The use of real time PCR for arboviruses diagnostics: integrative review

Licínio, Christiane O. L.; Ayres, Flávio M.

doi:10.5935/1676-2444.20210048

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BRIEFING COMMUNICATION • J. Bras. Patol. Med. Lab. 57 • 2021 • https://doi.org/10.5935/1676-2444.20210048 copy

The use of real time PCR for arboviruses diagnostics: integrative review

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ABSTRACT

Arboviruses are viral diseases transmitted by arthropods (arthropod-borne virus). Standing out dengue, zika virus, and chikungunya among the emergent and re-emergent arboviruses in recent years around the world. The similarity between the symptoms makes the clinical diagnosis ineffective, making difficult the prophylactic and preventive measures of new outbreaks. Molecular diagnosis using the real-time polymerase chain reaction (PCR) technique is one of the ways to diagnose such diseases. In this study, the literature on the diagnosis of arboviruses was compiled and evaluated. The objective was to answer the guiding question: Is the real-time PCR methodology effective in the diagnosis of arboviruses? Scientific articles of free access were searched in the databases Pubmed (50 articles) and Scielo (107 articles), between 2014 and 2019. The selection was done through the inclusion and exclusion criteria, only 20 articles remained. Among them, 85% cross-sectional studies, 10% systematic reviews, and 5% case studies. The period of publications was 50% in 2017, 35% in 2016, and 5% in 2014, 2015 e 2019, each. Regarding the viruses treated in the articles, 25% researched dengue and the same percentage for chikungunya, 20% researched about zika virus. The efficacy of the molecular diagnosis was published in 21% of the articles (sensitivity and specificity), 53% highlighted the limit of detection, 70% highlighted the absence of cross-reactions, and 80% highlighted the differentiation between viruses.

Article	Authors	Article title	Research design	Year of publication
1	Alva-Urcia et al.(¹1 Vasudevan RS. Dengue and zika: control and antiviral treatment strategies [Internet]. Vol. 1062. 2018. Available at: http://www.springer.com/series/5584. http://www.springer.com/series/5584... )	Emerging and reemerging arboviruses: a new threat in Eastern Peru	Cross-sectional study	2017
2	Kurosaki et al.(²2 Lorenz C, Aguiar BS, Azevedo TS, Chiaravalloti Neto F, Suesdek L. Impact of environmental factors on neglected emerging arboviral diseases. PLoS Negl Trop Dis. 2017; 1–19.)	Development and evaluation of a rapid molecular diagnostic test for zika virus infection by reverse transcription loop-mediated isothermal amplification	Cross-sectional study	2017
3	Giry et al.(³3 Li X, Gao X, Fu S, et al. Arboviruses and their related infections in China: a comprehensive field and laboratory investigation over the last 3 decades. Rev Med Virol. 2017; 1–21.)	Improved detection of genus-specific Alphavirus using a generic TaqMan^® assay	Cross-sectional study	2017
4	Edwards et al.(⁴4 Lopes N, Nozawa C, Linhares REC. Características gerais e epidemiologia dos arbovírus emergentes no Brasil. Rev Pan-Amazônica Saúde [Internet]. 2014; 5(3): 55–64. Available at: http://scielo.iec.pa.gov.br/scielo.php?script=sci_arttext&pid=S2176-62232014000300007&lng=en&nrm=iso&tlng=en. http://scielo.iec.pa.gov.br/scielo.php?s... )	Analytical and clinical performance of a chikungunya qRT-PCR for Central and South America	Cross-sectional study	2017
5	Giry et al.(⁵5 Alva-Urcia C, Aguilar-Luis MA, Palomares-Reyes C, et al. Emerging and reemerging arboviruses: a new threat in Eastern Peru. PLoS One. 2017; 12(11): 1–13.)	Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan^® assay	Cross-sectional study	2017
6	Luo et al.(⁶6 Gould E, Pettersson J, Higgs S, Charrel R, Lamballerie X. Emerging arboviruses: why today? 2017; 4(June): 1–13.)	Laboratory and molecular characterization of dengue viruses in a 2014 outbreak in Guangfo region, Southern China	Cross-sectional study	2017
7	Fortuna et al.(⁷7 Edwards T, del Carmen Castillo Signor L, Williams C, et al. Analytical and clinical performance of a Chikungunya qRT-PCR for Central and South America. Diagn Microbiol Infect Dis [Internet]. 2017; 89(1): 35–9. Available at: http://dx.doi.org/10.1016/j.diagmicrobio.2017.06.001. http://dx.doi.org/10.1016/j.diagmicrobio... )	Imported arboviral infections in Italy, July 2014-October 2015: a National Reference Laboratory report	Cross-sectional study	2017
8	Eppes et al.(⁸8 Giry C, Roquebert B, Li-Pat-Yuen G, Gasque P, Jaffar-Bandjee MC. Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan® assay. BMC Microbiol. 2017; 17(1): 1–10.)	Testing for zika virus infection in pregnancy: key concepts to deal with an emerging epidemic	Systematic review	2017
9	Corman et al.(⁹9 Romeiro MF, de Souza WM, Tolardo AL, et al. Evaluation and optimization of SYBR green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the flavivirus genus in Brazil. Rev Soc Bras Med Trop. 2016; 49(3): 279–85.)	Assay optimization for molecular detection of zika virus	Cross-sectional study	2016
10	Johnson et al.(¹⁰10 Hu SF, Li M, Zhong LL, et al. Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1-4. BMC Microbiol [Internet]. 2015; 15(1): 1–15. Available at: http://dx.doi.org/10.1186/s12866-015-0595-1. http://dx.doi.org/10.1186/s12866-015-059... )	Laboratory diagnosis of chikungunya virus infections and commercial sources for diagnostic assays	Cross-sectional study	2016
11	Patel et al.(¹¹11 Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HRH. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005; 5(2): 209–19.)	A field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the chikungunya virus	Cross-sectional study	2016
12	Pessôa et al.(¹²12 Donalisio MR, Freitas ARR, Freitas R, Von Zuben APB. Arboviroses emergentes no Brasil: desafios para a clínica e implicações para a saúde pública. Rev Saúde Pública. 2017; 51: 30.)	Investigation into an outbreak of dengue-like illness in Pernambuco, Brazil, revealed a cocirculation of zika, chikungunya, and dengue virus type 1	Cross-sectional study	2016
13	Chen et al.(¹³13 Giry C, Roquebert B, Li-Pat-Yuen G, Gasque P, Jaffar-Bandjee MC. Improved detection of genus-specific Alphavirus using a generic TaqMan® assay. BMC Microbiol. 2017; 17(1): 1–9.)	Development and evaluation of a sybr green-based real-time multiplex RT-PCR assay for simultaneous detection and serotyping of dengue and chikungunya viruses	Cross-sectional study	2016
14	Abd El Wahed et al.(¹⁴14 Chaves BA, Orfano AS, Nogueira PM, et al. Coinfection with zika virus (ZIKV) and dengue virus results in preferential ZIKV transmission by vector bite to vertebrate host. 2018; 218: 563–71.)	Recombinase polymerase amplification assay for rapid diagnostics of dengue infection	Cross-sectional study	2015
15	Xavier et al.(¹⁵15 Roth C, Delgado FG, Simon-Lorière E, Sakuntabhai A. Immune responses to dengue and Zika viroses — guidance for T cell vaccine development. Int J Environ Res Public Health [Internet]. 2018; 15(2): 1–12. Available at: http://www.mdpi.com/1660-4601/15/2/385. http://www.mdpi.com/1660-4601/15/2/385... )	Clinical and laboratory diagnosis of Zika fever: an update	Systematic review	2017
16	Souza et al.¹⁶⁾	Clinical and laboratory diagnosis of congenital zika virus syndrome and diaphragmatic unilateral palsy: case report	Case study	2016
17	Acosta et al.(¹⁷17 Balmaseda A, Stettler K, Medialdea-Carrera R, et al. Antibody-based assay discriminates Zika virus infection from other flaviviruses. Proc Natl Acad Sci [Internet]. 2017; 201704984. Available at: http://www.pnas.org/lookup/doi/10.1073/pnas.1704984114. http://www.pnas.org/lookup/doi/10.1073/p... )	False-negative dengue cases in Roraima, Brazil: an approach regarding the high number of negative results by NS1 Ag kits	Cross-sectional study	2014
18	Romeiro et al.(¹⁸18 Stettler K, Beltramello M, Espinosa DA, et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353(6301): 823–6.)	Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil	Cross-sectional study	2016
19	Galo et al.(¹⁹19 Bastos MLA, Araújo RMO, Oliveira DS, Cavalcante ANM, Júnior GBS. Thrombotic thrombocytopenic purpura associated with dengue and chikungunya virus coinfection: case report during an epidemic period. [Internet]. Rev Inst Med Trop São Paulo. 2018; 1–4. Available at: https://doi.org/10.1016/j.orcp.2018.05.003. https://doi.org/10.1016/j.orcp.2018.05.0... )	Development of in-house serological methods for diagnosis and surveillance of chikungunya	Cross-sectional study	2017
20	Slavov et al.(²⁰20 Slavov S, Ferreira F, Rodrigues E, Gomes R, Covas D, Kashima S. Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit. J Vector Borne Dis. 2019; 56(2): 166–9.)	Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit	Cross-sectional study	2019

Article	Objective	Samples	Target virus of the research	Main results
1	Evaluate the prevalence of DENV, OROV, CHIKV, MAYV and ZIKV in patients with acute fever disease in the city of Puerto Maldonado (Peru)	139 human serum samples	DENV CHIKV ZIKV	41 (29.5%) positive for arboviruses; 13 (9.35%) positive for CHIKV; nine (6.48%) positive for DENV; seven (5.03%) positive for ZIKV. The differentiation between arboviruses is precarious by clinic only
2	Develop and evaluate a rapid molecular diagnostic test for ZIKV with the RT-LAMP methodology	120 suspect samples, 90 serum/plasma and 99 urine	ZIKV	100% agreement between RT-LAMP and qRT-PCR, the limit of detection of qRT-PCR being the most sensitive
3	Implement a new molecular method for detecting Alphavirus	Virus strains obtained from various laboratories	CHIKV	Positivity in CHIKV samples. LoD greater than the reference test in the same methodology. Twelve virus species were tested and there was no cross-reactivity
4	Develop and validate a qRT-PCR assay capable of detecting all CHIKV lineages	RNA from virus culture and serum samples from Guatemala and Ecuador in July 2015	CHIKV	Effective assay, mainly the strain circulating in South America. Compared to CDC USA PCR, 98% sensitivity and 100% specificity
5	Implement a syndromic approach based on the use of a multiplex qPCR assay to facilitate rapid diagnosis of dengue-like syndromes in Reunion Island	Virus strains from various laboratories and positive human plasma	CHIKV DENV	Evaluation and accuracy of the assay were satisfactory for CHIKV and DENV, offering reliable answers. It has been shown to be a cheaper option than simple tests for the same pathogens
6	Investigate the usefulness of simple laboratory tests as predictive markers of confirmed DENV diagnoses, as well as to analyze the genotype and phylogenetics of the circulating strain in the dengue epidemic in Guang-Dong in 2014	1044 patients: 875 confirmed DENV cases (862 positive by NS1 and 13 by PCR) and 169 negative	DENV	Simple laboratory tests (leukopenia, thrombocytopenia, aminotransferases and activated thromboplastin time) are useful markers in the diagnosis of DENV
7	Describe the results of analyzes of infections by arboviruses imported from Italy carried out by the National Reference Laboratory for Arboviruses (NRLA) at the Italian National Institute of Health from July 2014 to October 2015	Inpatient serum samples (n = 180)	DENV CHIKV ZIKV	Greater number of cases of DENV. There was an increase in CHIKV cases, and the first case of ZIKV was detected. In the latter, the PRNT technique was decisive for identification; the virus was negative in serology and PCR techniques (which proved to be sensitive and specific). However, only for a short period of time, limited to viremia in the body
8	Review laboratory testing methodology to explore virus presence and immune response	Blood, serum, urine, amniotic fluid, CSF, saliva, semen, breast milk and vaginal mucosa	ZIKV	Viremia level is low in the serum, from two to 10 days, which makes molecular diagnosis difficult and increases the number of false negatives. The case study demonstrated permanence of the virus in red blood cells for 81 days, suggesting that the use of whole blood may increase sensitivity
9	Examine the diagnostic performance of real-time PCR for detection of ZIKV	Serum and urine from individuals who travelled to Brazil, Dominican Republic and Suriname between 2015 and 2016	ZIKV	Low level viremia has been shown to cause false negatives. This study found no difference between blood and urine viremias, but suggests the use of the two samples combined. Assays with NS1 are more specific and sensitive, while assays with NS3 and NS5 have low sensitivity and difficulty in specific probes, respectively
10	Describe the results found by CDC USA on the efficiency of diagnostic kits for CHIKV	Serum and plasma	CHIKV	For PCR, the NS2 target is more sensitive, but there is no publication on its validation. Other methodologies were effective and had no cross-reactions
11	Develop and evaluate a real-time PCR assay to potentially be a point of care diagnostic tool for detecting CHIKV	Human serum and plasma	CHIKV	The assay was shown to have a clinical sensitivity and specificity of 100%, a low limit of detection and only a cross- reaction with a little-known virus (O’nyong’nyong). With the use of a differentiated primer, the cross-reaction was extinguished
12	Investigate and identify viral etiology and advise health authorities on implementing control measures to contain an outbreak	77 human plasma samples, collected at Hospital Severino de Souto Siqueira, in Recife, PE, between 25 and 31 May 2015	DENV CHIKV ZIKV	31 (40.2%) patients with ZIKV infection; nine (11.7%) patients infected with DENV; one (1.3%) patient positive for CHIKV IgM, but negative in the PCR test. Coinfection by ZIKV/DENV was proven in 2.6% of patients. The study highlights the need for differentiation between viruses to improve health control measures
13	Develop a specific, sensitive and robust assay in the RT-LAMP methodology for diagnosis and differentiation between DENV serotypes 1-4	190 serum samples	DENV serotypes 1-4	RT-LAMP results were satisfactory; this methodology was more effective than traditional PCR and real-time PCR. Results (RT-LAMP, RT-PCR, qRT-PCR): sensitivity 98.9%, 84.2% and 90.5%, respectively; specificity 100%, 93% and 100%, respectively
14	Develop two RT-RPA assays to detect DENV 1-4	Human samples from outbreaks in Senegal and Thailand	DENV serotypes 1-4	Results (RT-RPA and qRT-PCR): sensitivity 98% RNA Thailand and 72% RNA Senegal; 98% RNA Thailand and 94.4% RNA Senegal. Specificity 100% and 100%, respectively
15	Update the clinical and laboratory diagnosis of fever by VZIK	blood, saliva and urine. Amniotic fluid, CSF, placenta and umbilical cord in cases of neonatal infection	VZIK	qPCR detects acute infection within seven days of symptom onset. Blood is less sensitive than urine (15 days) and saliva. Third day of manifestation the best time for testing. In neonates with clinical symptoms and negative PCR, perform serology
16	Report a case of unilateral diaphragmatic paralysis in a neonate with a confirmed diagnosis of congenital zika by amniotic fluid examination using RT-PCR and by CSF serology	Amniotic fluid and CSF	DENV CHIKV ZIKV	Positive RT-PCR in amniotic fluid at week 29 of gestation. Negative in maternal and newborn serum after delivery (acute infection during pregnancy). IgM in newborn serum confirms intrauterine infection (IgM does not cross placental barrier)
17	Suggest that NS1 Elisa tests have low sensitivity and produce false negative results and cross reactions with other arboviruses		150 human serum samples which tested DENV negative by Elisa	DENV	33 Elisa negative samples were positive for qRT-PCR, ie 22% false negatives were truly positive; 75% were from DENV-4. Correlation with other studies in which secondary DENV infection produces false negatives in NS1 kits
18	Evaluate and optimize a diagnostic test using the qRT-PCR methodology for the Flavivirus genus	410 serum samples with acute fever, DENV negative by RT-PCR	DENV	qRT-PCR is more sensitive and specific than traditional PCR, in addition to allowing virus quantification. There was no amplification of other Flavivirus, indicating that there is no cross-reaction
19	Develop and evaluate serological methods for diagnosis and research of CHIKV in Nicaragua	260 acute and convalescent serum samples	CHIKV	qRT-PCR was used as the gold standard. Seven PCR positive samples were serologically negative; four PCR negative and serology positive samples may indicate serology cross- reactivity. Low sensitivity of serology in the acute phase, being high for PCR in the same phase
20	Report the detection of DENV and ZIKV by qRT-PCR in a pre-symptomatic blood donor, in Ribeirão Preto, São Paulo	A blood sample (plasma) from the donor	DENV ZIKV	Detection of ZIKV infection concurrently with DENV infection. 13 million copies/ml of ZIKV and 5 million copies/ml of DENV-4 were found in the same blood donor plasma

Article	Sensitivity	Limit of detection	Specificity	Cut-off value	Differentiation between viruses	Cross-reaction
1	HSe	NI	HSp	NI	Yes	No
2	NI	10 cp/assay	NI	< 35	Yes	No
3	AS	40 cp/assay	HSp	< 20.96	Yes	No
4	98.4%	19.6 cp/assay	100%	< 37	Yes	No
5	HSe	NI	HSp	< 36	Yes	No
6	NI	NI	NI	< 38	NI	NI
7	HSe	NI	HSp	NI	Yes	No
8	NI	NI	NI	NI	NI	NI
9	HSe	10 cp/assay	HSp	NI	Yes	No
10	HSe	5.3 cp/assay	HSp	NI	NI	NI
11	100%	80 cp/assay	100%	< 36	Yes	No
12	HSe	100 cp/ml	HSp	< 40	Yes	No
13	90.5%	100 cp/ml	100%	NI	Yes	No
14	94.4%-98%	10 cp/µl	100%	< 38	Yes	No
15	HSe	NI	HSp	NI	NI	NI
16	NI	NI	NI	NI	Yes	NI
17	NI	NI	NI	< 39	Yes	NI
18	HSe	100 cp/ml	HSp	NI	Yes	No
19	NI	NI	NI	NI	Yes	No
20	NI	NI	NI	NI	Yes	No

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[1] CORRESPONDING AUTHOR: Christiane Oliveira Lima Licínio. e-mail: chrislima28@hotmail.com