A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Results
Sensitivity of two different RT-LAMP colorimetric setups. The main components of the RT-LAMP colorimetric reaction are two enzymes (a reverse transcriptase (RT) and a strand displacement polymerase), a colorimetric dye (phenol red) and a primer set (typically composed of six primers) 12 . To detect SARS-CoV-2 using RT-LAMP, we took advantage of the primer set previously validated in vitro by Zhang et al. 24 and tested, on clinical specimens from large cohorts of COVID-19 patients, by several other authors [16][17][18][19] . The primer set (N-A) targeted the N gene, which encodes the nucleocapsid protein and has the most abundant expression of subgenomic mRNA during infection [32][33][34][35] .
We tested two different assay formats. In one format, we used the WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs), which includes all the reagent components with the exception of the primers. In the other, we purchased the separate enzymes (RTx and Bst 2.0) from New England Biolabs, while the reaction buffer with the colorimetric dye (phenol red) were prepared in-house as described by Tanner et al. 12 . The analytical sensitivities of these two setups were evaluated and compared by assaying in parallel tenfold serial dilutions of an in vitro transcribed N-gene RNA standard (IVT RNA), starting from 10 5 copies down to 10 copies (per 20 μL reaction), at tenfold intervals (Fig. 1A). Color changes from pink (negative) to yellow (positive) were registered after a 30-min incubation period at 65 °C, as we found that for extended periods (up to 60 min), negative controls often turned yellowish. The amplification of the IVT RNA was confirmed by agarose gel electrophoresis (Fig. 1B). Ten replicates were analyzed per assay format (Fig. 1C) and IVT RNA dilutions were simultaneously analyzed by RT-PCR (Fig. 1D). The limit of detection (LoD) was reliably found to be between 100-1000 viral copies for the assay using the WarmStart Colorimetric LAMP 2 × Master Mix (Fig. 1A), whereas for that using the separate components the LoD was consistently one Log10 lower (10-100 copies). However, for half of the replicates, a tenfold lower LoD was achieved for both test formats (Fig. 1C). Such stochastic detection efficiency has been reported by others (1), and therefore we defined 100-1000 (WarmStart Colorimetric LAMP 2 × Master Mix) and 10-100 (reaction with separate components) as the robust limits of detection.
For the same serial dilution range, the RT-PCR assay was able to consistently detect down to 10 copies per reaction (mean Ct = 35.22) (Fig. 1D). Compared to RT-PCR, the RT-LAMP assay, depending on the test setup, detected up to ten-or one 100-fold less copies of viral RNA. As the RT-LAMP format using the separate components was consistently more sensitive, we decided to choose this setup in subsequent assays.
Sensitivity and specificity of the colorimetric RT-LAMP assay in detecting viral RNA from the nasopharyngeal fluid. We investigated whether the RT-LAMP assay, using separate components, could be used to accurately detect SARS-CoV-2 in clinical samples. For that purpose, we tested a set of surplus RNA samples extracted from the nasopharyngeal (NP) fluid of 177 individuals who were previously tested for COVID-19, using the standard clinical RT-PCR testing. The samples comprised 126 RNA samples that tested positive (RT-PCR positive, Ct ≤ 40) and 51 samples that tested negative (RT-PCR negative, Ct ≥ 40). As shown in Fig. 2A, after incubation for 30 min at 65 °C, a pink to yellow color change was visualized in all RT-LAMP reactions estimated to have more than 100 RNA molecules present in the reaction (RT-PCR positive, Ct ≤ 32, Fig. 1C), which is in agreement with the observed experimental sensitivity (Fig. 1A). We found two false positives, i.e. two RT-PCR www.nature.com/scientificreports/ negative samples that scored positive in the RT-LAMP assay (Table 1). Thus, the overall specificity of the assay was 96.1% (CI 87-99%) and the sensitivity for samples with Ct ≤ 32 was 100% (CI 94.7-100%). For lower viral load, as measured by RT-PCR (Ct > 32), the assay showed a decrease in diagnostic sensitivity (Table 1, Fig. 2B). Overall, these results indicate a robust performance of the colorimetric RT-LAMP assay across a broad range of purified RNA samples.
Sensitivity of the colorimetric RT-LAMP assay in detecting SARS-CoV-2 in saliva samples. We next optimized our RT-LAMP assay for direct detection of SARS-CoV-2 in saliva samples. To reduce the risk associated with handling samples containing infectious viral particles, saliva was self-collected into a tube and placed at 95 °C for 30 min, for inactivation. This simple heat inactivation procedure has been shown to enable an effective genetic detection of SARS-CoV-2 by other authors 30,31 . After a brief centrifugation step that significantly improved assay reliability (data not shown), the supernatant was diluted with TE, to buffer basal pH differences in saliva, and immediately analyzed or stored at − 80 °C. Lalli et al. have shown that TE is LAMPcompatible and does not affect the assay sensitivity 29 .
We determined the LoD of the assay using both the IVT RNA standard and viral SARS-CoV-2 particles spiked into healthy human saliva to simulate clinical samples. We were able to detect 100 IVT RNA copies (Fig. 3A) and 24 SARS-CoV-2 viral particles (Fig. 3B) per reaction in only 30 min after inactivation, using our RT-LAMP protocol. Since at this sensitivity the assay would detect the typical viral load of SARS-CoV-2 found in the saliva of COVID-19 patients (100-1000 genomes per μL) 36 , we proceeded to test the clinical samples. www.nature.com/scientificreports/ Saliva and matched NP swab specimens of 49 individuals infected with SARS-CoV-2 (as previously determined by RT-PCR) were collected and analyzed by RT-LAMP (saliva) and RT-PCR (NP fluid). In addition, 15 saliva samples of healthy donors were tested by RT-LAMP. Saliva samples were self-collected as described above, and individuals were asked not to eat or drink before testing. A set of 10 of the 49 COVID-19-positive patients was asked to induce salivation by placing the tongue on the salivary sublingual glands. For this group, we could  Table 1). The vertical lines indicate the corresponding 95% confidence intervals.   www.nature.com/scientificreports/ (CI 70-93%) for saliva samples with matched NP swabs with Ct ≤ 28 (Fig. 4C). Reaction volumes, but not saliva amounts, were scaled up to increase the assay sensitivity (Fig. 4B). All saliva samples that were falsely negative by direct RT-LAMP were positive after RNA extraction (Fig. 4B). This step increases by 4-9 times the estimated cost of the assay (1€). Inspired by the work of Yaffe et al. 37 , to keep RT-LAMP affordable, we tested whether we could use silica columns routinely used in molecular biology laboratories to purify bacterial plasmids (mipreps), to extract viral RNA from saliva samples. As shown in Fig. 4D, false negative samples were found to be positive after RNA purification using this method, with an estimated cost per RT-LAMP test of 2€.

Development of an in-house-made colorimetric RT-LAMP.
Aiming to establish a colorimetric RT-LAMP test fully independent of commercial suppliers, we produced the two enzymes needed for the assay and benchmarked them against commercial alternatives using IVT RNA of SARS-CoV-2.
As for the strand displacement polymerase, the gene encoding the large (Klenow) fragment of Geobacillus stearothermophilus was synthesized, with codon optimized for expression in E. coli, and inserted into the pET28 + vector. After a simple 2-step purification protocol, we ended up with 250 μL of Bst LF, at a concentration of 7.6 mg/mL. We next determined the LoD of the assay combining 1 μL of the purified Bst LF, 50-fold diluted (0.15 μg per 20 μL reaction), the in-house-made colorimetric reaction buffer, and RTx (New England Biolabs). This semi-commercial assay consistently detected 1-10 copies of the SARS-CoV-2 N gene per reaction (Fig. 5A). We found that, under our colorimetric conditions, Bst LF outperformed Bst 2.0 (New England Biolabs) ( Fig. 1A and 5A). The amount of the produced Bst LF was enough to perform 12,500 tests at that analytical sensitivity (1-10 copies).
Alternatives to the commercial RTx were also explored. We started by testing several non-thermostable reverse transcriptases (from NZYtech and Roche), but it was not possible to detect LAMP products with an acceptable sensitivity (less than 10 6 viral IVT RNA copies per reaction, data not shown). We also expressed and purified the MashUP RT enzyme (clone available at https:// pipet tejoc key. com) that, when combined with Bst 2.0, was able to detect down to 10 IVT viral RNA copies (Fig. 5B), a LoD similar to the one obtained with the commercial enzyme (Fig. 5A). The MashUP purification consists of a single-step protocol, and sufficient enzyme was obtained to perform 500 assays (0.5 μL corresponding to 3.4 μg/μL were used directly in the reaction).
Finally, we combined the produced enzymes (Bst LF and MashUP) with the homemade colorimetric reaction mixture and assessed (i) the LoD of the assay (Fig. 5C) and, as proof of concept, (ii) whether this setup could identify SARS-CoV-2 N-gene sequences in the RNA extracted from the NP fluid and saliva of COVID-19 patients (Fig. 5D,E). Our in-house-made assay successfully detected SARS-CoV-2 viral sequences in all the three COVID-19 patients' samples (Fig. 5D). Moreover, when using patients' saliva, processed as described above, instead of NP RNA, the assay was also capable of identifying SARS-CoV-2 infected patients (Fig. 5E). Corroborating the work www.nature.com/scientificreports/ of Alekseenko et al. 38 , these results clearly indicate that, using simple expression and purification protocols and home-made buffers, it is possible to establish a colorimetric assay, fully independent of specific supply chains, that efficiently detects SARS-CoV-2 RNA sequences from clinical specimens.
A new colorimetric method for detection of RT-LAMP amplification products. The strong and evident color shift observed with phenol red renders this pH-sensitive dye much preferred for end-point colorimetric detection of LAMP products. However, when the phenol red method is used with crude samples, interference of the sample pH with the assay readout is often observed. Indeed, when establishing the direct RT-LAMP saliva protocol, we had to discard one sample due to the initial acidification of the reaction, as a strong color shift to yellow was observed immediately after sample addition into the reaction mixture. Although several colorimetric indicators are available for detection of LAMP products 3,11-15 , the pale color shift they produce, which is difficult to distinguish by the naked eye, has certainly restrained their wide use. To overcome these limitations, we developed a new colorimetric detection method based on the complexometric indicator, murexide (MX), which forms a complex with divalent zinc (Zn 2+ ) 39 . In the absence of Zn 2+ , MX has a pink color, whereas in the presence of the divalent cation it turns yellow. Because pyrophosphate (PPi) forms a strong complex with zinc, we reasoned that the PPi released during DNA polymerization would displace Zn 2+ cations from MX, inducing a color change from yellow to pink. By mimicking the reaction components in a test tube containing the Zn-MX complex, an evident color shift from yellow to pink was observed immediately after PPi addition (Fig. 6A). Unfortunately, we found that Zn, but not MX, strongly inhibited the LAMP reaction (data not shown), making it impossible to use Zn-MX in a one-step colorimetric assay. Therefore, after an incubation period at 65 °C for 30 min, the tubes were opened and MX (0.5 mM) and ZnCl 2 (2.5 mM) were added to the reaction. To avoid carryover problems due to the post-amplification opening of the tubes, this step was performed in a separate room.
Using the in-house produced enzymes, we first compared the sensitivity of Zn-MX with that of phenol red using IVT RNA (Fig. 6B,C). Like phenol red, Zn-MX showed an evident color difference depending on the presence (pink) or absence (yellow) of LAMP amplification. Moreover, the method enabled the clear detection of SARS-CoV-2 in crude saliva samples of nine COVID-19 positive patients (Fig. 6D), whereas with phenol red the viral genetic material was only identified in eight of these samples (Fig. 6E).

Discussion
Widespread testing, preferably based on different supply chains, is required to curtail the ongoing pandemic. To address that need, we have in this work evaluated a LAMP-based colorimetric test to rapidly detect SARS-CoV-2 in RNAs extracted from patient's NP fluids, using a single tube protocol. The assay also allows for detection of the virus directly from patient´s saliva with minimal processing and increased protection of the testing personnel. We also showed that using simple expression and purification protocols together with homemade buffers, it is possible to establish an inexpensive colorimetric assay, fully independent of specific supply chains, that efficiently detects SARS-CoV-2 RNA.
While not as sensitive as the reference diagnostic method for COVID-19, RT-PCR, the simplicity, turnaround time and low associated costs of our test make it an attractive and efficient tool for infection control. According to existing literature, the LoD of the test is sufficient to identify individuals with viral titers high enough to transmit the virus (300-1000 viral copies per μL) 27,40,41 . This test sensitivity is understood to be adequate for surveillance and screening of the asymptomatic population. The availability of such a testing solution is therefore of great importance, as infectiousness peaks occur before or at the symptoms onset 42 . Indeed, the rapid evolution of COVID-19 has been partly attributed to transmissions occurring through people who are presymptomatic or asymptomatic 43 ; efforts to implement a strategy enabling communities to test asymptomatic individuals require urgent attention and testing tools to support it.
Several authors have recently shown that the use of different primer sets boosts RT-LAMP sensitivity, possibly due to better primer efficiency and/or higher target abundance. Also different saliva treatment protocols, combining certain chemicals and proteinase K, have been shown to improve SARS-CoV-2 detection in saliva samples 16,21,22,28,29,31 . Thus, we reason that there is still room to improve the sensitivity of our test.
As expected, RNA extraction greatly improved the saliva test sensitivity, by increasing the concentration of the viral sequences in the sample. Many other authors have reported similar findings 21,22,44,45 and extensive efforts have been made to establish alternative protocols that enable RNA enrichment using fast and inexpensive methodologies 21,22 . Here we showed that RNA extraction using common plasmid DNA extraction columns is an economical way to concentrate and purify viral RNA from saliva samples.
To eliminate the impact of acidic saliva samples on the test readout, we have developed a new colorimetric reading, independent of changes in the pH of the LAMP reaction. The method uses a divalent zinc salt (such as ZnCl 2 ) and the complexometric indicator murexide to form a transient complex (Zn-MX). The presence of PPi, a by-product of the reaction, is indicated by the indicator displacement method, since Zn 2+ forms a more stable complex with PPi and thus releases murexide. As the presence of zinc inhibits the amplification reaction, the metal can only be added at the end of the reaction, thus requiring the tubes to be opened post-amplification. This procedure poses the threat of carryover contaminations, very common in LAMP reactions 46,47 , which leads to false positives. We therefore do not anticipate that the Zn-MX method, in its current formulation, can be used routinely in a molecular diagnostic laboratory. However, the molecular saliva-based tests currently available for COVID-19, whose workflow already demands opening the LAMP reaction tube, may certainly benefit from our method 48 . Additionally, the method can be safely used in closed systems using microfluidic diagnostic cartridges, similar to the one recently described by Ganguli et al. 49  www.nature.com/scientificreports/ Overall, this study, while addressing some of the testing bottlenecks imposed by the current pandemic, reinforces RT-LAMP as a powerful method for sensitive and inexpensive molecular diagnosis of COVID-19 that can be easily deployable in limited resource settings.

Materials and methods
Sample collection, processing and storage. Clinical specimens were collected at Hospital das Forças Armadas and processed in Laboratório de Bromatologia e Defesa Biológica (Unidade Militar Laboratorial de Defesa Biológica e Química). Saliva specimens (~ 1 mL) were self-collected into sterile tubes (50 mL or 1.5 mL). Patients were asked not to eat or drink before testing. NP swab-matched samples were collected in parallel and placed in 3 mL Universal Viral Transport Media. Tubes containing clinical specimens were decontaminated with an alcohol-based solution and identified. After collection, samples were kept at 4 °C for 2-4 days or processed immediately. Samples were inactivated by incubation at 95 °C for 5 min (NP swabs) or 30 min (saliva samples). Salivas were centrifuged at 5000g for 5 min and 200 μL of the supernatant were diluted in TE 10 × (1 ×, final concentration) and frozen at − 80 °C until analysis. The saliva pellets were also frozen. www.nature.com/scientificreports/ RNA extraction from clinical samples. Total viral RNA was extracted from 140 μL of NP deactivated samples using Viral RNA Mini Kit (QIAGEN) and eluted in 60 μL of RNAse free water, to ensure the RNA elution buffer has no impact of pH in RT-LAMP reactions. As for saliva samples, total RNA (from the pellets) was isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer's instructions or the LogSpin method 37 as described by the authors. Briefly, the pellet was mixed by vortexing with 250 μL a guanidine-based solution (8 M guanidine-HCl, 20 mM MES hydrate and 20 mM EDTA). The mixture was centrifuged at 16,000g for 5 min and the supernatant was mixed with 250 μL of 100% ethanol, and loaded into the ZR plasmid miniprep columns (ZYMO Research). The column was washed twice with 450 μL of 3 M Na-Acetate and 320 μL of 70% ethanol. RNA was eluted in 30 μL of water.

SARS-CoV-2 RNA standard.
To prepare the SARS-CoV-2 RNA standard, the N gene was amplified from the plasmid 2019-nCoV_N_Positive Control (Integrated DNA Technologies) with a T7-promoter-containing primer (5′-TAA TAC GAC TCA CTA TAG Gatgtctgataatggaccccaaaa-3′) and the reverse primer (5′-ttaggcctgagttgagtcagc-3′), then the product was in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis Kit, NEB), according to the manufacturer's instructions. Template DNA was removed using Turbo DNase (Invitrogen) and RNA was then purified using the RNeasy Mini Kit (QIAGEN). Standard RNA copy numbers were calculated from concentration measured using Take3 from Epoch from Biotek and confirmed using a Ultro-spec2100pro (Amersham Biosciences). Zinc-murexide colorimetric method. All reagents obtained from commercial sources in analytical grade. Analytical solutions were prepared in ultrapure grade water from a Milli-Q system, as follows: MOPS buffer pH = 7.4 at 20 mM, magnesium chloride (MgCl 2 ) at 47.5 mM, zinc chloride (ZnCl 2 ) at 47.1 mM, sodium pyrophosphate (Na 4 P 2 O 7 ) at 50 mM, ATP at 25 mM, and murexide (MX) at 0.5 mM. The MX solution was prepared immediately before use or otherwise kept frozen. Samples (1 mL), simulating the starting conditions of the RT-LAMP assay, contained 8 mM of magnesium chloride and 1.4 mM of ATP, buffered at pH = 7.4 with 10 mM of MOPS. To these samples were added a few drops of a MX solution to attain a suitable color intensity, which turned the samples violet, indicating that MX was in the free form. Addition of ZnCl 2 at 8 mM to the samples rendered them orange, indicating a change of the indicator to its complexed form. Finally, titration of pyrophosphate into the samples caused a color change back to pink from ca. 16 mM, pointing to a release of the indicator caused by the binding of zinc to pyrophosphate. These color changes demonstrated that MX is a suitable colorimetric indicator to detect pyrophosphate in presence of magnesium (Supplementary Figure S1).

Virus isolation and spike experiments. SARS-Cov
Expression and purification of Bst1 klenow. The gene encoding the klenow fragment of Bst1 (UniProt sequence P52026, residue 291-876) was synthesized (codon optimized for expression in E. coli) and inserted into the pET28 + vector with nucleotides encoding an N-terminal 6HisTag and a TEV cleavage site (Genescript). The resulting plasmid was used for transformation of E. coli BL21 (DE3) pLysS. Overnight pre-cultures (10 mL) were grown at 37 °C and used to inoculate 1 L Power Broth (Molecular Dimensions) with 100 µg mL −1 ampicillin and 50 µg mL −1 kanamycin. The culture was grown at 37 °C until OD 600 reached 0.7-0.9. At this point, the culture was moved to 18 °C and expression was induced by adding 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After overnight expression, the cells were harvested by centrifugation at 7548g for 30  www.nature.com/scientificreports/ NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 1 mg/mL DNase I, 1 mg/mL lysozyme and one tablet EDTA free proteinase inhibitor (Roche)] and subjected to multiple freeze/thaw cycles (alternating room temperature water bath and liquid nitrogen). The lysate was cleared by centrifugation at 48,385g for 30 min at 4 °C and the supernatant was carefully removed and added to a 5 mL HisTrap HP purification column (Cytiva), previously equilibrated in buffer A (150 mM NaCl, 50 mM Tris-HCl pH 7.5). The protein was eluted over a 10 CV gradient from 5 to 100% buffer B (buffer A with 0.5 M Imidazol). Fractions containing Bst1 Klenow were identified by SDSPAGE, pooled and dialyzed overnight in 2 L buffer A in the presence of TEV (1:20) at 4 °C. The dialyzed and TEV cleaved protein was thereafter added to a 5 mL HisTrap column and eluted in the Flow Through (due to the removal of the HisTag). The HisTag free Bst1 Klenow was thereafter desalted through a HiTrap desalting column (Cytiva) followed by a final purification step on a 5 mL HiTrap Heparin HP column (Cytiva), to remove eventual residual DNA bound to the protein. The protein was eluted over a 10 CV gradient in buffer B2 (buffer A and 1 M NaCl). Fractions containing BstKlenow was identified by SDSPAGE, pooled, concentrated to 7.6 mg/ mL by Amicon Ultra-15 concentration filter units (10 kDa cut off, Millipore) and stored at − 80 °C.
Expression and purification of MashUP reverse transcriptase. The MashUp RT plasmid (kindly provided by https:// pipet tejoc key. com), which encodes a modified Feline Leukemia Virus Reverse Transcriptase (RT) and plasmid pGTf2 that encodes for a chaperon were co-transformed into E. coli BL21 (DE3) competent cells and plated on L-Broth (LB) agar (NZYTech) plates containing 50 μg/mL kanamycin and 30 μg/mL chlorophenicol. Overnight cultures were inoculated with fresh transformants and grown at 37 °C, 150 RPM in LB selective medium. Subsequently, the overnight culture was diluted 100 × in Terrific Broth (TB). The cells were grown at 37 °C, 150-170 RPM until OD 600 nm reach 0.8-1.0. Then, temperature was lowered to 18 °C and protein expression induced with 0.5 mM IPTG and 5 ng/mL tetracycline, for the RT and chaperone, respectively, and grown additionally for 18 h at 18 °C. The cells were harvested by centrifugation at 4500×g for 10 min at 4 °C and resuspended in MashUp-RT lysis buffer (25 mM Tris-HCl pH 8, 300 mM NaCl, 10% glycerol, 40 mM imidazole, 0.5% Triton X-100), supplemented with one tablet of Complete EDTA-free protease inhibitor cocktail (one unit per 1 L). Cells were disrupted by French press and the extract was clarified by centrifugation at 100,000×g, 90 min at 4 °C. The supernatant was loaded into an IMAC column equilibrated with lysis buffer. The column was washed with the same buffer and the adsorbed proteins were eluted from the column with 25 mM Tris-HCl pH 8, 300 mM NaCl, 10% glycerol, 500 mM imidazole, 0.5% Triton X-100. Protein was concentrated in an Ammicon ultrafiltration device with a 30 kDa cutoff. Total protein present in the sample was quantified by BCA assay (6.8 mg/mL) using albumin as a standard.
Ethics statement. The Director of the Hospital das Forças Armadas (HFA) approved all experimental procedures, which were carried out following the guidelines of the HFA Ethics Committee. The study was conducted in accordance with the European Statements for Good Clinical Practice and the declaration of Helsinki of the World Health Medical Association. Informed consent was obtained from all participants.