Evaluation of a commercial real-time PCR kit for dengue diagnosis in samples. collected during an outbreak in Goiânia, Central Brazil, in 2005

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1 JCM Accepts, published online ahead of print on 4 April 2007 J. Clin. Microbiol. doi: /jcm Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Evaluation of a commercial real-time PCR kit for dengue diagnosis in samples collected during an outbreak in Goiânia, Central Brazil, in 2005 José Eduardo Levi* a, Adriana Fumie Tateno a, Adriana Freire Machado a, Débora Camillo Ramalho a, Vanda Akico Ueda Fick de Souza a, Adriana Oliveira Guilarde b, Valéria Christina de Rezende Feres c, Celina Maria Turchi Martelli b, Marília Dalva Turchi b, João Bosco Siqueira Júnior b, Cláudio Sérgio Pannuti a. a Laboratório de Virologia, LIM-HC da FMUSP e Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo - Brazil. b Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goiás, Goiânia Brazil. c LACEN-Go, Secretaria de Estado da Saúde de Goiás, Goiânia Brazil. *Corresponding author. Mailing address: Virology Lab, Institute of Tropical Medicine, University of São Paulo, Rua Dr. Enéas de Carvalho Aguiar 470, 2 o andar, CEP , São Paulo-SP, Brazil. Phone: ; fax: dudilevi@usp.br RUNNING TITLE: PERFORMANCE OF A DENGUE REAL-TIME KIT IN OUTBREAK SAMPLES

2 2 ABSTRACT In the last two decades dengue re-emerged in Brazil as a significant public health problem. Clinicians demand a diagnostic test presenting a high sensitivity, applicable during the early symptomatic phase. We aimed to test two distinct molecular methods from suspected dengue cases during an outbreak in Central Brazil. Acute-phase serum specimens from 254 patients suspected of having dengue were collected during 2005 in the city of Goiânia, Central Brazil. Samples were blinded evaluated by real-time and multiplex-pcr in addition to routine IgM serology and virus culture. Overall, acute dengue was confirmed by serology, multiplex-pcr or virus isolation in 80% patients (203/254). Another four patients presented a real-time positive result as the unique marker of dengue. Higher real-time-pcr positivity and viral loads were observed in the early symptomatic phase of disease (<=5 days) when compared to samples collected after this period. Multiplex and real-time PCR assays presented a high kappa agreement (0.85). According to multiplex-pcr, sixty samples harbored DEN-3, four samples DEN-2 and one sample displayed a pattern compatible with DEN 2/3 double infection. The dengue real-time kit was found to be practical and adjustable for high throughput, displaying the best performance in the early symptomatic phase of dengue cases, valuable for confirming dengue diagnosis timely.

3 3 INTRODUCTION Dengue re-emerged in Brazil in the decade of 1990 as a significant public health problem, with thousands of cases, introduction of new serotypes (dengue 2 and 3) and hemorrhagic cases including casualties (7,8,10). The confirmatory diagnosis of dengue is routinely performed by an IgM test collected one week after the onset of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase. In theory, molecular tests are able to fulfill this window period, by directly assessing the presence of viral RNA in plasma/sera from suspicious subjects. Actually, RT-PCR was found extremely practical in the recent SARS outbreak, when medical staff could quickly avoid the adoption of quarantine measures for dengue RNA positive patients (1). Several RT-PCR methods for dengue RNA detection have been described in the literature, both for conventional and real-time PCR. In-house methods usually present excellent performance but are hard to be transferred to other laboratories with success. This variability in results derives from the different suppliers, thermocyclers and technicians skills among laboratories. Commercial kits represent an alternative to guarantee certain homogeneity in results, what is of importance when establishing testing procedures to be adopted by many centers. Also, the quality control of commercial kits, when good manufacture practices (GMP) are rigorously observed, is impossible to be mimicked by homebrew assays. We aimed to compare a commercial dengue real-time kit with an in-house multiplex test in a series of suspected dengue cases, recruited in a large urban area in Central Brazil, with co-circulation of DEN 1, DEN 2 and DEN 3 (2), in order to evaluate the performance of molecular assays in dengue diagnosis.

4 4 MATERIALS AND METHODS 2.1 Patients and samples: Patients presenting to a reference hospital and public health clinics in Goiânia (~1,200,000 population), Central Brazil, with symptoms compatible to dengue, according to WHO guidelines (14) were enrolled from January to June Two hundred and fifty-four patients, mean age of 31 years old (SD = ± 15 years), 57.4% females, were clinically evaluated. Patients had blood collected during acute and convalescent phases for laboratory diagnosis. Virus isolation was performed in blood samples (< 7 days of onset of symptoms) and serological tests in paired serum samples collected at the onset of symptoms (acute phase) and two weeks after the initial symptoms (convalescent sample) using in-house dengue IgM antibody capture MAC-ELISA (6) at the local public reference laboratory (LACEN-GO, Brazil). Plasma was separated immediately after blood drawing and kept frozen at 70 o C until analysis. Samples from suspected dengue patients, collected during the symptomatic phase, were coded and sent to the Virology laboratory, Instituto de Medicina Tropical da Universidade de São Paulo, São Paulo Brazil, for molecular analysis. Both laboratories were blind to laboratorial and epidemiological data from the patients enrolled. 2.2 Virus isolation: Virus isolation was performed using a monolayer of C6/36 Aedes albopictus cells (5). Dengue virus isolates were identified by an indirect fluorescent antibody test using serotype-specific monoclonal antibodies (3). 2.3 RNA Extraction: RNA was extracted in duplicate from 140µL of plasma employing the Qiagen Viral RNA kit (Qiagen, Germany). Elution was performed in 60µL according to manufacturer instructions.

5 5 2.4 cdna synthesis and multiplex RT-PCR: cdna was synthesized from 22µL of RNA, as above, plus 2,5µM of random hexamers (N 6, Amersham, Brazil), 1mM DTT, 1U/µL RNAse Inhibitor (Invitrogen, Brazil) and 2,5U of M-MLV-RT (Invitrogen, Brazil). This mixture was incubated for 5 minutes at 65 o C then 30 minutes at 37 o C and RT inactivated by a final incubation of 5 minutes at 95 o C. PCR was performed as described (4). Briefly, 5 µl of cdna was added to 20µL of PCR mix consisting of: Primers D1, TS1 and TS2 at 0.5 µm and TS3 and TS4 at µm; MgCl 2 3mM; 1x PCR Buffer; dntps 200µM and 1.25 units of Platinum Taq Polymerase (Invitrogen, São Paulo, Brazil). Thermocycler (Eppendorf Mastercycler Gradient, Hamburg, Germany) profile was as follows: 94 o C 2 minutes then 40 cycles of 94 o C for 45 seconds; 55 C for 1 minute; 72 o C for 1 minute, and a final extension at 72 o C for 7 minutes. Fragments of different lengths are obtained from dengue sorotypes as follows: 482bp (dengue-1); 119bp (dengue-2); 290bp (dengue-3) and 389bp (dengue-4). Total RNA extracted from C6/36 cell cultures infected with the respective dengue serotypes were used as controls. PCR products were run on 2% agarosis gels stained with ethidium bromide and documented on an UV apparatus. 2.5 Real-Time PCR: Duplicates of 10 µl of RNA eluate were directly applied to a denguereal time commercial kit (RealArt artus/qiagen, Germany) in a final volume of 25 µl and run on the ABI 7300 real-time equipment (Applied Biosystems, Brazil). This method consists of a one-step PCR capable to detect and quantify all four serotypes in one single TaqMan-like assay using FAM as the reporter fluorophore. An Internal Control is added to the samples previously to extraction. This molecule serves as a molecular target for amplification in a parallel assay with a distinct fluorophore (VIC). This allows for the identification of extraction failures and/or PCR inhibition. Dengue viral load is obtained by plotting the sample Ct in a curve generated by external quantified Dengue-RNA standards

6 6 also provided in the kit. This curve is generated in every run. The mean value of the duplicate was adopted. Statistical Analysis Data analysis was performed using SPSS, version 13.0 (SPSS Inc, Chicago, IL, USA). Exploratory data analysis with box-plot graphs was applied to evaluate viral load stratified by days of the onset of symptoms. Mann-Whitney U test was performed to compare the median values of viral load by days of symptoms and p value 0.05 was considered statistically significant. The concordance of the positive and negative results between molecular tests was calculated using Kappa index. All patients or legal guardians gave informed consent and the study received the approval of the Regional Ethical Committee.

7 7 RESULTS A laboratory diagnosis of dengue virus infection was established either by a positive IgM-capture Elisa in the first and/or second sample, virus isolation or multiplex PCR amplification. According to this criteria two-hundred and three cases were found positive (203/254=80%). Overall, real-time PCR was positive in 79 patients (31.1%); 57.8% (37/64) on samples obtained within 3 days of disease onset, decreasing to 39.5% (32/81) for samples obtained 4-5 days and to 9.2% (10/109) after 6 days of disease onset. Sensitivity of real-time PCR was 37% (75/203) and specificity 92% (47/51) when considering the four samples presenting real-time PCR result as the only laboratorial evidence of dengue as falsepositives. Those four samples were collected from one to four days of disease onset. The performance of the four dengue diagnostic techniques in the acute-phase sample, stratified by days of the onset of symptoms is illustrated in Figure 1. Multiplex-PCR was positive in 65 patients (25.6%), sixty samples harboring DEN-3, four samples DEN-2 and one sample showed a pattern compatible to DEN 2/3 double infection. Sixty-three samples were reactive and 173 were non-reactive by both PCR assays, with a Kappa agreement of 0.85 (Table 1). Virus culture identified DEN-3 in 43 samples and DEN-1 in one sample. One hundred and sixty samples were negative, two were not evaluated due to cell culture contamination and forty-eight were not tested due to specimens collected after 6 days of symptoms onset, giving a positivity percentage of 21.5% (44/204). A clear trend for higher virus recovery rate in early days of symptoms was observed, as shown in Figure 1. The combination of IgM serology and real-time-pcr accounted for at least 90% of the positive results among the 203 confirmed cases (all positive tests) regardless of the day of symptoms (Figure 2). Four samples displayed virus isolation as the only laboratorial marker, including a dengue 1 isolate. When comparing real-time PCR and virus isolation a concordance of 0.80 was found (164/204), being thirty-seven samples positive for both and one hundred and twenty-seven

8 8 negative for both. Seven samples were culture positive while real-time PCR negative, and thirty-three exhibited the opposite pattern i.e. real-time positive and virus culture negative. Viral loads ranged from 23 to 955,714,286 copies/ml with a decreasing viremia trend in samples drawn after the 5 th day of disease onset (Figure 3). Median of viral loads showed 5,854,075 copies/ml in the first day of symptoms, 440,142 copies/ml in day two and three and 1,929 from day 6 on. There was a significant difference on the median viral load from the first five days of onset of symptoms compared to samples obtained after five days (Mann-Whitney U test, p<0.05). DISCUSSION The dengue real-time kit was found to be practical and adjustable for high throughput. The in-house multiplex-pcr presented a slightly lower analytic sensitivity, but showed to be a valuable tool for epidemiological studies, allowing characterization of circulating dengue serotypes. The real-time kit displayed some analytical features rated as desirable in the routine laboratorial diagnosis. One of them was the reproducibility, as measured by the standard deviation between replicates in the same run, which was always lower than 1 Ct. A second aspect was the absence, in this series, of gray zone Cts, allowing us to confidently assign a sample as positive even for a high Ct/low viral load. The presence of an internal control also avoids false-negative results, and we didn t observe a single case of inhibition of its signal, except in samples with a very high viral load, when this is expected to occur. The multiplex-pcr has the advantage of serotyping, which may be relevant for patient management and for public health as well. It is considerably cheaper than the real-time kit, since it is performed with homebrew reagents. An important drawback of it was the need to repeat several tests due to inconclusive bands of low intensity. In this

9 9 instance we decided to repeat the assay in a monoplex format, i.e. containing primers only for the suspected (band size) dengue serotype. All repetitions showed that the first results were indeed false-positives. The identified serotypes are consistent with the laboratory surveillance of dengue virus in the same city, where the majority of dengue cases were attributable to dengue 3 in recent years (2). This dominance of dengue 3 was preceded in time by DEN-1 outbreaks and occasional dengue type 2 cases, so it is reasonable that a few cases of these two serotypes are still detected. As we performed both molecular assays in parallel, and beginning from the same RNA extract, the performance of both could be accurately compared, leading to our conclusion of the slightly higher analytical sensitivity of the real-time kit, as samples containing less than 10,000 copies/ml of dengue RNA were not consistently reactive in the multiplex assay. Of note, four samples were reactive exclusively by real-time. They presented viral loads of 228, 857, 994 and 2,318 copies/ml respectively. All them were collected in the first 4 days of symptoms, so it is reasonable to admit that they represent cases of low viremia at the collection date, below the sensitivity of our multiplex-pcr assay and hadn t developed IgM yet. We couldn t obtain a follow-up sample from them, which would probably reveal a positive IgM. Our results demonstrate the diagnostic value of molecular assays for dengue, provided that samples are drawn at the early days of symptoms, in between days 1 3. In this period, the additional value of IgM is low. After this critical phase, IgM is more useful than PCR, attributable to the short course of viremia, and the lag between this and the development of acute phase antibodies (IgM). A similar study was carried in Brazil by Poersch and co-workers (9), comparing nested-pcr and qualitative real-time PCR for the diagnosis of dengue in the acute phase. In

10 10 concordance to our results, they reported a superior sensitivity for real-time PCR, advocating its use by the health system in combination to IgM for late samples. The dengue real-time kit showed a high performance in the early symptomatic phase of dengue cases, valuable for confirming dengue diagnosis timely. It needs to be established whether viral load could be a determinant indicator of disease outcome. The goal of our work is to offer a fast (6 hours), reliable and affordable test for dengue in order to give support to clinical decisions, improving the quality of the management of confirmed cases and avoiding investigative procedures for negative cases. Overall, this may represent a significant economy of resources for the health system. In this work, samples were processed a posteriori, i.e. they were collected in between January and June of year 2005 and actually analyzed 6 months later, except for the IgM that, according to the protocol currently in use in our country, is performed one week after fever onset. Though the costs of molecular diagnostic tests are usually higher than serological tests, processing batches of samples allows the achievement of scale economy. We foresee the use of realtime PCR in a few specialized centers, strategically located and receiving samples from surrounding cities. If properly planned, number of samples won t be the limiting factor, as it happens today for IgM serology, for which the samples in queue surpass the installed laboratorial capacity, leading to a delayed diagnosis. The level of laboratory sophistication required is the same as we have already in Brazil for HIV viral load and resistance testing, so the system may benefit from the equipments and human resources now in place. Due to the cost, the commercial kit here presented may not be affordable by the Brazilian health system. Developing or adapting in-house real-time PCR tests described in the literature is a possibility we are pursuing now. The key step that demands further development is the RNA extraction. Manual extraction, as adopted in this work, is time

11 11 consuming, expensive and prone to contamination. We are seeking for automated or semiautomated methods in microplate format, allowing the simultaneous extraction of up to 96 samples, completing the process from sera/plasma to RNA in less than 1 hour. If conjugated to a fast one-step real-time PCR protocol of about 2 hours, we can foresee a daily routine of 96 tests with a turnaround time of 4 hours, which can cope with the demand of populated regions, experiencing dengue outbreaks from time to time. Acknowledgements This work was supported by grants / and from Pronex/CNPq/Sectec and CNPq /01 from the Brazilian Ministry for Science and Technology. Dr. Martelli received a CNPq Scholarship (307963/2004-7).

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14 14 TABLE 1 Comparison of multiplex and real-time PCR results on 254 patients with suspected acute dengue. Real-Time PCR Mx-PCR Pos Neg Total Pos Neg Total Kappa agreement = 0.85

15 15 % No. Samples Real-Time PCR (63.2) (55.6) (39.5) (9.1) (9.4) Multiplex PCR (57.9) (48.9) (33.3) (2.6) (9.4) Viral Isolation (52.6) (35.6) (21.0) (1.3) nd IgM serology (0.0) (15.6) (70.4) (88.3) (71.9) Real-Time PCR Multiplex PCR Virus isolation IgM serology Figure 1 Comparison of the performance of four diagnostic techniques (Real-Time PCR, Multiplex-PCR, Viral isolation and IgM serology,) for suspected dengue cases according to days of symptoms onset. (%), nd = not done. Days

16 Days Real-Time PCR IgM serology Real-Time + IgM Figure 2 Comparison of the performance of IgM, Real-Time PCR and both in a selected subset of 207 patients with confirmed dengue diagnosis (includes 4 cases of isolated real-time positivity, see results section). Absolute numbers of positive results are on top of each column. 7 3

17 17 Real-Time PCR Log scale Days 6-7 >7 Figure 3 Viral load of real time-pcr positive samples according to days of dengue symptoms. Box plot, dashed line represents medians and empty circles outliers. Values in log scale.