Diagnosis

 Clinical diagnosis and patient management were performed according to the local standard of care and were recorded on a standard assessment form. Symptomatic dengue was defined as acute febrile illness plus a positive IgM or RT-PCR with a follow-up positive IgG test in the serosurvey. Inapparent dengue infection was defined as having a positive dengue test (IgM, or RT-PCR) but no febrile illness. Confirmed cases of CHIKV infection were defined based on considered reactivity to IgM enzyme-linked immunosorbent assay (ELISA) and the presence of viral RNA in clinical samples. Patients whose specimens tested positive for anti-chikungunya IgM only by ELISA were designated as probable cases. Ten milliliters of intravenous blood samples were obtained from 77 eligible patients. These samples were collected aseptically into tubes with ethylenediaminetetraacetic acid. All samples were temporarily kept at −20 °C at the local hospital before being transported to our laboratory at the São Paulo Institute of Tropical Medicine, where they were stored at −80 °C until analysis.

Plasma samples were obtained from 77 suspected dengue patients attending the main hospital in the city. Laboratory assays, such as real-time reverse transcription polymerase chain reaction, virus cDNA sequencing, and enzyme-linked immunosorbent assay, were employed to identify the infecting virus and molecular phylogenetic analysis was performed to define the circulating viral genotypes.

RNA of Zika virus (ZIKV) and Dengue virus (DENV) or IgM antibodies (Abs) to DENV or chikungunya (CHIKV) were detected in 40 of the 77 plasma samples (51.9%). DENV was found in 9 patients (11.7%), ZIKV was found in 31 patients (40.2%), CHIKV in 1 patient (1.3%), and coinfection of DENV and ZIKV was detected in 2 patients (2.6%). The phylogenetic analysis of 2 available partial DENV and 14 ZIKV sequences revealed the identities of genotype 1 and the Asiatic lineage, respectively.

Consistent with recent reports from the same region, our results showed that the ongoing outbreak is caused by ZIKV, DENV, and CHIKV. This emphasizes the need for a routine and differential diagnosis of arboviruses in patients with dengue-like illness. Coordinated efforts are necessary to contain the outbreak. Continued surveillance will be important to assess the effectiveness of current and future prevention strategies.

Laboratory Diagnosis and Virus Identification

Detection of DENV Abs (IgM and IgG) and CHIKV Abs (IgM) 

Anti-dengue virus IgM Abs in plasma samples were detected by an IgM-capture ELISA kit (dengue virus IgM capture DxSelect; Focus Diagnosis, CA) according to the manufacturer's protocol. Anti-dengue virus IgG Abs were detected by ELISA using an IgG indirect ELISA kit (Dengue IgG indirect ELISA; Panbio Ltd, Queensland, Australia) according to the manufacturer's protocol. The assays for detection of anti-dengue virus IgG Abs were used only in the follow-up serosurvey of patients who had detectable anti-dengue virus IgM Abs. All plasma samples were also screened for CHIKV IgM Abs using IgM IIFT from EUROIMMUN AG. Plasma was considered positive only when the optical density exceeded 1.1 times that of the negative control.

Extraction of Viral RNA and cDNA Synthesis 

Total RNA was extracted from 140 μL serum samples by using the QIAamp viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The RNA was extracted in 60 μL of elution buffer and was used immediately to synthesize the cDNA with the Superscript III (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, a master mix consisting of 0.1 mmol/L deoxynucleoside triphosphates, 150 ng of random hexamers, 5 μL of 10 × buffer, 10 mmol/L dithiothreitol, 2.5 U of RNaseOUT recombinant RNase inhibitor (Promega, Madison, WI), and 5 U of Superscript III (Invitrogen, Carlsbad, CA) reverse transcriptase enzyme was prepared. A 10-μL aliquot of extracted RNA was added to this mixture, and the reaction was incubated at 25°C for 5 minutes, 42 °C for 50minutes, and then 95°C for 3 minutes. The resultant cDNA was stored at −20°C for future use.

Real-time RT-PCR for DENV 

Dengue RNA genomes from all 4 serotypes were examined by a previously published real-time PCR method.26 Briefly, A total of 7.5 μL of extracted RNA was used as a template in a 15-μL reaction volume using SuperScript III Platinum SYBR green 1-step quantitative RT-PCR (qRT-PCR) with the ROX Kit (Applied Biosystems, Austin, TX) and 0.4 μmol/L of pan-dengue primers.27 The samples were amplified in an ABI Prism 7300 sequence detection system (PE Applied Biosystems, Foster City, CA). The qRT-PCR assay consisted of a 10-minute reverse transcription step at 60°C and then 1 minute for Taq polymerase activation at 95 °C, followed by 45 cycles of PCR at 95°C without holding time, 60°C for 3 s, and 72°C for 10 s.

2 MATERIALS AND METHODS2.

1 Assays and systems

The cobas CHIKV/DENV test is a qualitative in vitro test for the detection of CHIKV RNA and DENV RNA, serotypes 1 through 4 in human plasma. The cobas CHIKV/DENV is designed to test for CHIKV and DENV RNA either alone or simultaneously. The cobas Zika test is a qualitative in vitro test for the detection of ZIKV RNA. Both tests allow plasma from donations of whole blood and blood components to be tested individually or in pools composed of individual samples.37, 38

The cobas CHIKV/DENV and cobas Zika tests are based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection on the cobas 6800/8800 systems. The cobas 6800/8800 systems are highly automated testing platforms consisting of sample supply, transfer, processing, and analytic modules. Reagents are ready to use, requiring no preparation, and can be stored on the instrument. Minimal operator hands-on time is required once reagents, consumables, and samples are loaded onto the instrument. The cobas 6800/8800 software performs automated data management that assigns test results that can be reviewed directly on the system screen and printed as a report or transmitted to a laboratory information system.39 Up to 384 test results can be generated on the cobas 6800 and up to 960 test results on the cobas 8800 within an 8-hour work shift.40

2.2 Clinical prevalence and specificity testing

Deidentified ethylenediaminetetraacetic acid plasma samples were collected from routine blood donations in Vietnam between October and December 2018, and stored at −30°C for a maximum of 4 months before testing at the National Institute of Hematology and Blood Transfusion in Hanoi, Vietnam. Ethylenediaminetetraacetic acid plasma samples were collected in Bangkok and eastern and southern Thailand, between May and November 2019, and stored at −40°C or −60°C for a maximum of 40 days before testing at the Faculty of Medicine, Siriraj Hospital, Mahidol University, or at the Faculty of Medicine, Ramathibodi Hospital, Mahidol University in Bangkok. Each of the laboratory sites tested a minimum of 3000 samples by individual donation testing (IDT) using cobas CHIKV/DENV and a minimum of 3000 samples by IDT using cobas Zika. The ethics committee or institutional review board for each site approved the study protocols and materials.

Each site tested the samples once by IDT with the respective cobas test and samples nonreactive were considered RNA negative for the targeted viruses. Samples reactive with cobas CHIKV/DENV were further tested to confirm reactivity (Figure 1). Additional testing included repeat testing by cobas CHIKV/DENV neat and in a 1:6 dilution format, and testing by an alternative NAT using an in vitro diagnostic test (RealStar Chikungunya RT-PCR Kit 2.041 or RealStar Dengue RT-PCR Kit 2.042) based on the reactive target reported in the cobas CHIKV/DENV test. An enhanced sample input volume was used for the RealStar assays based on prior studies comparing the sensitivity of the RealStar assays to cobas CHIKV/DENV, and each concentration was tested in multiple replicates.43 Samples with one or more reactive replicates on the target-specific Altona test were considered confirmed for the target. DENV serotypes and CHIKV genotypes were not determined. Samples reactive by cobas Zika were further tested, including a repeat test by cobas Zika neat and in a 1:6 dilution, by an alternative NAT in duplicate, and anti-Zika IgM test as described in Galel et al44 (Figure 2). Samples reactive by alternative NAT or anti-Zika IgM confirmed the presence of ZIKV. Discordant results between initial and additional testing for CHIKV, DENV, or ZIKV were tested by heminested PCR (HnPCR) performed on the amplification product of the initial testing to resolve the status of the sample.

2.3 Multiassay testing

Approximately 1500 deidentified ethylenediaminetetraacetic acid plasma samples were collected from infectious disease screened (including Zika), nonreactive volunteer donations from the continental United States. Samples were unscreened for CHIKV or DENV. No known Zika-, CHIKV-, or DENV-positive samples were included because the purpose of the study was solely to evaluate testing throughput on the cobas 8800 system. Samples were tested by IDT with cobas CHIKV/DENV and cobas Zika for use with the cobas 6800/8800 systems according to the manufacturer's instructions.37, 38

After centrifugation, a maximum of 460 samples were continuously loaded onto a cobas 8800 system. Each sample was pipetted by the instrument onto a processing plate for concurrent testing with cobas CHIKV/DENV and cobas Zika. Each processing plate contained up to 46 samples plus 2 controls per assay for a total of 96 tests. Time was captured at the beginning of processing of the first samples (start time), time to first available test result, and time to last available test result. Total processing time was evaluated to measure the maximum number of test results available in an 8-hour work shift.

Duplex nucleic acid test for the detection of chikungunya and dengue RNA viruses in blood donations

Performance of cobas CHIKV/DENV, a qualitative RNA detection assay for use on the cobas 6800/8800 Systems, was evaluated at two sites (Roche Molecular Systems, Inc. [RMS], and the American Red Cross [ARC]). Analytical sensitivity, genotype inclusion, correlation with other assays, and reproducibility used clinical CHIKV- or DENV-positive samples and secondary standards for DENV Types 1 to 4 and for three CHIKV genotypes (Asian; East Central South African; and West African); each secondary standard was traceable to international reference panels or reagents. Evaluation of analytic specificity assessed other microorganisms for interference and cross-reactivity; clinical specificity was determined by individually testing 10,528 volunteer blood donations from the continental United States.

Forty two representative sequences of the non-structural 5 (NS5) region of ZIKV including those from Rhesus monkeys from Uganda, early human isolates from Nigeria, different African and Asian mosquito isolates, the strain from Micronesia in 2007 and sequences from isolates circulating in different parts of the world from 2013, 2014 and 2015 were used for oligo design. More than 300 representative sequences of the non-structural protein 4 (nsP4) gene of the CHIKV including Eastern, Central, and South African isolates, the Asian lineage from different countries, and the Indian Ocean lineage were used for oligo design. The 3′ untranslated region (3′UTR) from Dengue viruses 1–4 (DENV-1 to DENV-4) were retrieved and one set of primers and a probe were designed to detect all four serotypes with equal efficiency. Hydrolysis probes were designed with minor groove binding proteins and labeled with the fluorescent reporters NED, VIC and FAM for the detection of ZIKV, CHIKV and DENV viruses, respectively (Applied Biosystems (ABI), Foster City, California). Sequences from the targeted viruses were contrasted with sequences from other flaviviruses and alphaviruses to avoid cross detection. All primers were purchased from the University Core DNA services, (University of Calgary, Calgary, Alberta). Primers were also designed in the regions flanking the detection region for the amplification of a longer fragment including the detection region to generate plasmid clones used in the preparation of in vitro transcribed RNA. The sequences and source of the oligonucleotides used are provided in Table 1. Primers and probes were designed using Primer Express® v3.0 from ABI and all sequence alignments were performed using Clustal W (BioEdit).

2.2. Real-time RT-PCR assay

A one-step RT-PCR was performed using TaqMan® Fast Virus One-Step RT-PCR Master Mix (ABI), 0.8 μM each of sense and antisense primers and 0.2 μM of the probes. Five microliters of the extracted RNA was combined with 5 μl of the master mix and the reverse transcription step was performed at 50 °C for 5 min followed by incubation at 95 °C for 20 s. Amplification included 45 cycles of denaturation at 95 °C for 3 s, followed by annealing, extension and data acquisition at 60 °C for 30 s on the 7500 Fast Real-Time PCR system (ABI).

2.3. Preparation of RNA transcripts for sensitivity studies

The cloning primers listed in Table 1 were used for the amplification of longer PCR products which were cloned using the TOPO® TA Cloning Dual Promoter Kit (Life Technologies, California, USA). RNA transcription was performed using RiboMAX™ SP6 RNA Production System or T7 RiboMAX™ Express (Promega, Madison, WI, USA) using standard protocols. The transcribed RNA was spectrophotometrically quantified for the calculation of copy numbers.

2.4. Extraction of viral nucleic acid

Viral RNA from the different specimen matrices including plasma, serum, urine, cerebral spinal fluid (CSF) and amniotic fluid was extracted using the easyMAG® automated extractor (BioMerieux, Quebec, Canada), according to manufacturer’s instructions. The sample input volumes for plasma, serum and CSF were 200 μl and the output volume was 55 μl. For urine and amniotic fluid the input and output volumes were 200 μl and 110 μl respectively. Tissue samples (placenta and brain) were extracted on the QIAcube automated extractor using the QIAamp DNA Mini QIAcube kit (Qiagen, Ontario, Canada) from 60 μl of homogenized tissue as input and nucleic acids were eluted into 200 μl.

2.5. Analytical sensitivity, exclusivity, reproducibility and dynamic range of RT-PCR

Ten and two-fold serial dilutions of quantified in vitro RNA were used to determine the analytical sensitivity by testing the dilutions in triplicate on three independent runs. The 95% limits of detection (95% LOD) were calculated by probit analysis using Microsoft Excel followed by rounding up the copy number. The range of viral loads tested in copies/reaction was 5.77 × 108 to 5.77 × 10−2 for DENV-1, 7.83 × 107 to 7.83 × 10−2 for DENV-2, 9.75 × 107 to 9.75 × 10−2 for DENV-3, 6.32 × 107 to 6.32 × 10−2 for DENV-4, 5.53 × 108 to 5.53 × 10−2 for ZIKV and 5.24 × 107 to 5.24 × 10−2 for CHIKV. Linear regression fitting of the log viral load versus Ct allowed for the calculation of Ct values corresponding to the 95% LOD.

Exclusivity of the assay was determined by testing high viral load samples of several RNA and DNA viruses, including other arboviruses, and viruses with overlapping clinical symptoms. The viruses tested were Coxsackie A16, adenovirus serotype 4, Varicella-Zoster, St. Louis encephalitis, Powassan, West Nile (lineage 1a), Kunjin (West Nile virus lineage 1b), Murray Valley encephalitis, Japanese encephalitis, Snowshoe hare, Jamestown Canyon, La Crosse, Cache Valley, Western equine encephalitis, and Eastern equine encephalitis viruses.

The reproducibility of the triplex assay was evaluated using high and low viral load aliquots of cultured virus and clinical samples; all viral cultures were obtained from the National Microbiology Laboratory (NML, Winnipeg, Manitoba, Canada). The intra and inter-assay reproducibility was calculated using samples with initial Ct values of 24.60 and 33.32 for ZIKV, 25.23 and 32.51 for CHIKV, 27.16 and 33.64 for DENV-1, 26.03 and 32.38 for DENV-2, 23.87 and 33.79 for DENV-3, 25.69 and 31.55 for DENV-4; three independent runs were performed with each specimen tested in triplicate on every run.

2.6. Clinical specimens

Specimens submitted from 2011 to 2016 to the ProvLab for the investigation of one or more of ZIKV, CHIKV and DENVs from travel related cases were re-tested by the triplex assay.

Seventy-nine samples including amniotic fluid (n = 1), CSF (n = 2), plasma (n = 29), serum (n = 29), and urine (n = 18) were tested for ZIKV. Two real-time RT-PCR assays published by the Centers for Disease Control (CDC, Atlanta, USA) targeting the membrane and envelope regions of the ZIKV genome [27] were used as gold standard assays for comparison. In addition, to validate the use of placental tissue, brain tissue and amniotic fluid, ZIKV culture was spiked at different viral loads ranging from Ct values of 25.84–33.25 into negative specimen matrices (n = 5), extracted and tested.

Fifteen samples from 2014 to 2016 were tested for CHIKV, including serum (n = 11), plasma (n = 3) and CSF (n = 1). Samples from 2011 to 2016 (n = 133) were tested for DENV, including amniotic fluid (n = 1), CSF (n = 7), plasma (n = 40), serum (n = 81) and urine (n = 4). The RealStar® Chikungunya RT-PCR Kit and Dengue RT-PCR Kit 2.0 from Altona Diagnostics (Hamburg, Germany) are CE-IVD marked in vitro diagnostic tests that were used as gold standards.

2.7. Co-infections

To assess competitive inhibition of target extraction and detection in cases of co-infections, combinations of low and high viral loads with Ct values ranging from about 21 to 32 were spiked into samples. Cultured ZIKV and a strong positive patient specimen for DENV-1 were used to spike negative plasma samples. For CHIKV, RNA was spiked into the extracts as needed. All extracts were tested in triplicate by the in-house assay.

Methods for Diagnosis

A wide variety of laboratory diagnostic methods are available to aid in the diagnosis of DENV and CHIKV infections. The premise of these tests is detection of the virus, viral components (antigens or nucleic acid), or the host immunologic response to the virus [10]. Therefore, selection and interpretation of testing depends on the kinetics of viremia and antibody response, which differ between primary and secondary infections. Other factors influencing test choice include the purpose of testing and availability of resources. Each type of test offers unique advantages and disadvantages, and a combination of tests may be employed in order to increase diagnostic confidence. For a summary of available tests for DENV and CHIKV infection, see Tables 1 and 2, respectively.

. Overview of Currently Available Tests

The acute febrile phase of infection corresponds to the period of viremia, which lasts typically from 5 days after onset of fever for both DENV and CHIKV. During this time, diagnosis rests on isolation of the virus, viral RNA, or viral antigen from the specimen. Isolation of DENV or CHIKV can be performed via mosquito inoculation or cell culture; CHIKV isolation can also be accomplished by intracerebral inoculation of mice [16]. Virus may be recovered from serum, plasma, whole blood, or tissues collected at autopsy. Mosquito inoculation is the most sensitive isolation method but is impractical for routine diagnosis due to the highly specialized requirements and high maintenance costs [3]. Cell culture is in wider use, with preference given to the mosquito cell line C6/36 (cloned from A. albopictus) or AP61 (cloned from A. pseudoscutellaris) [9, 16]. Other less sensitive options include mammalian cell cultures such as Vero, LLC-MK2, and BHK-21 [3]. The resultant virus isolate may be further characterized during subsequent in vitro studies, such as genome sequencing, virus neutralization, and infection studies [3]. Virus isolation is highly specific and has a theoretical detection limit of a single viable virus, although, in practice, the sensitivity is only approximately 40.5% in cell line-based virus isolation. It also requires highly trained operators, a dependence on sample integrity and a short viremia period, thus providing a narrow window of opportunity from illness onset. Virus isolation followed by an immunofluorescence assay for confirmation requires days to weeks [9, 16]. Therefore, despite its advantages, this approach is not widely used in routine diagnostic laboratories and may serve more use in surveillance purposes. A more recent development in viral isolation is described by Patramool et al., who used anionic polymer-coated beads to isolate DENV and CHIKV [27]. This may prove a useful strategy to monitor the status of circulating mosquitos in regions at risk for outbreaks with these arboviruses. Compared to traditional isolation techniques, this method provides reduced cost, good sensitivity, and rapidity, which is conducive to simultaneous analysis of a large number of samples [27].

Compared to virus isolation, viral nucleic acid detection techniques performed on acute-phase specimens offer better sensitivity with a much more rapid turnaround time. Viral nucleic acid can be detected for a few additional days beyond the period of viremia. Detection of viral nucleic acid can be accomplished by reverse transcriptase polymerase chain reaction (RT-PCR), real time RT-PCR, or isothermal amplification methods. All of these methods involve three basic steps: viral RNA extraction, amplification, and detection and characterization of the amplified product [9]. There is a wide variety of specimen types that can be tested with RT-PCR, including blood, serum, plasma, and fresh or formalin-fixed paraffin-embedded tissues. For DENV, urine and saliva have been found to be suitable specimen types as well [3]. Testing urine samples by real-time RT-PCR provides a larger window of detection that extends well past the viremia period; DENV RNA may be detected in urine up to day 16, compared to day 8 for blood specimens [28]. The ability to test urine and saliva is advantageous in patients for whom blood samples are difficult to obtain, such as in newborns and patients with hemorrhagic syndromes [14].

RT-PCR using primers designed for structural and nonstructural domains has been found to be useful in the rapid diagnosis of CHIKV. The combination of RT-PCR/nested PCR has proved efficient for specific detection and genotyping of CHIKV. Loop-mediated isothermal amplification (LAMP) assays can be rapidly carried out at a single temperature in a water bath, with visually detectable results, and comparable sensitivities to conventional PCR [17].

Detection of viral antigens is another diagnostic methodology available for DENV infection. Nonstructural protein 1 (NS1) antigen is a highly conserved glycoprotein produced during the virus replication process, and a soluble form of NS1 accumulates in high concentrations in the serum of patients with both primary and secondary DENV infections [29, 30]. Several commercial assays, consisting of both rapid tests and enzyme-linked immunosorbent assay (ELISA) kits, are available for the detection of the NS1 antigen. Serum is the most common sample type. DENV NS1 can also be detected in urine samples during the acute phase of DENV infection, which provides an opportunity for the development of a rapid noninvasive test [11]. Lastly, NS1 antigen may be detected in the cerebrospinal fluid (CSF) of patients with neurological symptoms [12]. A downfall is that these tests do not differentiate between dengue serotypes, as NS1 is highly conserved by all serotypes. Additionally, these tests are most successful during the acute phase of illness and lose sensitivity once the period of viremia ends. The sensitivity of NS1 has also been found to be lower in DENV secondary infections, which is thought to be due to assay interference by anti-NS1 antibodies which are present more frequently in secondary infections [10, 29]. An antigen-based commercial detection assay is not widely available for CHIKV, and the ones described thus far in the literature have unclearly established performance characteristics [21, 22].

After the period of viremia, the methods described thus far become much less sensitive for diagnosis. At this point, the best diagnostic strategy entails detection of antibodies indicative of host immune response to the virus. However, the caveat is that individuals in endemic areas often have immunologic levels to these viruses. Serologic methods include ELISA, indirect immunofluorescence assays (IFA), hemagglutination inhibition (HI), and microneutralization (MNt) [1]. ELISA and IFA are rapid and sensitive techniques for detecting virus-specific antibodies and can distinguish between IgG and IgM. For techniques that cannot make this distinction (HI and MNt), it is required to compare paired serum samples (acute and convalescent phases) to establish recent infection.

For DENV, serologic methods are most commonly employed, in particular IgM capture ELISA [4]. IgM antibodies are detectable in 50% by days 3–5 after onset, 80% by day 5, and 99% by day 10 after initial symptoms. They may persist for months; hence DENV IgM antibodies are a reliable marker of recent but not necessarily acute infection [29]. IgG antibody response develops a few days after the onset of IgM antibodies, and IgG may persist for many years [29]. Serologic confirmation of infection requires demonstration of a fourfold rise in antibody titer between acute and convalescent phase sera, or by demonstration of IgM antibodies specific for the virus [16]. Patterns of antibody response differ between primary and secondary infections, with primary dengue invoking stronger and more specific IgM response than in secondary, which have stronger and more rapid IgG response. Prior vaccination against other Flavivirus (Japanese encephalitis virus; Yellow-fever virus) or prior infection with nondengue flaviviruses (including West Nile) can potentially influence antibody responses measured in some assays [4]. The recent introduction of rapid diagnostic kits that offer combined detection of NS1 and IgM/IgG antibodies was an effort to create a point-of-care test with better performance characteristics [13]. Evaluation of some of these combined tests has revealed diagnostic sensitivity of 89–93% and specificity of 75–100% [3, 13].

A combination of molecular and IgM antibody detection assays is recommended for diagnosis of CHIKV infection. Some advocate adopting an algorithmic approach, wherein the IgM capture ELISA is used as an initial screening tool followed by the use of rapid molecular assays in CHIKV IgM negative samples, to facilitate rapid diagnosis during outbreaks [18].

4.2. Simultaneous Testing for DENV and CHIKV

Because infection with DENV and CHIKV should be on the differential diagnosis together at the initial patient presentation, tests that screen for these viruses simultaneously are preferred to test for them separately. CHIKV and DENV are not readily differentiated serologically due to cross-reactivity of their serocomplexes, so there is a reliance on molecular detection methods for this purpose [19]. A one-step duplex conventional RT-PCR assay for distinguishing DENV and CHIKV has been reported [20]. Saha et al. developed a highly sensitive and specific, rapid one-tube duplex RT-PCR assay which provides a result within 110 minutes [19]. Two authors have described a one-step multiplex real-time RT-PCR assay that can simultaneously detect and quantitate RNA for all DENV serotypes and CHIKV. Cecilia et al. report a sensitivity of 100% for DENV and 95.8% for CHIKV, while the specificity was 100% for both viruses when compared to conventional RT-PCR [24]. Pongsiri et al. report an assay sensitivity of 97.65% and specificity of 92.59% when compared to conventional RT-PCR [31]. Real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a sensitive alternative to real-time PCR for use in field applications [18]. A RT-LAMP method has been described in which a reverse transcription and amplification was designed in one step with two tubes under the same reaction conditions for the rapid identification and quantitative detection of RNA for CHIKV and DENV, respectively [32]. This assay has a sensitivity of 100% and specificity of 95.25%. The LAMP reaction can be ended within one hour under isothermal conditions and does not require sophisticated instruments, making this method adaptive to field diagnosis. Additionally, the use of a turbidimeter allows for quantitative detection of viral load [32]. For RT-PCR assays described above, the one-step process reduces the chance of contamination and there is lack of cross-reactivity between related Flavivirus groups and DENV [19].

4.3. Sending Out Samples

Within the United States, CHIKV testing is performed at the Centers for Disease Control and Prevention (CDC), a limited number of select state health departments, and one commercial laboratory. The CDC’s Arbovirus Diagnostic Laboratory at the Division of Vector-Borne Diseases (DVBD) is located in Fort Collins, CO. Test results are normally available 4 to 14 days after specimen receipt, but reporting times may be longer during summer months when arbovirus activity increases. Initial serological testing is performed using IgM capture ELISA and IgG ELISA. If the initial results are positive, further confirmatory testing is performed which may delay the reporting of final results. All results are sent to the appropriate state health department.

The CDC Dengue Branch, located in San Juan, Puerto Rico, provides DENV testing free of charge to submitting physicians and state and private laboratories. A “Dengue Case Investigation Form” must accompany the specimen. One potentially problematic issue with sending samples to this laboratory is that an international shipping license is required. Another challenge, especially for underdeveloped countries, is specimen preservation during shipment. The CDC recommendation is that the serum specimen is frozen immediately after separation and sent on dry ice, or alternatively kept refrigerated and sent in cold packs.

4.4. Future Test Developments

Other diagnostic methodologies may be available for future use in the laboratory diagnosis of DENV and CHIKV infection. One technique becoming an increasingly popular serological option in arbovirology is microsphere-based immunoassay (MIA). This technology is based on detection by flow cytometry of antigen or antibody attached to microspheres or beads. This is a much more rapid test than MAC-ELISA and also has the potential for performance in multiplex [33]. Similarly, microarray technology, which focuses on detection of nucleic acid fragments corresponding to different pathogens, is useful to screen a sample for the many pathogens on a wide differential diagnosis for infectious symptoms in a given region [10]. Finally, mass spectrometry could be applied to this field of diagnosis, proving especially useful in determining viral serotypes and genotypes during an outbreak [9].

Chikungunya virus infection should be considered in patients with acute onset of fever and polyarthralgia, especially travelers who recently returned from areas with known virus transmission. Laboratory diagnosis is generally accomplished by testing serum or plasma to detect virus, viral nucleic acid, or virus-specific immunoglobulin (Ig) M and neutralizing antibodies.

Testing

Viral culture may detect virus in the first 3 days of illness; however, chikungunya virus should be handled under biosafety level (BSL) 3 conditions. During the first 8 days of illness, chikungunya viral RNA can often be identified in serum.

Chikungunya virus antibodies normally develop toward the end of the first week of illness. Therefore, to definitively rule out the diagnosis, convalescent-phase samples should be obtained from patients whose acute-phase samples test negative.

Serological test

A sample of your blood is collected and is tested for the presence of anti - Chikungunya antibodies. Enzyme-linked immunosorbent assays (ELISA) test is done to determine the presence of IgG and IgM antibodies. Check the following table and find out what the values mean.

Chikungunya antibodies (IgG and IgM)

ComponentReference rangeInterpretationChikungunya IgG antibody0.79 or lessNo significant levels of Chikungunya IgG antibody detected.0.80 – 1.09*Possible presence of Chikungunya IgG antibody. Repeat testing within 10 – 14 days required.1.10 or greaterDefinite indication for presence of chikungunya IgG antibody; suggests past or current infection.Chikungunya IgM antibody0.79 or lessNo significant levels of Chikungunya IgM antibody0.80 – 1.09* Possible presence of Chikungunya IgM antibody. Repeat testing within 10 – 14 days required1.10 or greaterDefinite indication for presence of chikungunya IgM antibody; suggests past or current infection.

Antiviral test: RT – PCR (Reverse transcriptase - polymerase chain reaction)

This test is useful to detect the presence of the genes of the chikungunya virus in the blood sample of the patient.