## Introduction

Zika virus (ZIKV) is an enveloped virus belongs to the genus Flavivirus in the family Flaviviridae. Other notable viruses in this genus are West Nile virus (WNV), dengue virus (DENV 1–4), yellow fever virus (YFV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Rocio virus (ROCV)1,2,3. The genome of ZIKV consists of a single-stranded positive-sense RNA molecule (+ ssRNA), with approximately 11 Kb in length and has a single open reading frame (ORF) which is flanked by 5′ and 3′ untranslated regions (UTRs), respectively4. ZIKV genome encodes three structural proteins [capsid (C), pre-membrane (prM) and envelope (E)] and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5)5.

ZIKV was first isolated in 1947 during a yellow fever study in the Zika forest, Uganda6. In the 60 years that followed its discovery, few cases of ZIKV infection were reported in humans and the virus has remained an obscure pathogen. In April 2007, the ZIKV caused an outbreak of disease in Yap State, Federated States of Micronesia, and subsequently, spread rapidly across the Pacific causing outbreaks in French Polynesia, the Cook Islands, Easter Island, and New Caledonia7,8,9,10,11,12,13. The ZIKV was then introduced to Brazil, possibly in 2013, and in May 2015, the first case of ZIKV infection was reported in Brazil14,15,16. The virus then spread rapidly through the Americas and became a global health concern, causing the largest ZIKV epidemic recorded to date14. Besides Zika fever, the virus was responsible for a severe outbreak of microcephaly and other congenital abnormalities in neonates born to mothers infected during pregnancy, as well of many cases of neurological disorders such as Guillain–Barré syndrome (GBS) in infected patients1,15,17,18,19,20.

ZIKV is primarily transmitted to humans through the bites of infected female mosquitoes from the genus Aedes, mainly Aedes aegypti and Aedes albopictus, although other species of mosquitoes may also be involved in the transmission chain21,22,23,24,25,26,27. In addition to vector-borne transmission, ZIKV can be transmitted through sexual intercourse and blood transfusions, as well as during pregnancy28,29,30,31. In most patients, infection by ZIKV is asymptomatic and, when present, the clinical manifestation is characterized by the presence of fever, rash, myalgia, arthralgia and headache, which overlaps significantly with the features of DENV and chikungunya (CHIKV) infections32. Since these clinical symptoms are shared with infections from other arboviruses, diagnosing ZIKV infection using clinical indications alone is difficult. Therefore, accurate diagnostic platforms are important to identify and differentiate the etiologic agent of illness1,33.

In the clinical laboratory, ZIKV infection can be diagnosed using serology and molecular methods. Serological assays available include plaque reduction neutralization tests (PRNT), enzyme-linked immunosorbent assays (ELISA), and lateral flow assays to detect IgM or IgG antibodies7,34. However, cross reactivity with other flaviviruses, such as DENV, in serological assays limits their utility in regions where other flaviviruses circulate. Thus, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard molecular method for ZIKV diagnosis and has been successfully use to detect ZIKV in serum, urine, saliva, semen and other body fluids7,35,36,37,38,39,40. Nevertheless, RT-qPCR is expensive, requires technical expertise, reliable access to electricity and utilizes sophisticated equipment for amplification and detection of viral genome. These drawbacks negatively impact the establishment of effective disease control programs caused by ZIKV, especially in low-resource settings. Moreover, the COVID-19 pandemic has worsened this situation in Brazil since most diagnostics resources have been directed for SARS-CoV-2 detection41.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, simple and robust tool for the rapid amplification of nucleic acid at a single and fixed temperature42. The assay has many advantages compared to other molecular methods including low cost, convenience, high sensitivity and specificity, making it a powerful method for point-of-care (POC) diagnostic43. Importantly, the isothermal nature of RT-LAMP reactions means that they can be performed without expensive equipment and, moreover, the colorimetric output means results can be determined with the naked eye43. Due to these advantages and potential for POC applications, many LAMP platforms for ZIKV have been developed since ZIKV emergence in the Americas44. However, many of these assays still require sophisticated equipment, expensive supplies or laborious protocols for ZIKV amplification and detection, which limits its applicability in low-resource settings.

We previously developed a one-step RT-LAMP assay for detection of ZIKV in mosquito samples that addressed the above-mentioned limitations of ZIKV LAMP assays45. Our strategy is based on a close-tube, one-step protocol and does not require RNA extraction from the biological specimens. With these features in-place, our main goal here was we develop and validate a one-step RT-LAMP assay for ZIKV detection in different human specimens, including serum, urine, saliva, and semen. Detection of the virus was achieved in as little as 20 min without RNA extraction or any pretreatment of the patient samples. We then validated the performance of this assay for using patient samples collected in Pernambuco State, Brazil, which was the epicenter of the last ZIKV epidemic. The results validated our assay for POC applications for human diagnosis and does not require expensive equipment and can be performed by an operator with minimal technological expertise. This RT-LAMP technology represents a rapid, sensitive, and specific assay for ZIKV diagnosis and has the potential to improve diagnostic capabilities and surveillance actions in remote areas and countries with limited laboratory infrastructure.

## Results

### Optimization of ZIKV RT-LAMP reaction conditions

To optimize ZIKV RT-LAMP assay performance for human samples, we screened reaction conditions such as temperature, incubation time, Mg2+ concentration, dNTPs concentration, enzyme concentration and primer sequences. We also evaluated whether all primers, including the external (F3 and B3), internal (FIP and BIP) and loop (LF and LB) primers were required for successful amplification of target RNA. Initially, we performed a time course series and found that ZIKV genome amplification (105 PFU) occurred with incubation time as short as 20 min (Fig. 1A), but 40 min of incubation provided more consistent results for low titer samples. Positive amplification was detected at incubation temperatures ranging from 59 to 72 °C (Fig. 1B). Based on these results, we carried out the other assays at the temperature of 72 °C for 40 min to maximize the chance of virus detection (sensitivity) and the specificity, respectively. Based on reagent titrations, the optimal concentrations for the Mg2+, dNTPs, and Bst 3.0 DNA polymerase were determined to be 8 mM, 1.8 mM, and 4U of the enzyme, respectively (Fig. 1C–E). All six primers were required for amplification of the RNA target, since removal of either backward and forward inner (FIP/BIP) or loop primers (LF/LB) failed to produce a positive reaction (Fig. 1F). These optimized parameters were used for subsequent experiments described below.

### Detection of ZIKV in virus-spiked human samples

To evaluate the ability of the RT-LAMP assay to detect ZIKV in clinically relevant specimens, we spiked urine, serum, saliva and semen obtained from healthy, consenting, adult volunteers with a high viral load (1 × 106 PFU/mL) followed by a 1:1000 dilution in water to achieve a low viral load (1 × 103 PFU/mL). Samples were infected for 1 h at 37 °C and then directly assayed by RT-LAMP assay without RNA extraction using the same volume of human biofluids. ZIKV was detected in all spiked samples, regardless of the viral dose. In semen samples, the high and low viral load gave similar results as detected by naked eye and UV irradiation, but the low viral load spike gave better amplification then the high viral load as detected by agarose gel analysis. As expected, non-template control (NTC) samples (water) and negative control (human biological sample uninfected) tested negative (Fig. 2A–I). RT-LAMP results were compared to RT-qPCR, through which the cycle quantification (Cq) values46 were (13.6; 13.8; 13.0; 13.1) and (24.7; 24.6; 24.3; 24.5), for high viral and low viral load in urine, serum, saliva and semen, respectively.

### Analytical specificity of ZIKV RT-LAMP assay

In order to evaluate the specificity of the RT-LAMP primers to detect only ZIKV, we assayed human serum spiked with key endemic arboviruses circulating in Brazil, including DENV-1 (PE/97-42735), DENV-2 (PE/95-3808), DENV-3 (PE/02-95016), DENV-4 (PE/10-0081), YFV (17DD), and CHIKV (PE2016-480) (Table 1). RT-LAMP specifically detected ZIKV as determined by naked eye analysis, visual observation under UV light or agarose gel electrophoresis (Fig. 3). These results were confirmed by RT-qPCR, in which the Cq value for the ZIKV sample was 12.4 (Fig. 3). A common problem with highly sensitive RT-LAMP assays is cross-contamination. To prevent this, we added 1 μL of 1:10 dilution of SYBR Green I dye diluted in RNase-free water to the center of the tube lid caps before the reaction and mixed afterwards47. This reduces the potential for the introduction of contamination and in work performed here, no contamination was seen observed using the one step, closed tubes RT-LAMP strategy (data not shown).

### Analytical sensitivity of ZIKV RT-LAMP assay

To evaluate the analytical sensitivity (limit of detection—LOD) of the assay, RT-LAMP was performed in human serum spiked with varying concentrations (105 PFU to 10–7 PFU) of ZIKV. Spiked samples were directly assayed by RT-LAMP without RNA extraction. RT-LAMP was able to detect a broad range of virus concentration (from 105 to 10−6 PFU). Then, viral RNA of same dilutions were extracted and assayed by the gold standard RT-qPCR test. The analytical sensitivity of RT-qPCR was only observed down to 101 PFU with a Cq value of 34.2 (Fig. 4). The experiments were independently repeated 10 times to allow probit regression analysis to accurately determine the limit of detection of RT-LAMP. The limit of detection of RT-LAMP at 95% probability was − 1.07 log10 PFU of ZIKV with confidence interval from − 1.93 to 0.49 (Table 2 and Fig. S5), which is 100-fold more sensitive than RT-qPCR for ZIKV. Similar analytical sensitivity results were also obtained in urine, saliva and semen (data not shown).

### Diagnostic performance and cost of the RT-LAMP assay for detection of ZIKV in patient samples

We next validated the RT-LAMP assay using clinical samples obtained from patients with suspected arbovirus infection. A total of 100 serum samples double blinded (20 positive and 80 negative for ZIKV by RT-qPCR) (Table S1) were used in the experiment. The Cq value in these samples ranged from 21.0 to > 40.0 and samples with Cq values of ≤ 38.0 in triplicate wells were considered positive for ZIKV by RT-qPCR. The RT-LAMP assay detected ZIKV in 25 samples, including five samples which had been determined negative by gold standard method, whereas 75 were deemed negative by ZIKV RT-LAMP (Fig. 5).

The diagnostic performance of this assay for detection of ZIKV was inferred by statistical analysis of several parameters using RT-qPCR assay as reference test. The RT-LAMP assay had a clinical sensitivity of 100% (95% CI 83.16% to 100.00%) and clinical specificity of 93.75% (95% CI 86.01% to 97.94%). The overall ZIKV prevalence in the samples was 20.00% (95% CI 12.67% to 29.18%). The positive predictive value of this assay was 80.00% (95% CI 63.13% to 90.33%) and negative predictive value was 100%. The overall accuracy of the ZIKV RT-LAMP assay was 95.00% (95% CI 88.72% to 98.36%) (Table 3), highlighting the diagnostic positive features of the RT-LAMP assay for rapid detection of ZIKV in patient samples.

To confirm the identity of positive samples by RT-LAMP for ZIKV detection in human samples, we sequenced a positive sample of human serum by the Sanger method. Analyzes obtained from the sequenced amplicons and BLAST analysis demonstrated that ZIKV RT-LAMP amplicons match 100% with virus circulating in Brazil (Fig. 6), proving the specificity of the RT-LAMP for detection only ZIKV. Together, these results suggested that RT-LAMP protocol described here is highly specificity for detection of ZIKV.

### Statistical analysis

Graphs were generated using the GraphPad Prism Software version 5.01 for Windows (GraphPad Software, La Jolla, California, USA). Estimates of sensitivity, specificity, ZIKV prevalence, positive predictive value, negative predictive value and overall accuracy of the ZIKV RT-LAMP assay were calculated based on the results from 100 samples using the web-based software MedCalc’s Diagnostic Test Evaluation Calculator (https://www.medcalc.org/calc/diagnostic_test.php). This analysis was based on the results from 100 human samples previously tested by RT-qPCR. A probit regression analysis’ was performed to calculate the limit of detection of the ZIKV RT-LAMP using MedCalc software (version 19.2.0, MedCalc Software, Ostend, Belgium).

### Ethical statement

This study was approved by the Fiocruz Pernambuco Institutional Review Board (IRB) under protocol number CAAE 67404117.7.0000.5190 and was conducted following the ethical principles for medical research involving human subjects developed by World Medical Association Declaration of Helsinki. Informed consent was obtained from healthy volunteers, but was waived by the IRB for diagnostic samples suspected of ZIKV. Human serum samples were collected from patients who presented clinical symptoms compatible with Zika in the State of Pernambuco, Brazil.