Molecular and serological techniques to detect co-circulation of DENV ...

Journal of Clinical Virology 82 (2016) 108–111

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Short communication

Molecular and serological techniques to detect co-circulation of DENV, ZIKV and CHIKV in suspected dengue-like syndrome patients Mauro Jorge Cabral-Castro a , Marta Guimarães Cavalcanti b , Regina Helena Saramago Peralta c , José Mauro Peralta a,∗ a

Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil Departamento de Doenc¸as Infecciosas e Parasitárias, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Departamento de Patologia, Faculdade de Medicina, Universidade Federal Fluminense, Niterói, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 27 February 2016 Received in revised form 21 July 2016 Accepted 24 July 2016 Keywords: Differential diagnosis Dengue virus Zika virus Chikungunya virus Arboviruses Viral RNA

a b s t r a c t Background: Arboviruses are important emerging viruses worldwide. The signs and symptoms of Zika virus (ZIKV) infection are similar to those presented by infections with dengue virus (DENV) and chikungunya virus (CHIKV). Furthermore, diagnosis of ZIKV infection is particularly challenging in dengue endemic regions and with co-circulation of DENV, CHIKV, and ZIKV, making diagnosis based solely on clinical and epidemiological data unreliable. As these three viral infections share similar clinical manifestations, differential diagnosis is crucial. Objectives: In this study, diagnoses of ZIKV, CHIKV and DENV infections were investigated in 30 patients with suspected dengue fever residing in the area of co-circulation of these three arboviruses. Study design: The study included whole blood and/or serum samples obtained from 30 patients with suspected dengue fever. All patients were tested for DENV infection as well as for CHIKV and ZIKV infections. Assays for detecting anti-DENV IgM and DENV RNA by semi-nested RT-PCR and ZIKV and CHIKV RNA by real-time RT-PCR were performed. Results: DENV RNA was not detectable in any of the clinical samples, whereas ZIKV RNA was detectable in 17 samples (56.7%). Co-infection by ZIKV and CHIKV was documented in one case. Of the 17 ZIKVpositive individuals, 8 showed reactivity for anti-DENV IgM, which suggested recent DENV infection, cross-reactivity or co-infection. Conclusion: Our findings confirm that accurate laboratory testing is of paramount importance for differential diagnosis in areas of simultaneous transmission of different arboviruses with similar clinical presentations. © 2016 Elsevier B.V. All rights reserved.

1. Background Arboviruses are important emerging viruses transmitted by Aedes aegypti. Dengue virus (DENV) infection has been well established in Brazil since 1985, and Zika virus (ZIKV) and Chikungunya virus (CHIKV) infection have recently occurred, with an elevated ratio of ZIKV infection [1]. Although ZIKV was first isolated in 1947 from the blood of a sentinel monkey in Zika Forest, Uganda [2], ZIKV infections in humans were rare until 2007, when an outbreak was reported in Yap, Micronesia. Initial diagnoses via laboratory tests with rapid analysis suggested that DENV was the causative agent.

∗ Corresponding author. E-mail address: [email protected] (J.M. Peralta). http://dx.doi.org/10.1016/j.jcv.2016.07.017 1386-6532/© 2016 Elsevier B.V. All rights reserved.

However, new immunological and molecular analyses, including nucleic acid sequencing, have been performed on samples from the outbreak, and the results indicated approximately 90% identity to ZIKV. Therefore, ZIKV is considered to be the causative agent of the Yap epidemic [3,4]. According to the Pan American Health Organization, autochthonous circulation of ZIKV in the Americas was confirmed in February 2014 [1,5], and the Brazilian Ministry of Health has confirmed autochthonous circulation of ZIKV in 19 Brazilian states since December 2015 [6,7]. First autochthonous cases of CHIKV infection in Brazil were detected in Oiapoque (Amapá) [8,9]. In 2015, Brazilian Health Ministry reported a total of 20,661 suspected cases of chikungunya fever [6]. ZIKV infection is characterized as a mild febrile disease that includes headaches, maculopapular rash, conjunctivitis and arthralgia. ZIKV syndrome

M.J. Cabral-Castro et al. / Journal of Clinical Virology 82 (2016) 108–111

is similar to both dengue and chikungunya infection [10,11]. Moreover, diagnosis of ZIKV infection is particularly difficult in dengue endemic areas and areas of DENV, CHIKV and ZIKV co-circulation. These facts make diagnosis based only on clinical and epidemiological data unreliable. In addition, there are problems with laboratory diagnosis, which is used for identifying the etiologic agent, including cross-reactivity in the methods applied for detecting antibodies, especially among Flaviviruses. A period of low viremia in some cases also complicates the detection of viral RNA and viral isolation [10–12]. Thus, differential diagnosis is crucial because these three viral infections share similar clinical manifestations, and laboratory confirmation by immunological and molecular tests is important for accurate differential diagnosis of these arboviruses. Regardless, immunological methods for detecting antibodies against ZIKV may present cross-reactivity with other flaviviruses, particularly DENV, which may hinder a precise diagnosis [10,12–14]. 2. Objectives In this study, the diagnosis of ZIKV, CHIKV and DENV infections was investigated in 30 patients with suspected dengue fever residing in the co-circulation area of these three arboviruses.

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diagnostic kit (Dengue Virus IgM Capture DxSelect TM , FOCUS Diagnostic, USA) according to the manufacturer’s recommendations. This kit is approved by the Brazilian Regulatory Agency and is used in Public Health Laboratories. Viral RNA was extracted from 250 ␮L of WB and/or serum using TRIZOL LS Reagent (Ambion TM , Life Technology, USA) according to the manufacturer’s recommendations. The extracted RNA was stored at −70 ◦ C until use. cDNA synthesis was performed with random primers by reverse transcription using the ImProm-IITM Reverse Transcriptase kit (Promega, USA) following the manufacturer’s recommendations. DENV cDNA was amplified by semi-nested PCR using primers D1 and D2 in the first step and serotype-specific primers in the second step, as previously described [13]. The amplified products were fractionated by electrophoresis through 1.5% agarose gels, stained with GelRedTM 1X (Biotium) and visualized on an ultraviolet transilluminator. For ZIKV and CHIKV testing, samples were analyzed using real-time RT-PCR with specifics primers and probes, as previously described [3,14]. For ZIKV, two sets of primers and two probes for two different target sequences were used in two distinct reactions [3]. The results are expressed as Ct values, which are inversely proportional to the concentration of viral RNA in each sample. Values of Ct were considered positive up to 38.5 cycles [3]. In all assays two negatives and two positives controls were included.

3. The study 4. Results From April 2015 to January 2016, whole blood (WB) and/or serum samples were obtained from 30 patients with suspected dengue fever who were seeking medical assistance at the University Hospital Clementino Fraga Filho/Federal University of Rio de Janeiro, located in Rio de Janeiro (Brazil). The patients were randomly selected for testing for CHIKV and ZIKV infections in addition to DENV infection. All samples analyzed were anonymized. The WB aliquots were immediately stored in a solution of RNA Later, which rapidly permeates tissues and cells to stabilize and protect cellular RNA (AmbionTM , Life Technology, USA) for molecular analysis according to the manufacturer’s recommendations. The serum samples were immediately stored at −70 ◦ C until use. Assays for the detection of IgM antibodies against DENV in serum or plasma were performed using a commercially available

Because of the co-circulation of DENV, CHIKV and ZIKV in Brazil and the difficulty of clinical diagnosis due to the similarity of the symptoms caused by these arboviruses, differential diagnosis is necessary to identify the etiologic agent of infection. Differential diagnostics were applied for detecting DENV, CHIKV, and ZIKV RNA in 30 patients. Amplification of DENV RNA was not detected in the samples from any of the patients, whereas ZIKV RNA was amplified with samples from 17 (56.7%) patients (Table 1). Additionally, antiDENV IgM was detected in 9 of the 30 (30%) patients, 8 of whom also showed the presence of ZIKV RNA. Additionally, 47.1% (8/17) of the ZIKV-confirmed samples showed anti-DENV IgM reactivity, which could result from recent DENV infection, ZIKV and DENV coinfection or cross-reactivity. Amplification of both CHIKV and ZIKV

Table 1 Comparative results between patients suspected of dengue like-syndrome. Patients

Date

Sample

Anti-DENV IgM

RT-PCR DENV

Real time RT-PCRc CHIKV (Cte )

Pat 02 Pat 04 Pat 05 Pat 07 Pat 08 Pat 10 Pat 11 Pat 12 Pat 13 Pat 14 Pat 15 Pat 20 Pat 22 Pat 23 Pat 24 Pat 26 Pat 29 Pat 30

2015 April 28 2015 Jun 11 2015 Jun 12 2015 Jun 30 2015 Jul 14 2015 Jul 17 2015 Jul 21 2015 Aug 19 2015 Aug 25 2015 Aug 25 2015 Nov 17 2015 Dec 15 2015 Dec 28 2016 Jan 1 2016 Jan 5 2016 Jan 6 2016 Jan 6 2016 Jan 7

WBa Serum WB Serum Serum Serum Serum Serum Serum WB WB Serum Serum WB WB WB WB Serum

Reactive NRd NR NR NR NR NR Reactive NR Reactive Reactive Reactive Reactive Reactive Reactive NR Reactive NR

NAb NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

31.7 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

a b c d e

Whole blood (WB) was used for RNA extraction and its respective plasma was used for ELISA IgM anti-DENV. Not amplified. ® GoTaq Probe 1-Step RT-qPCR System (Promega, USA). Not Reactive. Ct considered positive below cycles 38.5.

Real time RT-PCRc ZIKV (Cte ) ZIKV (835–911)

ZIKV (1086–1162)

36.6 37.5 37 26.1 38.1 33 38.5 38.4 35.3 36.8 NA 35.2 NA 38.4 NA 27.8 33.3 28

37.1 NA NA 25.9 36.8 33 NA NA 36.2 35.8 NA 34.7 38.3 NA 36.8 32.2 36.6 28.5

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was found for only one patient, who was also anti-DENV IgM positive (Table 1 and Supplementary data). All 30 samples from the patients were collected within the first ten days of the onset of symptoms. Of the 17 ZIKV-infected patients, 10 showed cDNA amplification for both sets of primers/probes, whereas ZIKV sequence 1 (835–911) was amplified in 5 and ZIKV sequence 2 (1086–1162) in two. Amplification with only one of the primer/probe sets suggests a low concentration of ZIKV RNA [3].

accurate differential diagnosis of ZIKV and DENV infections is required to prevent misdiagnosis. Conflict of interest None. Funding FAPERJ, CAPES and CNPq, Brazil.

5. Discussion

Ethical approval

Clinical diagnosis of ZIKV infection is not specific and can be confused with other diseases, particularly DENV and CHIKV infections. Thus, differential diagnosis with molecular methods is essential for confirming ZIKV infections, yet such confirmation presents some challenges. The findings of our study demonstrate that ZIKV infection predominated over dengue in the group of patients analyzed, based on laboratory-confirmed cases. Nine of the 30 patients were reactive for anti-DENV IgM, but no patient showed DENV RNA amplification by semi-nested RT-PCR. Therefore, cross-reactivity between anti-DENV IgM and anti-ZIKV antibodies likely occurred in these cases. These results are consistent with the literature on the possible cross-reactivity between DENV and ZIKV and other flaviviruses when immunological methods are used for IgM detection [3,4,15]. Furthermore, anti-DENV IgM/IgG reactivity without NS1 detection should not be employed as the sole laboratory confirmation of dengue infection in areas of ZIKV and DENV co-transmission. The occurrence of false positive reactivity for dengue in individuals with ZIKV infection may be overwhelming in groups that are at risk of the disease, such as pregnant women. Because the probable causal relationship with microcephaly is a major concern, accurate differential diagnosis of ZIKV and DENV infections must be a priority [16,17]. With the high possibility of IgM and IgG cross-reactivity in immunoassays, particularly between DENV and ZIKV, differential diagnosis via detection of viral RNA is critical for identifying the causative agent of these infections. ZIKV RNA was amplified in 17 of the 30 patients, and CHIKV RNA was amplified in only one patient. This patient was a 27-year-old woman who developed a moderate fever (38.5 ◦ C), erythematous rash and pruritus without arthralgia and/or arthritis. All WB and serum samples were obtained at ten days after onset of symptoms. After a period of four days with mild symptoms and almost no cutaneous involvement, the above patient had a reappearance of the rash and pruritus without fever for two days (Supplementary data). Furthermore, tests for detecting DENV, CHIKV, and ZIKV viral RNA are typically indicated up to 3–6 days after the onset of symptoms, though detection can occur for up to 10 days after onset of symptom as documented for CHIKV infection [1,12]. The findings of nine patients presenting anti-DENV IgM reactivity and ZIKV RNA detection by real-time PCR indicate possible cross-reactivity in the current diagnostic methods. Nonetheless, the possibility of co-infections by these three viruses (DENV, CHIKV, and ZIKV) cannot be ruled out because of the vector distribution and epidemiological situation in the country. Co-infections by DENV and ZIKV [18] and by DENV and CHIKV [19] have previously been reported in New Caledonia, Oceania, and Gabon, Central Africa, respectively, and more recently a possible co-infection with DENV, CHIKV and ZIKV was detected in Colombia [20]. Using molecular techniques, we documented the occurrence of ZIKV infection in a proportion of patients initially suspected of being infected with DENV. Additionally, due to the possible serological cross-reactivity (anti-DENV IgM/IgG) among flaviviruses,

Ethical approval was provided by the ethics in research committee of the HUCFF/UFRJ (CAAE 02920212.8.3001.5279). Acknowledgments Part of this study received financial support from Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2016.07.017. References [1] Pan American Health Organization. Neurological Syndrome, Congenital Malformations, and Zika Virus Infection. Implications for Public Health in the Americas. 2015 Dec [cited 2015 Dec 15], 2015 http://www.paho.org/hq/ index.php? option=com docman&task=doc view&Itemid=270&gid=32405&lang=en. [2] G.W.A. Dick, S.F. Kitchen, A.J. Haddow, Zika virus. I. Isolations and serological specificity, Trans. R. Soc. Trop. Med. Hyg. 46 (1952) 509–520. [3] R.S. Lanciotti, O.L. Kosoy, J.J. Laven, J.O. Velez, A.J. Lambert, A.J. Johnson, et al., Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007, Emerg. Infect. Dis. 14 (8) (2008) 1232–1239. [4] M.R. Duffy, T. Chen, W.T. Hancock, A.M. Powers, J.L. Kool, R.S. Lanciotti, et al., Zika virus outbreak on yap island, federated states of Micronesia, N. Engl. J. Med. 360 (2009) 2536–2543. [5] J.J. Waggoner, B.A. Pinsky, Zika virus: diagnostics for an emerging pandemic threat, J. Clin. Microbiol. 54 (2016) 860–867. [6] Brazil Ministry of Health. Monitoring of Cases of Dengue, Chikungunya Fever and Fever by Zika Virus to Epidemiological Week 52, 2015. [cited 2016 Jan 1], 2016 http://portalsaude.saude.gov.br/images/pdf/2016/janeiro/15/svs2016be003-dengue-se52.pdf. [7] N.S. De Carvalho, B.F. De Carvalho, C.A. Fugac, B. Dórisc, E.S. Biscaia, Zika virus infection during pregnancy and microcephaly occurrence: a review of literature and Brazilian data, Braz. J. Infect. Dis. (2016), http://dx.doi.org/10. 1016/j.bjid.2016.02.006 http://dx.org/. [8] M.R. Nunes, N.R. Faria, J.M. de Vasconcelos, N. Golding, M.U. Kraemer, L.F. de Oliveira, et al., Emergence and potential for spread of Chikungunya virus in Brazil, BMC Med. 30 (13) (2015) 102. [9] N. Rodrigues Faria, J. Lourenc¸o, E. Marques de Cerqueira, M. Maia de Lima, O. Pybus, L. Carlos Junior Alcantara, Epidemiology of Chikungunya virus in bahia, Brazil, 2014–2015, PLoS Curr. (2016), http://dx.doi.org/10.1371/currents. outbreaks.c97507e3e48efb946401755d468c28b2. [10] G.S. Campos, A.C. Bandeira, S.I. Sardi, Zika virus outbreak, Bahia, Brazil, Emerg. Infect. Dis. 21 (10) (2015) 1885–1886. [11] E.B. Hayes, Zika virus outside africa, Emerg. Infect. Dis. 15 (9) (2009) 1347–1350. [12] E.A. Kelser, Meet dengue’s cousin, zika, Microbes Infect. (2015) 1–4. [13] R.S. Lanciotti, C.H. Calisher, D.J. Gubler, G.J. Chang, A.V. Vorndam, Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction, J. Clin. Microbiol. 30 (3) (1992) 545–551. [14] R.S. Lanciotti, O.L. Kosoy, J.J. Laven, A.J. Panella, J.O. Velez, A.J. Lambert, et al., Chikungunya virus in US travelers returning from India, 2006, Emerg. Infect. Dis. 13 (5) (2007) 764–767. [15] A.H. Fagbami, Zika virus infections in Nigeria: virological and seroepidemiological investigations in Oyo State, J. Hyg. 83 (2) (1979) 213–219.

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