In the present study, a RT-LAMP assay was developed for isothermal detection of ZIKV RNA in simulated clinical specimens. The RT-LAMP assay could detect all the ZIKV strains used in this study without cross-reacting with a number of other common arthropod-borne viruses including all four DENV serotypes, JEV, LGTV, SINV, CHIKV and GETV. The RT-LAMP assay was sensitive, specific and useful for broad coverage detection of both the Asian and African ZIKV lineages.
Recently, several research groups have developed RT-LAMP assays for the detection of ZIKV by targeting the different regions of the ZIKV genome with excellent sensitivity and specificity [31-43]. They however, designed the primers for the RT-LAMP assays based only on a single lineage of ZIKV, which is either Asian [33-37, 39-40] or African [32] lineage. Many studies designed the RT-LAMP primers based primarily on the sequences of the Asian ZIKV since the recent epidemics in the Pacific islands and Americas [5-7, 11] were caused by ZIKV from the Asian lineage [53]. Nonetheless, the possibility of re-emergence of the African lineage should not be excluded. Chotiwan et al. (2017) suggested using two sets of RT-LAMP primers, one for Asian ZIKV and another one for the African ZIKV [31]. The two-tube RT-LAMP assay, however, would definitely increase the cost of diagnosis. On the other hand, Kurosaki et al. (2017) combined two sets of RT-LAMP primers (total 11 primers) in single tube for simultaneous detection of Asian and African ZIKV [41]. Nonetheless, the use of more than six primers is often not preferable in RT-LAMP assay to avoid false positive due to a primer self-amplification [54]. Recently, Escalante-Maldonado et al. (2019) and Bui et al. (2020) developed RT-LAMP assay using a set of six primers based on the 64 and 130 ZIKV sequences retrieved from GenBank, respectively [42-43]. Here, we designed a set of six RT-LAMP primers without critical mismatches with at least 95% of the 463 ZIKV genome sequences for broad coverage detection of both the Asian and African ZIKV lineages in a single-tube assay.
The detection limit of the RT-LAMP assay (~4 copies of ZIKV RNA) was comparable to that of ZIKV RT-LAMP assays previously reported; the detection limits ranged from 1 to 111 copies of ZIKV RNA [31, 32, 35]. In this study, the ZIKV RNA copy/infectious particle ratio in the simulated clinical specimens ranged from 102 to 103. Similar ZIKV RNA copy/infectious particle ratios have also been previously reported [55, 56]. Thus, the RT-LAMP assay is a potentially useful diagnostic tool for detecting viremic patients who are potentially contagious. Early detection of the viremic patients would permit immediate execution of proper disease control measures such as deploying insecticidal spraying and activating community-based mosquito control activities. Early diagnosis would also help clinicians in providing proper supportive treatment and counselling of Zika patients without needing to prescribe unnecessary medications.
Zika virus has been detected in human blood, saliva [57, 58], urine [59-61], semen [62, 63] and breastmilk [64]. Among all types of specimens, blood, saliva and urine are the common specimens used for Zika diagnosis. It has been reported that the viral titers in the patient’s blood, during the acute infection, ranged from 103 to 106 RNA copies/ml [65]. The viremic phase of ZIKV infection, however, is short with less than 2 to 5 days after fever onset [57, 59, 60]. The use of saliva has been shown to improve the detection of ZIKV RNA but did not enlarge the window of detection [57]. In contrast, shedding of ZIKV in urine persists for ~2 weeks [60] and sometimes it can be up to >20 days after fever onset [59]. The use of urine specimen, therefore, is a practical way to enlarge the window of ZIKV detection. Here, we demonstrated that the RT-LAMP assay detected the simulated clinical specimens (serum, saliva, and urine) with viral load of as low as 10 PFU/ml, which is equivalent to approximately 103 to 104 RNA copies/ml. This detection sensitivity would be sufficient to diagnose viruric patients if their urine samples are collected within 10 days after fever onset considering viruric patients usually has viral load of >103 RNA copies/ml during the first 10 days of ZIKV infection [59].
There are, however, several limitations to our study. The performance of the RT-LAMP assay has not been validated on any real clinical samples. The evaluation was done in a very small cohort of simulated clinical samples (n = 24), in which the virus titres could be controlled. Therefore, there is no assurance of similar performance in filed diagnostic settings.