Development of a low-resource rapid Zika virus diagnostic test.

Zika virus (ZIKV), a member of the Flaviviridae family, was first isolated in 1947 from the serum of a rhesus monkey in Uganda (Dick et al., 1952). In 2007, an outbreak on Yap Island (Hayes, 2009) resulting in the first instance of ZIKV transmission outside of Africa and Asia was reported. ZIKV outbreaks have subsequently been reported in multiple locations including French Polynesia (Roth et al., 2014), Papua New Guinea (Chang et al., 2016), New Caledonia (Dupont-Rouzeyrol et al., 2015), and Brazil (Wen et al., 2017). Spanning throughout nearly all seven continents, ZIKV remains a priority disease by World Health Organisation (WHO) (TwistDx^™, 2023b). Although predominantly transmitted via mosquitos (Aedes albopictus and aegypti) (Azar and Weaver, 2019), blood transfusion and sexual transmission have also been reported (Gregory et al., 2017). Historically ZIKV transmission was primarily observed in remote, developing countries (Paixão et al., 2016). However, in 2019, the first locally acquired cases of ZIKV transmission were reported in southern Europe (Brady and Hay, 2019), initiating the prevalence and spread of ZIKV in developed nations. Most ZIKV infections manifest in mild, flu like symptoms (Mumtaz et al., 2016). In severe cases, medical complications such as Guillain Barre (Hendel-Paterson et al., 2016) and severe thrombocytopenia (Sharp et al., 2016) have been reported. ZIKV infection mechanisms also allow for cross placental infections, resulting in microcephaly in developmental infants (Wen et al., 2017).

With no approved vaccine or therapeutics (Da Silva et al., 2018), rapid and accurate detection of ZIKV is a crucial component in predicting and monitoring potential outbreaks (Heukelbach et al., 2016). The majority of ZIKV-positive individuals, including pregnant women are also asymptomatic (Paixao et al., 2018). Crucially delayed diagnosis among pregnant women is particularly concerning due to the inherent risk of congenital abnormalities. Current ZIKV detection and diagnostic strategies utilise both nucleic acid amplification tests (NAATs) (Gourinat et al., 2015) or antibody-based detection-based techniques (Kadkhoda et al., 2017). Due to antigenic cross-reactivity between Zika antigens and other flaviviruses (Stettler et al., 2016), serology based testing such as Enzyme Linked Immunosorbent Assay (ELISA) are less favoured. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) therefore, still remains the ‘gold standard’ for detection and diagnostics among arboviruses (Dong et al., 2012). While accurate and sensitive, a draw-back for RT-qPCR is the need for specialised equipment and trained personnel that restricts these tests to centralised laboratories.

A diagnostic platform that enables in-field or point-of-care (POC) detection without the need for highly trained personnel or specialised equipment offers beneficial attributes to both clinical diagnostics and surveillance of not just ZIKV, but arboviruses alike (Ahmed et al., 2022). Isothermal NAAT’s address all these issues and have been described as potential alternatives to RT-qPCR for viruses such as Zika (Cheikh Tidiane Diagne et al., 2020). As of recent years, innovation and proliferation of various isothermal amplification tests have taken significant strides within the field of rapid diagnostics (Xue et al., 2020). Recombinase aided amplification (RAA) (Jiangsu Qitian Gene Biotechnology Co., Ltd, 2023) is a promising isothermal technique that utilises similar molecular mechanisms as Recombinase Polymerase Amplification (RPA) (TwistDx^™, 2023a). For RNA viruses such as ZIKV, a reverse transcriptase enzyme is added to the RAA reaction. Comparable to RT-qPCR, reverse transcriptase-recombinase aided amplification (RT-RAA) has been shown to be both rapid (Xue et al., 2020) and clinically sensitive (Wang et al., 2020). However, NAAT test uptake for low-resource detection of disease is hampered by the lack of field-friendly sample preparation techniques, instead requiring purification of RNA using magnetic beads or column-based laboratory technologies (Pollak et al., 2022).

Here we describe a rapid, isothermal ZIKV diagnostic (Iso-ZIKV-Dx). Our test format combines a unique low-resource sample preparation reagent with RT-RAA amplification, and lateral flow detection (LFD) to enable ZIKV detection in urine in under 30 min without the need for expensive nor advanced instrumentation. As such, our Iso-ZIKV-Dx exhibits promising diagnostic capabilities suitable for low-resource settings.

In designing our primers and probes, we performed stringent bioinformatic analysis to ensure our primers and probes target as many strains and isolates of ZIKV from all existing lineages as possible. We mapped out all known ZIKV sequences from NCBI covering both Asian and African lineages of ZIKV and constructed a phylogenetic tree using the most unique 105 sequences (Figure 1).

Due to the lack of approved vaccines or specific therapeutics for ZIKV, rapid detection remains crucial in predicting and controlling potential outbreaks. ZIKV outbreaks often occur in rural and remote areas, making it necessary for ZIKV diagnostics to be suitable for use in resource-limited settings, whilst still meeting the requirement for sensitive and accurate detection of ZIKV infection. Isothermal NAAT techniques are a potential solution, especially when combined with safe and simple sample preparation methods, as they could be easily deployed in low-resource settings with minimal training requirements for healthcare workers. In this study, we evaluated a novel rapid ZIKV diagnostic (Iso-ZIKV-Dx) which combines a low-resource sample preparation, RT-RAA test, and LFD. We used a phylogenetically divergent and highly virulent strain (MR766) of ZIKV (Shao et al., 2017). In this study we demonstrated the analytical sensitivity of our RT-RAA test to be 500 copies/μL when using synthetic ZIKV RNA, and confirmed the test did not detect other co-circulating viruses. It should be noted that our synthetic ZIKV RNA generated from RNA transcripts of the NS1 gene whilst the co-circulating viruses consisted of total viral RNA. The rapid Iso-ZIKV-Dx detected 3.56 × 10⁷ TCID₅₀/mL, equivalent to 34.28 copies/reaction RT-qPCR in synthetic urine spiked with ZIKV MR766 and offers improved safety for low-resource testing as the virus is inactivated in the very first step of the procedure. The novel sample preparation reagent, TNA-Cifer Reagent E, has previously been shown to inactivate other pathogens, such as DENV (Calvert et al., 2017), Nipah virus (Rossini et al., 2017) and Hendra virus (Jiangsu Qitian Gene Biotechnology Co., Ltd, 2023), ensuring operator safety in the event of other potential pathogens within the clinical sample. Our data emphasises the benefits of our uncomplicated sample preparation protocol, which proved effective in detecting ZIKV in urine samples. It is noteworthy that ZIKV RNA is known to have limited stability at room temperature, making it challenging to detect within a specific time frame (Tan et al., 2017). Of note, a comparative analysis on current RT-qPCR tests for ZIKV reported discrepancies in detection sensitivity and specificity amongst Asian versus African lineages (de Moraes et al., 2018) with particular emphasis on the difficulty observed among African lineages specifically. Therefore, the primers and probes used in this study were strategically designed to target highly conserved regions of the NS1 gene among both ZIKV lineages as illustrated via our bioinformatic alignment analysis of the most unique NS1 gene sequences.

RT-qPCR is still considered the most reliable technique for diagnosing ZIKV in clinical pathology, and is widely recognised as the gold standard method for detection. However, due to the RNA stability issues at room temperature reported in urine (Tan et al., 2017), methods such as RT-qPCR that require sample storage favour clinical matrices such as plasma, serum or whole blood. For comparison, in serum and plasma, ZIKV RT-qPCRs typically demonstrate detection limits between 1 and 64 copies/reaction (n = 31) (Pessôa et al., 2016). A comparative study of RT-qPCR versus RT-LAMP using urine to detect ZIKV, however, demonstrated a detection limit of 6 copies/reaction (n = 8) for RT-qPCR (Calvert et al., 2017). Our rapid Iso-ZIKV-Dx test showed similar levels of sensitivity, detecting as low as 34 copies/reaction in ZIKV-spiked synthetic urine. The Range of reported ZIKV concentration in urine was observed to be between 0.7 and 220⁶ copies/mL (Gourinat et al., 2015). The vastness of this range is thought to be due to the variability in ZIKV RNA stability, further supporting the need for a rapid sample processing method. Non-invasive clinical sample collections such as urine (Lamb et al., 2016) offer significant advantages for isothermal, POC compatible methods such as RT-RAA, RT-RPA, and RT-LAMP, due to the prolonged period of ZIKV RNA detectable throughout the infection period (Rossini et al., 2017). As such, our rapid Iso-ZIKV-Dx test could be valuable in overcoming the obstacle of testing urine samples by potentially detecting ZIKV directly at the point of sample collection. One study using RT-LAMP reported a LOD of 6.6 copies/reaction in urine (n = 63) (Castro et al., 2018), whilst another study achieved 6 copies/reaction in urine (n = 178) (Calvert et al., 2017). Although both RT-qPCR and RT-LAMP offer slightly improved sensitivity compared to the Iso-ZIKV-Dx test, their application as POC diagnostics in low-resource settings is limited. RT-qPCR requires highly skilled personnel, expensive machinery and laboratory-based sample preparation. While RT-LAMP is known for its simplicity, sensitivity and speed which resulted in wide scale application in low resource settings, it still needs heating machines capable of attaining temperatures of 55–65°C. In contrast, RT-RAA, similarly to RT-RPA can be performed at near-ambient temperature (37–40°C) and moreover has a higher tolerance to PCR inhibitors (Li et al., 2020). Both have previously required laboratory-based purification of RNA, whereas our Iso-ZIKV-Dx test does not require any sophisticated laboratory equipment, providing potential the entire procedure, from sample to result, be performed in low-resource near-patient settings. The ideal rapid POC test should provide a range of detection and diagnostic capabilities, as defined by the REASSURED criteria – Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable to end-users (Land et al., 2019). Our data suggests that the Iso-ZIKV-Dx (sample preparation, RT-RAA, LFD) has potential to meet the REASSURED criteria, providing real-time connectivity through direct analogue reading of the result which could be performed at or near the patient in a low-resource clinic. Insofar as our assay has met pre-clinical and REASSURED criteria, our next phase of evaluation will involve clinical and in-field evaluation of the assay to evaluate clinical efficacy and performance among pathology of ZIKV infections. As our study only involved live MR766 strain of ZIKV, our hope would be to evaluate our ZIKV test with multiple strains within these clinical trials.

Due to manual steps in our assay, it should be acknowledged that screening of large-scale clinical samples would not occur as rapidly as standard RT-PCR assays. However, unlike RT-PCR, our rapid ZIKV test has been designed to be field deployable, requiring minimal resources and minimally trained staff. As a result, the time from sample preparation to diagnostic result is considerably faster than when compared to transportation and processing within central laboratory. Moreover, this particular attribute of our ZIKV test renders it a potential early warning screening tool in the face of a sudden outbreaks. A second potential challenge for our ZIKV test, and indeed many in-field nucleic acid amplification assays (Tang et al., 2016), is the risk of post amplification cross-contamination. A potential solution to this issue is the substitution of dTTP with dUTP in the amplification reactions. This approach has been shown to reduce cross-contamination in LAMP assays (Kil et al., 2015). However, to the authors knowledge, the feasibility of implementing such measure in recombinase-aided amplification assays has yet to be assessed. Therefore, a more feasible alternative is to utilise ‘U-Star’ disposable cartridges (TwistDx^™, 2023b), which would reduce cross contamination occurrences by limiting external exposure of lateral flow strips to the environment.

Rapid detection of arboviruses such as ZIKV play a crucial role in limiting outbreaks that have already emerged or are currently on going. Additionally, surveillance and monitoring can serve to identify early signs of the pathogen and thereby prevent outbreaks through vector control. The study of vector competency and transovarial transmission of arboviruses such as ZIKV thus remain a particular area of focus. Due to factors such as climate change and increased urbanisation (Li et al., 2017) vector competency continues to broaden, further implicating the emergence of novel and existing viruses. A tool with in-field capabilities that provides robust and rapid results could be highly beneficial in epidemiology for monitoring ZIKV. To that end, further studies are warranted to determine if our Iso-ZIKV-Dx is compatible for detection of virus in mosquitoes, as was similarly performed for the TNA-Cifer Reagent E combined with RPA-LFD for detection of dengue virus in Aedes aegpytii mosquitoes. This would allow our Iso-ZIKV-Dx test to be extended beyond diagnostics alone, in potentially offering strategies toward aiding in surveillance and monitoring of viral vectors (Pollak et al., 2023b) and possibly reservoir hosts for arboviruses such as ZIKV.

In conclusion, we developed a rapid, isothermal test for ZIKV that only requires incubation at 39°C and produces a real-time result in 30 min. Pre-clinical evaluation suggests that our Iso-ZIKV-Dx could offer promising innovations for diagnostics in ZIKV endemic areas. We demonstrate that our ZIKV test was able to detect as low as 500 copies/μL of synthetic ZIKV RNA and also, does not detect any other co-circulating arbovirus. We successfully established a simple sample preparation procedure which demonstrated that we were able to completely inactivate one of the highest virulent strains of ZIKV (MR766, African lineage) in 5 min at room temperature, whilst at the same time, extracting sufficient quantities of viral RNA for detection. By combining our simple sample preparation procedure with RT-RAA and Lateral Flow Strip detection, we successfully developed a robust rapid isothermal ZIKV diagnostic. Our data illustrated that our ZIKV test operates five times faster than ‘gold standard’ RT-qPCR and exhibited a detection limit of 34 RNA copies/reaction in spiked synthetic urine. Furthermore, due to the simplistic nature of our ZIKV test, we surmise that the applications of our Iso-ZIKV-Dx extend beyond detection alone, and could potentially provide innovations toward surveillance and monitoring for ZIKV and arboviruses alike.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

RB: conceptualization, data curation, formal analysis, investigation, methodology, resources, supervision, validation, visualization, writing – original draft, and writing – review and editing. NP, JM, and DM: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, and writing – review and editing. JH-P: resources and writing – review and editing. All authors contributed to the article and approved the submitted version.