Diagnostic Options and Challenges for Dengue and Chikungunya Viruses

4.1. 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].