Abstracts

Dengue is currently regarded as the most important mosquito-borne viral disease, in terms of both geographical distribution and number of cases reported annually. Recent studies have estimated a risk population of 3.97 billion people, and of the major vector-borne diseases the cases of dengue have been prevailing over those of malaria in the Americas, Asia and much of Africa⁵5. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496(7446):504-7.,⁶6. Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein JS, Hoen AG, et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012;6:e1760..

The etiological agent, the Dengue Virus (DENV), belongs to the genus Flavivirus of the family Flaviviridae, and the main mosquito vector to transmit the disease to humans is the predominantly urban species Aedes aegypti¹¹11. Gubler DJ. Epidemic dengue and dengue hemorrhagic fever: a global public health problem in the 21st Century. In: Scheld WM, Armstrong D, Hughes JM, editors. Emerging Infections 1. Washington: ASM Press; 1998. p. 1-14..

The disease has a wide clinical spectrum - ranging from a flu-like illness to severe dengue - and, therefore, it is not possible to determine the clinical manifestations that can ensue at the time of dengue infection. Although, there is no specific treatment for dengue, early diagnosis may guide appropriate clinical management and, thereby, prevent severe complications and/or death²⁴24. World Health Organization. Dengue: guidelines for diagnosis, treatment, prevention and control. Geneva: World Health Organization; 2009. Available from: http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf
http://whqlibdoc.who.int/publications/20... .

The state of Roraima, located in the Brazilian Amazon region, has shown a high incidence of infection over the past decade¹1. Acosta POA, Cordeiro JS, Granja F, Siqueira TCS, Brito FEG, Freitas AG, et al. Dengue in the northernmost part of Brazil from 1999 to 2011: characterization of circulating DENV strains. Dengue Bull. 2012;36:50-63. and is currently recognized as a hyperendemic area, as well as a port-of-entry for serotypes and genotypes of dengue viruses into Brazil. DENV-4 reemerged in Roraima in 2010²2. Acosta POA, Maito RM, Granja F, Cordeiro JS, Siqueira T, Cardoso MN, et al. Dengue virus serotype 4, Roraima State, Brazil. Emerg Infect Dis. 2011;17:1979-80.,¹⁹19. Naveca FG, Souza VC, Silva GAV, Maito RM, Granja F, Siqueira T, et al. Complete genome sequence of a dengue virus serotype 4 strain isolated in Roraima, Brazil. J Virol. 2012;86:1897., 28 years after it was last detected in the state, and now all four dengue serotypes are circulating¹1. Acosta POA, Cordeiro JS, Granja F, Siqueira TCS, Brito FEG, Freitas AG, et al. Dengue in the northernmost part of Brazil from 1999 to 2011: characterization of circulating DENV strains. Dengue Bull. 2012;36:50-63.. In recent years, the state has seen an increase in severe forms of the disease, probably due to secondary infections and/or higher virulence of circulating strains¹1. Acosta POA, Cordeiro JS, Granja F, Siqueira TCS, Brito FEG, Freitas AG, et al. Dengue in the northernmost part of Brazil from 1999 to 2011: characterization of circulating DENV strains. Dengue Bull. 2012;36:50-63.. The World Health Organization (WHO) currently recommends early laboratory tests for the diagnosis of the dengue virus infection using NS1 antigen detection in human serum²⁴24. World Health Organization. Dengue: guidelines for diagnosis, treatment, prevention and control. Geneva: World Health Organization; 2009. Available from: http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf
http://whqlibdoc.who.int/publications/20... , and this is mostly adopted in Roraima. The highly immunogenic non-structural glycoprotein NS1 contains approximately 353 amino acids (46 kDa) and is more conserved among the flaviviruses. Although, NS1 does not form part of the virus structure, it is required for viral RNA replication. It is also displayed on the surface of infected cells and is secreted into the bloodstream, during the acute phase of the disease. This latter characteristic makes it sensitive enough to diagnose a patient on the very first days of infection⁴4. Avirutnan P, Punyadee N, Noisakran S, Komoltri C, Thiemmeca S, Auethavornanan K, et al. Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis. 2006;193:1078-88.,¹⁷17. Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S, Green S, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis. 2002;186:1165-8..

In 2012, 998 serum samples from presumptive diagnosis of dengue were tested with the use of the Bio-Rad Platelia™ Dengue NS1 antigen capture kit at the Central Public Health Laboratory in Roraima (LACEN/RR). Of these samples, 778 (78%) tested negative, i.e. they were ruled out for dengue infection through laboratory diagnosis. Based on this, two questions arise: have these patients been infected by other arboviruses? Does the dengue NS1 antigen test give rise to false-negative results?

To find answers to these questions, it was first decided to assess the possibility of false-negative results for the samples ruled out by the dengue virus NS1 antigen detection. In order to carry out this evaluation, the most sensitive TaqMan - MGB probed real-time quantitative reverse transcription PCR (TaqMan qRT-PCR) technique was applied.

Of the 778 cases initially discarded in 2012 as not being dengue, 150 samples were randomly selected from patients who, in addition to fever, had in their clinical records one or more common signs of dengue such as arthralgia, headache, retro-orbital pain, chills, myalgia, and rash. The average time of sample collection was 1-5 days after the onset of the symptoms.

Viral RNA was extracted directly from serum by the Axygen® AxyPrep™ Body Fluid Viral DNA/RNA Miniprep kit, and subsequently subjected to the qRT-PCR assay (GURUKUMAR et al., 2009)¹²12. Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh PS, Shah PS, et al. Development of real time PCR for detection and quantitation of dengue viruses. Virol J. 2009;6:10:1-8.. This technique is able to detect the genome of any of the four dengue serotypes, but unable to identify specific serotype DENV infection. Fluorescence reading was performed on a StepOne™ Real-Time PCR system. All reactions were performed under the same conditions with the use of ROX as a passive reference dye for normalization of data and VIC as reporter. Samples with Ct < 39 were considered to be dengue positive and underwent serotyping by semi-nested RT-PCR, as described by LANCIOTTI et al. (1992)¹⁵15. Lanciotti RS, Calisher CH, Gubler DJ, Chang G-J, Vorndam V. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30:545-51. and an immunochromatographic assay that allows qualitative and differential detection for IgG and IgM antibodies (Dengue Test Bioeasy)⁸8. Dengue IgG/IgM test Bioeasy. Available from: http://trisul.com.br/arquivos/dengue_(bula).pdf
http://trisul.com.br/arquivos/dengue_(bu... . IgG positive samples, collected until the 4^th day from the onset of symptoms, were characterized as secondary infection⁷7. de Souza VAUF, Tateno AF, Oliveira RR, Domingues RB, Araújo ES, Kuster GW, et al. Sensitivity and specificity of three ELISA-based assays for discriminating primary from secondary acute dengue virus infection. J Clin Virol. 2007;39:230-3..

Of the 150 samples tested, 33 (22%) were positive by qRT-PCR. The samples with the false-negative NS1 antigen were collected between 1-4 days after the onset of symptoms (Table 1) within this period, according to the literature and the manufacturer's instructions, dengue NS1 antigen tests are highly sensitive³3. Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol. 2002;40:376-81.,²⁰20. PlateliaTM Dengue NS1 Ag (Bio-Rad): detecção qualitativa ou semi-quantitativa do antígeno NS1 do vírus da dengue no soro ou plasma humano, pelo método imunoenzimático. Available from: https://www.bio-rad.com/webroot/web/pdf/inserts/CDG/pt/72830_12-2008.pdf
https://www.bio-rad.com/webroot/web/pdf/... .

Studies from different countries indicate a low sensitivity on the NS1 test in secondary infection[9,14,16,18.23], and as Roraima is a hyperendemic State to dengue, at first, it was considered that the main cause for this high number of false negative samples was due to secondary infection. Nevertheless, only eight (28%), of the 30 analyzed samples, showed positive IgG (Table 1).

In 37% of the IgG positive samples IgM antibodies were also detected. FELIX et al., on a dengue outbreak in 2010 in Santos-Brazil, found 64.2% IgM positive among a total of 260 samples with secondary infection¹⁰10. Felix AC, Romano CM, Centrone CC, Rodrigues CL, Villas-Boas L, Araujo ES, et al. Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010. Clin Vaccine Immunol. 2012;19:1972-6..

All four dengue virus serotypes were reported among the positive cases (Fig.1), and these findings are sufficient to classify the state of Roraima as a hyperendemic area. The most common serotype DENV-4 accounted for 75% of the tested serum samples. These data are consistent with that of the Roraima State Epidemiology and Surveillance Branch for this period, which shows that a further 90% of the samples sent for viral identification in 2012 were DENV-4²¹21. Roraima. Secretaria Estadual de Saúde. Departamento Vigilância Epidemiológica. Relatório: casos de dengue notificados no ano 2012. Sistema de Informação de Agravos de Notificação (SINAN). Boa Vista: SINAN; 2012.. According to the manufacturer's instructions the sensitivity of the assay does not differentiate between dengue serotypes²⁰20. PlateliaTM Dengue NS1 Ag (Bio-Rad): detecção qualitativa ou semi-quantitativa do antígeno NS1 do vírus da dengue no soro ou plasma humano, pelo método imunoenzimático. Available from: https://www.bio-rad.com/webroot/web/pdf/inserts/CDG/pt/72830_12-2008.pdf
https://www.bio-rad.com/webroot/web/pdf/... , however, a study conducted in the state of Sergipe, in Northeastern Brazil, found 48.7% false negative, among 119 samples tested for the NS1 antigen. All the negative samples were identified as DENV-4, and authors point to a low sensitivity of the Platelia dengue NS1Ag test in regions where this serotype is predominant²²22. Sea VRF, Cruz ACR, Gurgel RQ, Nunes BTD, Silva EVP, Dolabella SS, et al. Underreporting of dengue-4 in Brazil due to low sensitivity of the NS1 Ag test in routine control programs. PLoS One. 2013;8:e64056.. The DENV-4 strains tested in this study belong to genotype II, the same one that is circulating in the state of Roraima¹⁹19. Naveca FG, Souza VC, Silva GAV, Maito RM, Granja F, Siqueira T, et al. Complete genome sequence of a dengue virus serotype 4 strain isolated in Roraima, Brazil. J Virol. 2012;86:1897., and considering that the Bio-Rad kit uses a monoclonal antibody, it was suggested that this antibody is likely to have low-affinity to epitopes of NS1 from the DENV-4 genotype II, however, there are studies that did not find any difference in the NS1 amino acid sequence in DENV-2 false negative and positive samples for the NS1 Ag detection¹⁰10. Felix AC, Romano CM, Centrone CC, Rodrigues CL, Villas-Boas L, Araujo ES, et al. Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010. Clin Vaccine Immunol. 2012;19:1972-6..

After interrelating the NS1 test sensitivity to dengue serotypes, GUZMAN et al. 's study¹³13. Guzman MG, Jaenisch T, Gaczkowski R, Ty Hang VT, Sekaran SD, Kroeger A, et al. Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis. PLoS Negl Trop Dis. 2010:4:e811. evaluated the four dengue serotypes in samples from Southeast Asia and Latin America, which described the lower sensitivities by the Platelia Dengue NS1 Ag kit in DENV-2. Studies performed in Brazil, before DENV-4 reemerged, found the lowest sensitivity to serotypes DENV-2 and DENV-3¹⁰10. Felix AC, Romano CM, Centrone CC, Rodrigues CL, Villas-Boas L, Araujo ES, et al. Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010. Clin Vaccine Immunol. 2012;19:1972-6.,¹⁸18. Lima MRQ, Nogueira RMR, Schatzmayr HG, Santos FB. Comparison of three commercially available dengue NS1 antigen capture assays for acute diagnosis of dengue in Brazil. PLos Negl Trop Dis. 2010:4:e738..

The analysis presented here reinforces the importance of increasing the awareness of healthcare providers regarding the fact that patients tested negative by the NS1 antigen detection may be infected with the dengue virus, especially when the clinical symptoms and other laboratory data indicate such infection.

ACKNOWLEDGMENTS

The authors would like to thank the Federal University of Roraima. This work was supported by grants from Instituto Leônidas e Maria Deane and Santander Universidades.

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