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Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis

Open AccessPublished:January 02, 2022DOI:https://doi.org/10.1016/j.ijid.2021.12.361

      Highlights

      • Yellow Fever continues to cause disease in tropical and subtropical areas.
      • Yellow Fever may mislead with other diseases presenting the same clinical symptoms.
      • Our reaction was sensitive presenting LoD95% of approximately 1 virus per reaction.
      • The reaction did not present cross-reactions with other viruses.

      Abstract

      Introduction

      Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays.

      Objectives

      The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses.

      Methods

      We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency.

      Results and Discussion

      Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.

      Keywords

      Introduction

      The emergence and re-emergence of mosquito-borne arboviruses are of great public health impact resulting in numerous outbreaks worldwide (
      • Huang YJS
      • Higgs S
      • Vanlandingham DL.
      Emergence and re-emergence of mosquito-borne arboviruses.
      ). The yellow fever virus (YFV) is the prototype member of the genus Flavivirus and the requirement to complete its life cycle is the mosquito-borne transmission to some vertebrate hosts. In America, the transmission occur via saliva of infected mosquitoes of genus Aedes in the urban transmission cycle and genera Sabethes or Haemogogus in the sylvatic transmission cycle (
      • Gardner CL
      • Ryman KD.
      Yellow fever: a reemerging threat. (Special Issue: Emerging pathogens.).
      ).
      The YFV virion is approximately 40 nM in diameter with surface projections between 5 to 10 nm. The genome package has approximately 11 kb in a single-stranded, positive-polarity, infectious RNA molecule with a cap structure at the 5′ terminus. The 3′ terminal nucleotides form a very stable, highly conserved stem-loop structure, serving to stabilize the genome and finally, all the viral proteins. Three structural and 7 non-structural proteins are encoded in a single open reading frame (ORF) (
      • Gardner CL
      • Ryman KD.
      Yellow fever: a reemerging threat. (Special Issue: Emerging pathogens.).
      ).
      Infection by the YFV displays clinical manifestations ranging from mild non-specific illness to severe disease including high fever, chills, severe headache, jaundice, bleeding, failure of multiple organs, and shock (
      • Domingo C
      • Charrel RN
      • Schmidt-Chanasit J
      • Zeller H
      • Reusken C.
      Yellow fever in the diagnostics laboratory review-article.
      ;
      • Goldani LZ.
      Yellow fever outbreak in Brazil, 2017.
      ).
      The virus is endemic in tropical areas of Africa, Central and South America. Recently, outbreaks in the unvaccinated population were registered in Brazil with a spread rate of 0.12 km/day (
      • Giovanetti M
      • de Mendonça MCL
      • Fonseca V
      • Mares-Guia MA
      • Fabri A
      • Xavier J
      • de Jesus JG
      • Gräf T
      • Dos Santos Rodrigues CD
      • Dos Santos CC
      • Sampaio SA
      • Chalhoub FLL
      • de Bruycker Nogueira F
      • Theze J
      • Romano APM
      • Ramos DG
      • de Abreu AL
      • Oliveira WK
      • do Carmo Said RF
      • de Alburque CFC
      • de Oliveira T
      • Fernandes CA
      • Aguiar SF
      • Chieppe A
      • Sequeira PC
      • Faria NR
      • Cunha RV
      • Alcantara LCJ
      • de Filippis AMB
      Yellow Fever Virus Reemergence and Spread in Southeast Brazil, 2016-2019.
      ). This scenario resulted in a governmental decision for fractional YF vaccine doses in Brazil to cover the whole country population (
      • Domingo C
      • Charrel RN
      • Schmidt-Chanasit J
      • Zeller H
      • Reusken C.
      Yellow fever in the diagnostics laboratory review-article.
      ;
      • Monath TP
      • Vasconcelos PFC.
      Yellow fever.
      ;
      • Possas C
      • Lourenço-de-Oliveira R
      • Tauil PL
      • de Pinheiro FP
      • Pissinatti A
      • da Cunha RV
      • et al.
      Yellow fever outbreak in Brazil: the puzzle of rapid viral spread and challenges for immunisation.
      ;
      • Romano APM
      • Costa ZGA
      • Ramos DG
      • Andrade MA
      • de Jayme VS
      • de Almeida MAB
      • et al.
      Yellow Fever Outbreaks in Unvaccinated Populations, Brazil, 2008-2009.
      ).
      YF can be difficult to diagnose clinically, especially during the early stages of the disease. Severe YF cases may be misleading with the diagnosis being severe malaria, leptospirosis, viral hepatitis (especially fulminant forms), other hemorrhagic fevers, infection with other flaviviruses (such as dengue hemorrhagic fever), and poisoning ().
      A number of diagnostic methods and assays have been described for the detection of YFV infection, including viral culture, antigen detection, serological, and nucleic acid tests (
      • Domingo C
      • Charrel RN
      • Schmidt-Chanasit J
      • Zeller H
      • Reusken C.
      Yellow fever in the diagnostics laboratory review-article.
      ;
      • Marfin AA
      • Monath TP.
      Yellow Fever Virus.
      ). The most used nucleic acid-based methods are RT-PCR, RT-qPCR, and isothermal amplification assays. Furthermore, many assays are not able to identify all genotypes and the index of false-negative results can increase (
      • Domingo C
      • Charrel RN
      • Schmidt-Chanasit J
      • Zeller H
      • Reusken C.
      Yellow fever in the diagnostics laboratory review-article.
      ,
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ;
      • Escadafal C
      • Oumar Faye
      • Sall AA
      • Ousmane Faye
      • Weidmann M
      • Strohmeier O
      • et al.
      Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings.
      ;
      • Nunes MRT
      • Palacios G
      • Nunes KNB
      • Casseb SMM
      • Martins LC
      • Quaresma JAS
      • et al.
      Evaluation of two molecular methods for the detection of Yellow fever virus genome.
      ;
      • Waggoner JJ
      • Rojas A
      • Pinsky BA.
      Yellow fever virus: Diagnostics for a persistent arboviral threat.
      ;
      • Weidmann M
      • Ousmane Faye
      • Oumar Faye
      • Kranaster R
      • Marx A
      • Nunes MRT
      • et al.
      Improved LNA probe-based assay for the detection of African and South American yellow fever virus strains.
      ). Until the publication of this manuscript, only 4 RT-qPCR-based tests were described in literature to detect all genotypes (
      • Dash P
      • Boutonnier A
      • Prina E
      • Sharma S
      • Reiter P.
      Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.
      ;
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ;
      • Drosten C
      • Göttig S
      • Schilling S
      • Asper M
      • Panning M
      • Schmitz H
      • et al.
      ;
      • Weidmann M
      • Ousmane Faye
      • Oumar Faye
      • Kranaster R
      • Marx A
      • Nunes MRT
      • et al.
      Improved LNA probe-based assay for the detection of African and South American yellow fever virus strains.
      ).
      The aim of the present work was to develop a multiplex RT-qPCR assay for YFV detection. Conserved regions for all YFV genotypes were chosen as reaction targets and a human gene was implemented as an Internal Control. Comparative analysis of the present protocol was evaluated against diagnosis assays in reference to arbovirus monitoring laboratories in Brazil that use the molecular protocol recommended by the Pan American Health Organization (PAHO) (
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ). The compared parameters were analytical sensitivity and specificity, clinical performance, and cost per reaction.

      Material and Methods

      Specimens

      Specimens were obtained from reference laboratories in Brazil for the diagnosis of arboviruses (n = 204). We received results from 92 positive specimens for YFV, 20 positives for Zika virus (ZIKV), 21 positives for Chikungunya virus (CHIKV), 8 positive samples for dengue virus (DENV), cell cultures for dengue subtypes 1, 2, 3, and 4, and 59 negative samples.

      Prototype development

      Positive Control Construction

      The Positive Control was developed based on MS2 bacteriophage. A specific 5’UTR sequence of YF genome and part of human ribosomal RNA were cloned into the MS2 replicase gene of pET47b(+)-MS2. This vector was used to express a non-propagating MS2 particle. The use of MS2-like particles as a Positive Control was evaluated in an RT-qPCR assay described in the literature and the proposed multiplex prototype (
      • Borghetti IA
      • Zambenedetti MR
      • Requião L
      • Vieira DS
      • Aur M
      • R De C
      • et al.
      ;
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ;
      • Zambenedetti MR
      • Pavoni DP
      • Dallabona AC
      • Dominguez AC
      • de Poersch CO
      • Fragoso SP
      • et al.
      Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.
      ).

      Nucleic Acid Isolation

      Total RNA was extracted from 140 μL aliquots of different samples and MS2-YF control. The protocol was carried out using QIAamp viral RNA mini kit (QIAGEN®, Germany) following the manufacturer's recommendations and the RNA was eluted with 60 μL of elution buffer.

      RT-qPCR assay

      Multiplex RT-qPCR protocol was designed to simultaneously detect 5’ UTR of the YFV (
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ) and a conserved region of the human housekeeping gene 18S rRNA (
      • de Manta FSN
      • Leal-Calvo T
      • Moreira SJM
      • Marques BLC
      • Ribeiro-Alves M
      • Rosa PS
      • et al.
      Ultra-sensitive detection of mycobacterium leprae: DNA extraction and PCR assays.
      ;
      • Rampazzo RCP
      • da Solcà MS
      • Santos LCS
      • de Pereira LN
      • Guedes JCO
      • Veras PST
      • et al.
      A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.
      ). The RT-qPCR contained 5 μL of extracted RNA, Multiplex PCR Mastermix (IBMP, Brazil), 0.4 μL enzymes (Taq DNA Polymerase and Reverse Transcriptase) (IBMP, Brazil), 3.1 μL oligonucleotides mixture; 1.5 μL probes mixture. The pathogen probe was labeled with the reported dye FAM and the probe that hybridizes with the human gene was labeled with the HEX fluorophore. The reactions occurred in an ABI7500 Real-Time PCR System series (Thermo®, USA) set with the following protocol: 1 × 50°C/15 min; 1 × 95°C/10 min 45x [95°C/15s, 60°C/45s].

      RT-qPCR standards

      YFV standards were reconstituted as per the manufacturer's instructions to contain between 12,500-20,000 viral particles per microliter (Vircell Microbiologist®, Spain). Serial dilutions from 900 to 0.14 RNA viral particles per reaction were conducted in negative RNA plasm solution to build the calibration curve tested in RT-qPCR assays.

      Statistical analyses

      Analytical sensitivity was determined using a series of limiting dilutions of YFV RNA in RNA extracted from YFV-negative plasm. Each concentration was tested in 24 replicates. The LoD95% was calculated by fitting a probit model to the detection rate for each concentration tested, using custom R scripts (version 3.5.1). The Kappa coefficient was calculated using the diagnostic method of reference laboratories results with 95% confidence intervals (CI). The coefficient was used to test for agreement between the diagnostic methods, and Kappa results were interpreted according to Landis and Koch (
      • Landis JR
      • Koch GG.
      The Measurement of Observer Agreement for Categorical Data.
      ): 1.00–0.81 almost perfect, 0.80–0.61 substantial, 0.60–0.41 moderate, 0.40–0.21 fair and ≤0.20 slight agreement and Fishers's exact test and chi-Square were done using the GraphPad 8.0.

      Results and Discussion

      YF is difficult to diagnose, especially during the early stages and often overlaps with flaviviruses, Chikungunya, poisoning, viral hepatitis, severe malaria, or leptospirosis. All of these disorders have to be considered in differential diagnosis, and laboratory confirmation is essential to an early and specific diagnosis, for patient management and to adopt preventive measures to minimize severe cases and deaths (;
      • Wilder-Smith A
      • Gubler DJ
      • Weaver SC
      • Monath TP
      • Heymann DL
      • Scott TW.
      Epidemic arboviral diseases: priorities for research and public health.
      ).
      During 2014, primates died in metropolitan regions in Brazil. These epizootic events were part of YF's re-emergence in the country and were followed by hundreds of confirmed positive cases in humans during 2016-2021, leading to a public health emergency. As a response, the Brazilian Ministry of Health implemented preventive vaccination. Meanwhile, collaborative efforts between Reference Centers for arboviruses diagnosis allowed for the initiative to develop the Kit Febre Amarela-IBMP (IBMP, Brazil) (
      MS/SVS
      ;
      • Possas C
      • Lourenço-de-Oliveira R
      • Tauil PL
      • de Pinheiro FP
      • Pissinatti A
      • da Cunha RV
      • et al.
      Yellow fever outbreak in Brazil: the puzzle of rapid viral spread and challenges for immunisation.
      ;

      Rojas A, Diagne CT, Stittleburg VD, Mohamed-hadley A, Ar Y, Balmaseda A, et al. Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection 2018;98:1833–6. https://doi.org/10.4269/ajtmh.18-0024.

      ).
      We initially adapted a previously described RT-qPCR singleplex protocol (
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ) into a multiplex reaction that simultaneously detects endogenous 18S rRNA genes from human samples. The comparison of singleplex demonstrated that constitutive 18S addition rRNA gene did not affect the detection of YFV, even in very low amounts. Therefore, the improvement of reaction was also the Internal Control (IC), an endogenous gene, that guarantees the RNA quality and avoids false-negative results, especially because it did not alter analytical sensitivity when compared with singleplex reactions (data not shown).
      The linear regression and amplification plot can demonstrate the multiplex assay quality (Figure 1).
      Figure 1
      Figure 1(A) The linear regression plot for the YFV target. The regression line demonstrates R2 = 0.961. The amplification factor, calculated from the slope, was 1.86. The points plotted were mean ± SD of 900, 90, 30; 4.5; 2.25; 1.12, 0.56, 0.28, 0.14 virus/reaction. The continuous line represents the linear range, with which the regression equation was calculated. This regression line was extrapolated throughout the entire range (dashed line). Each point represents each technical replicate from 3 runs. (B) Amplification plot of points described in linear regression for YFV target and IC.
      Another limitation of previous tests for YF diagnosis is the lack of tools to support interpretation, such as the Positive Control. In the method presented here, a modified bacteriophage MS2 genome containing part of 5’ UTR of the YFV genomic RNA and a fragment of 18S human rRNA was used to simulate a positive sample from the extraction step up to the final analyses. Therefore, problems in RNA extraction, mix preparation, multiplex amplification, analyses, or a problem with the real-time system are accounted for when interpreting the results obtained with this protocol.
      The reactions described here cover all YFV genotypes, as described by PAHO reactions (
      • Domingo C
      • Charrel RN
      • Schmidt-Chanasit J
      • Zeller H
      • Reusken C.
      Yellow fever in the diagnostics laboratory review-article.
      ), although presented in a multiplex design with an endogenous human control. With this setup, the LoD95% determined for YFV was 0.932 viral genome copies per reaction, 95% CI. This analytical sensitivity allows for detection at higher Ct values and mitigates false-negative results (
      • Boga JA
      • Alvarez-Arguelles ME
      • Rojo-Alba S
      • Rodríguez M
      • de Oña M
      • Melón S.
      Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus.
      ;
      • Domingo C
      • Patel P
      • Yillah J
      • Weidmann M
      • Méndez JA
      • Nakouné R.
      ;

      Rojas A, Diagne CT, Stittleburg VD, Mohamed-hadley A, Ar Y, Balmaseda A, et al. Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection 2018;98:1833–6. https://doi.org/10.4269/ajtmh.18-0024.

      ;
      • Waggoner JJ
      • Rojas A
      • Pinsky BA.
      Yellow fever virus: Diagnostics for a persistent arboviral threat.
      ). Concentrations lower than the LoD95% were occasionally positive, indicating that the reaction conditions were optimized to the maximum (Figure 2).
      Figure 2
      Figure 2Determination of the RT-qPCR LoD95% by probit regression analysis. The determined LoD95% was 0.932 copies per reaction for yellow fever virus, 95% CI, which is equivalent to 79.9 virus per mL plasm. All assays contain Positive Control with amplification of both targets with Ct >30, plasm RNA, which shows amplification only for the Internal Control, and a Negative Control, which has no amplification for both targets.
      Three reference centers evaluated the YF IBMP Kit and compared it with our results. There were 92 positive samples and 112 negative samples using the protocols of reference centers. Using the proposal YF IBMP Kit the detection demonstrated 87 positive samples and 107 negative samples, we believe the 5 discrepancies were caused by the freezing and thawing of the RNA leading to sample degradation. In the context of cross-reactions, we analyzed samples known to be positive for other arboviruses such as DENV (n = 8) without serotype-specific test, ZIKV (n = 20), CHIKV (n = 21), and cultured samples used as standards in ZDC BioMol kit (IBMP, Brazil) for ZIKV, CHIKV, DENV1, DENV2, DENV3, and DENV4. All presented negative results for YFV detection. Moreover, the human 18S rRNA was detected in all material from patients which excludes the possibility of RNA degradation, operator mistakes, reagents quality, or problems with PCR instruments. Thus, the multiplex RT-qPCR presented here is widely considered an efficient tool for the detection of YFV especially because its reproducibility and the Kappa values confirmed almost perfect for all centers.
      The proposed YF IBMP Kit reagents presented 15 months of stability and no impact in the results of a known sample and/or control after 6 cycles of freeze-thaw. Its performance could be compared with PAHO reference methods presenting benefits to users as minimizing mistakes and predicting RNA quality from the extraction step to the final analyses. This kit also could be an alternative to guarantee compatible and comparable results in different centers enabling accurate epidemiological analysis and keeping users safe. This In Vitro Diagnostic (IVD) Kit – obtained the ANVISA (Brazilian Health Regulatory Agency) registry (80780040002) and has been used by the General Coordination of Public Health Laboratories (CGLAB/DAEVS/SVS/MS) as a method for YF detection since 2019.

      Conflict of interest

      At the time of submission, RCPR, MRZ, FA, TJ, MKT, LGM, and FKM were employees at IBMP, which manufactures and commercializes the test described in this study.

      Acknowledgments

      We thank Coordenação Geral de Laboratórios de Saúde Pública (CGLAB).

      Funding Source

      The Funding Source was Instituto de Biologia Molecular do Paraná (IBMP).

      Ethical Approval statement

      We did not use human samples. The Reference Laboratories were responsible for these comparison experiments.

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