standard forpoliovirus detection, the sensitivity of singleplex and multiplex real-time PCR were 94.5% and 92.7%, respectively, while the specificitywas 64% for both the PCR assays. If the PCR methods are consideredthe gold standard for detection, the sensitivity of culture was 85% andthe specificity varied from 84.2% to 80%.The quantity of virus as estimated by Ct values differed betweenculture positive and negative samples. Culture positive samples hadsignificantly lower Ct values (singleplex PCR: median [27.6], range[16.4-39.8]; multiplex PCR: median [26.1], range [15.0-37.0]) thansamples that were negative in culture (singleplex PCR: median [35.6],range [26.7-39.1]; multiplex PCR: median [33.8], range [27.3-36.2]),(P < 0.05) (Fig. 1).Figure 2 illustrates the very high correlation (R2 = 0.931) betweenthe single and multiplex real-time PCR assays. The two real-timeassays also showed very good agreement for the detection of Sabinpolioviruses with a kappa of 0.831 (95%CI, 0.687-0.974). The medianCt values of the three poliovirus serotypes detected by single andmultiplex real-time PCRwere not significantly different ([S1: singleplex{29.3}, multiplex {28.8}; P = 0.6,S2: singleplex {24.8}, multiplex {21.5};P = 0.2,S3: singleplex {31.8}, multiplex {31.9}; P = 0.5]).4 | DISCUSSIONThe use of two real-time PCR assays for detection of single or multipleSabin polioviruses in stool samples from vaccinated children showedthat the assays were more sensitive than culture, although thedifference did not achieve statistical significance. This could be due tothe low sample size (n = 80) in this study, and hence, similar studies infuture involving larger sample sizes are necessary. The lowerspecificity (64%) seen in both the real-time PCR assays is becausethese assays were able to detect additional Sabin poliovirus positivesamples which were negative by culture. The higher sensitivity of bothreal-time PCR assays could be because the PCR also detects virus thatmay be defective or bound to antibody making it non-infectious, andthus, non-detectable by culture.The PCR assays were easy to use and the results were available in amuch shorter time than culture. The turn-around time for culture was4-7 days for positive samples and 14 days for negative samples. Incontrast, the turn-around time was 10-12 h for singleplex real-time PCRand only 6-7 h for the one-step multiplex real-time PCR. The substantialamount of time saved using real-time PCR assays could be crucial in theearlier detection of outbreaks in the post-eradication era, thus, givingsufficient time to prevent widespread transmission of polioviruses.7TABLE 1 Comparison of Sabin poliovirus detection by culture, single and multiplex real-time PCR in stool samples of children recently vaccinatedwith trivalent oral poliovirus vaccineCultureNo. of samples (%)Singleplex real-time PCRNo. of samples (%)Multiplex real-time PCRNo. of samples (%)Positive for Sabin poliovirus 55 (68.75%) 61 (76.25%) 60 (75%)Negative for Sabin poliovirus 25 (31.25%) 19 (23.75%) 20 (25%)S1 – 6 (7.5%) 8 (10%)S2 – 12 (15%) 11 (13.75%)S3 – 4 (5%) 4 (5%)S1 + S2 – 9 (11.25%) 12 (15%)S1 + S3 – 2 (2.5%) 4 (5%)S2 + S3 – 6 (7.5%) 3 (3.75%)S1 +S2 + S3 – 22 (27.5%) 18 (22.5%)S1, Sabin 1; S2, Sabin 2; S3, Sabin 3.FIGURE 1 Comparison of culture positive and negative sampleswith Ct values in single and multiplex real-time PCRFIGURE 2 Correlation between the single and one step multiplexreal-time PCR assays for detection of poliruses in the stool ofchildren given trivalent oral poliovirus vaccineGIRI ET AL. | 3The standard method of evaluating mucosal immunity topoliovirus is to challenge individuals with OPV and measure sheddingby culture, but few studies have used PCR.2,8 A Mexican study, whichused a novel real-time PCR assay, detected low levels of OPVexcretion after vaccination in stool and sewage samples and alsodifferentiated between revertant and non-revertant serotypes.9 Arecent study from India evaluating mucosal immunity to a dose ofinactivated poliovirus vaccine (IPV) in children previously primed withOPV found the positive predictive value of real-time PCR for culture inthe detection of a challenge dose of Sabin polioviruses in stool to be87% for serotype 1 and 89% for serotype 3, respectively.10 Somestudies have reported the use of PCR for environmental surveillancethough culture has been extensively used for such purposes.11 A studyfrom Cuba found PCR to be as sensitive as culture in L20B cell line forpoliovirus detection from waste water and stool samples.11Along with serotype specific singleplex real-time PCR assays forpoliovirusdetection,multiplexPCRassayshavebeendeveloped inthe lastfew years to simultaneously detect all three poliovirus serotypes, thus,reducing turn-around-time.6,12 A study by Buonagurio et al,13 comparingamultiplex reverse transcription PCR (RT-PCR) method to culture for thedirect detection of Sabin polioviruses in stool collected from first-doseOPV recipients showed that thePCRdetected poliovirus types 1, 2, and3in 67.2, 82.6%, and 53.8% specimens respectively compared to 55.4%,64.1%, and 27.7% by culture. The RT-PCR assay showed an increase of12%, 18%, and 50% in the detection rates of poliovirus types 1, 2, and 3compared to culture.13 Another recent study found the sensitivity andspecificityof amultiplexone-step real-timequantitativePCRassay for thedetection of Sabin poliovirus in stool samples to be 89% and 91%,respectively, compared to culture.6 Similar to our findings, the study alsoshoweda significantly higherquantityof thevaccinevirusdetectedby themultiplex PCR in culture positive samples compared to samples whichwere culture negative.6 Another study by Laassri et al, also found aquantitative multiplex real-time PCR assay to be highly sensitive andspecific compared to culture for Sabin poliovirus detection in clinicalsamples.7Despite the present setbacks in eradicating poliomyelitis world-wide, it is expected that the circulation of wild polio viruses (presentlyWPV1 only) will stop in the near future.7 However, for maintaining apolio free world, continuous surveillance for vaccine virus is necessaryas circulation of OPV strains could lead to more outbreaks of vaccinederived polioviruses.7 Real-time PCRassays can play a crucial role in thesurveillance and early detection of vaccine virus circulation globally.7CONFLICTS OF INTERESTNone.REFERENCES1. Sutter RW, Platt L, Mach O, Jafari H, Aylward RB. The new polioeradication end game: rationale and supporting evidence. J Infect Dis.2014;210:S434–S438.2. Tebbens RJD, Pallansch MA, Chumakov KM, et al. Expert review onpoliovirus immunity and transmission. Risk Analysis. 2013;33:544–605.3. World Health Organization. Polio Laboratory Manual. WHO/IVB/04.10. Geneva; 2004.4. van der Avoort HGAM, Hull BP, Hovi T, et al. Comparative study offive methods for intratypic differentiation of polioviruses. J ClinMicrobiol. 1995;33:2562–2566.5. Kilpatrick DR, Yang CF, Ching K, et al. Rapid group-, serotype-, andvaccine strain-specific identification of poliovirus isolates by real-timereverse transcription-PCR using degenerate primers and probescontaining deoxyinosine residues. J Clin Microbiol. 2009;47:1939–1941.6. Taniuchi M, Begum S, Uddin MJ, et al. Kinetics of poliovirusshedding following oral vaccination as measured by quantitativereverse transcription-PCR versus culture. J Clin Microbiol. 2015;53:206–211.7. Laassri M, DiPiazza A, Bidzhieva B, Zagorodnyaya T, Chumakov K.Quantitative one-step RT-PCR assay for rapid and sensitive identifi-cation and titration of polioviruses in clinical specimens. J VirolMethods. 2013;189:7–14.8. Laassri M, Lottenbach K, Belshe R, et al. Effect of different vaccinationschedules on excretion of oral poliovirus vaccine strains. J Infect Dis.2005;192:2092–2098.9. Troy SB, Ferreyra-Reyes L, Huang CH, et al. Use of a novel real-timePCR assay to detect oral polio vaccine shedding and reversion in stooland sewage samples after a Mexican national immunization day. J ClinMicrobiol. 2011;49:1777–1783.10. John J, Giri S, Karthikeyan AS, et al. Effect of a single inactivatedpoliovirus vaccine dose on intestinal immunity against poliovirus inchildren previously given oral vaccine: an open-label, randomisedcontrolled trial. Lancet. 2014;9953:1505–1512.11. Lago PM, Gary HE Jr, Perez LS, et al. Poliovirus detection in wastewater and stools following an immunization campaign in Havana,Cuba. Int J Epidemiol. 2003;32:772–777.12. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR:optimization and application in diagnostic virology. Clin Microbiol Rev.2000;13:559–570.13. Buonagurio DA, Coleman JW, Patibandla SA, Prabhakar BS, TatemJM. Direct detection of Sabin poliovirus vaccine strains in stoolspecimens of first-dose vaccinees by a sensitive reverse transcription-PCR method. J Clin Microbiol. 1999;37:283–289.SUPPORTING INFORMATIONAdditional Supporting Information may be found online in thesupporting information tab for this article.How to cite this article: Giri S, Rajan AK, Kumar N, et al.Comparison of culture, single and multiplex real-time PCRfor detection of Sabin poliovirus shedding in recentlyvaccinated Indian children. J Med Virol. 2017;9999:1–4.https://doi.org/10.1002/jmv.247934 | GIRI ET AL.

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children

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