DENV serotyping by serological methods have been impeded by cross-reactivity of antibodies induced by different DENV serotypes. A DENV envelope protein-based Ig-M ELISA capable of differentiating DENV serotypes during the acute phase of dengue was reported recently. However, its clinical performance needs to be further validated and existing IgM from previous infection remains a challenge in data interpretation. Several CE-IVD marked multiplex real-time RT-PCR methods capable of serotyping are available for laboratory settings. A Luminex-based single DNA fragment amplification assay capable of multiplex DENV serotyping was reported recently.
Rapid on-site detection and serotyping of DENV can potentially alert front-line health professionals of invasion of a new or long-time absent serotype, allowing timely implementation of intervention strategies. Although parallel NS1 antigen and IgM/IgG rapid testing has improved dengue diagnosis at points of need/points of care (PON/POC) in recent years, both tests do not provide serotype information. In addition, they are generally less sensitive and specific than those of ELISA and nucleic acid testing (NAT) methods. Their sensitivity could be further lowered during secondary infection since the levels of detectable NS1 could be reduced by existing antibody, and levels of IgM are not as high as in primary infection and can be undetectable in some cases.
Real-time RT-PCR assays and the Luminex-based assay allow serotyping of DENV with great sensitivity. However, their execution and/or data interpretation require skilled technicians and relatively expensive equipment that are not commonly available in remote areas or developing countries; long-distance transportation of specimens is another major obstacle.
To facilitate timely near-patient serotyping of DENV at low-resource settings, rapid, easy, mobile, NAT methods of high sensitivity and serotype-specificity are still needed to bring accurate detection/serotyping of DENV to PON/POC. On-site serotyping of DENV by a combination of isothermal amplification and DNA sequencing with a portable sequencer was recently demonstrated to be feasible. However, its immediate clinical applications at PON/POC are limited by the costs and technical skills required.
A limited number of methods, including the reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) and the TaqMan probe-based RT-insulated isothermal PCR (RT-iiPCR) methods, became available recently with potential to enable easy near-patient detection and serotyping of DENV. LAMP can be performed with a simple incubator and generally requires only a very simple nucleic acid extraction step. One DENV serotyping RT-LAMP panel including four singleplex DENV-1, 2, 3, and 4 reactions targeting the 2A, NS4B, NS4A, and 3’UTR markers was reported to generate signals visualised by naked eyes or a laboratory fluorescence-monitoring device in 25 minutes. Its clinical performance was demonstrated preliminarily to be similar to that of a real-time RT-PCR. Another RT-LAMP panel also included four reactions all targeting the NS1 gene; SYBR Green I signals can be detected visually or with a field-deployable Genie II flourometer (OptiGene, UK) in about 35 minutes. Preliminary study showed that this assay and CDC real-time RT-PCR assay complemented NS1 to increase the diagnostic coverage of febrile patients to similar degrees.
Another singleplex DENV serotyping RT-PCR panel that works on a mobile PCR system has been evaluated preliminarily to have performance comparable to that of CDC Real-time RT-PCR. The DENV-1, 2, 3, and 4 reagents, targeting the NS5, E, prM, and prM genes, respectively, are based on the TaqMan probed-based iiPCR technology. Automated amplification and detection of iiPCR are achieved consistently in a capillary tube in a simple, insulated heater/detector. CE-marked compact iiPCR devices are commercially available, including a field-deployable model (POCKIT, GeneReach Biotech) and a hand-held series (POCKIT Micro) with built-in rechargeable batteries. Results are detected and interpreted automatically to generate qualitative results within one hour. Its performance has been verified and validated to be comparable to that of several reference laboratory methods (real-time PCR, virus isolation) with various markers and sample types. A CE-marked pan-DENV RT-PCR is already available to detect all four DENV serotypes in human plasma and serum on this platform to aid PON/POC identification of DENV. PCR testing requires nucleic acid extraction, and a compact automated nucleic acid extraction system has been bundled with these PCR devices in a durable suitcase to realise a mobile PCR laboratory. Furthermore, a fully automated, sample-in-answer-out, compact system (POCKIT Central Nucleic Acid Analyser) is available recently with the CE mark to further minimise human error risks and allow easy molecular bio-detection near PON/POC.
Progress in translating novel molecular technology into diagnostics for dengue infection has helped the development of relatively inexpensive, rapid, and simple NATs to meet the needs in early serotyping of DENV near PON/POC. These tools have potential to enable timely management, control, and monitoring of different DENV serotypes especially in under-served communities. Further verification and validation studies of these methods should be expedited to bring them to clinical settings.
References available on request.