Biological samples and experimental design
The clinical samples (urine and cervical scraping) were obtained from routine Papanicolaou (Pap) tests at the University Medical Center of the Autonomous University of the Morelos State (UAEM). All samples analyzed in the present study were obtained with informed consent and all experimental procedures were approved by the local ethics committee. During the collection of the biological samples, no additional or invasive procedures were performed on the patient. In this first experimental pilot stage, six samples analyzed were randomly taken from a batch of positive and negative HPV infections, 3 for each group. From every sample, the relevant clinical-epidemiological data were obtained, such as general data, exposure to risk factors for HPV infection, and frequency of consumption of maize and derivatives. To avoid bias in the result, FB1 levels (MARC author) and molecular detection of HPV (VR author) were performed by two different people in a blinded manner.
DNA extraction
The concentration of cells was performed by centrifugation. The cells contained in 1.5μL of the preservation buffer were centrifuged in a microcentrifuge at 5,000 g for 5 minutes. The supernatant was discarded. 300μL of nuclear lysis buffer (100mM NaCl, 10mM Tris HCl, 25mM EDTA and 0.5% SDS) and 30 μL proteinase K were added and incubated in a thermal block at 56 ° C for 12 hrs. Next, 300μL of 5M ammonium acetate was added to precipitate the proteins and the tubes were left at -20 °C for 30 minutes. Subsequently, they were centrifuged at 5,000 g for 30 minutes. After, 600μL of isopropanol was added, incubated for 12 hrs at -20 ° C and centrifuged at 5,000 g for 15 minutes, the supernatant was decanted. The precipitate (DNA) was washed with ethanol and centrifugation at 3,000 g for 5 minutes. The procedure was repeated three times. Finally, the ethanol was decanted, and the DNA was dissolved with 30μL of nuclease-free water. The DNA was quantified in a NanoDrop 3300.
Molecular detection and HPV genotyping
Molecular detection and genotyping of HPV was performed using genomic microarrays, using the CLART® HPV2 method (Clinical Array Technology Genomica). This system automatically detects 35 HPV genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89). The system is based on the amplification of specific fragments of the viral genome and its subsequent hybridization with specific probes for each genotype of HPV printed on the microarray. The detection was carried out by amplifying a fragment of 450 bp within the L1 region of the virus, highly conserved among the different types of HPV. However, this region has enough variations to differentiate each type of virus with specific probes. The detection system with CLART® human Papillomavirus 2 is based on the precipitation of an insoluble product in those areas of the microarray in which the hybridization of the amplified products with the specific probes takes place. During PCR, the amplified products are labeled with biotin. After amplification, these products are hybridized with their respective specific probes that are immobilized in specific and known areas of the microarray, followed by an incubation with streptavidin-peroxidase. For visualization, the PCR products are denatured at 95 °C for 10 minutes and the procedure recommended by the manufacturer is followed. The preparation of the microarrays, the hybridization process, and the incubation were carried out following the manufacturer's recommendations. At the end of the process the plate was placed in the CAR to take the images of all the wells and be analyzed automatically. The processing of the data was done automatically.
Detection of Fumonisins in urine
Samples pretreatment
Urine samples were treated as previously reported [10] with minor modifications, urine was centrifuged for 10min, the supernatant was diluted 1:1 with distilled water. A standard solution of FB1 (1mg/mL) was prepared and added to each sample as internal control (0.1 ng/mL). Samples were cleaned up into the C18 Oasis cartridges, MAX 3cc, as reported. Once the extraction solution (formic acid 2% in methanol) was individually obtained (2mL), it was dried in a vacuum centrifuge and then reconstituted in 200 μL of methanol / water in a 1:1 ratio. After the last step, the samples were placed in MS certificate vials. Positive and negative (control) samples were prepared by the same conditions.
UPLC-MS-QTOF analysis
The determination of FB´s or hydrolyzed derivatives was performed using the ultrahigh resolution liquid chromatography (UPLC) technique coupled to a mass spectrometer quadrupole-time of flight (MS-QTOF) in an Infinity II Agilent equipment with a G6545A Agilent QTOF spectrometer with an ESI source. Injection volume was 8 µL in a C18 column (SORBAX 2.7x100mm, 1.8 µM), a gradient elution at 25°C was carried out with a mobile phase at 0.3 mL/min of H2O (solvent A), and acetonitrile (solvent B) both with formic acid 0.1% from 25 to 75% of solvent B on 11 min. Initial conditions were recovered before each injection. Mass conditions were, temperature for gas and sheath gas at 180 and 350 °C respectively, voltages for capillary and fragmentor were 3000 and 100 V respectively, an acquisition in positive mode from 400-900 m/z was recorded. Positive, negative samples (control), and blank were analyzed by duplicate.
Data analysis
Data treatment was carried out on Profinder B.08 software using the extraction by formula for FB1 (C34H59NO15) besides its hydrolyzed forms PHFB1 (C28H53NO10) and HFB1 (C22H47NO5), as well as FB2 or its isomer FB3 (C34H59NO14) and its hydrolyzed (C28H53NO9, C22H47NO4), all aducts were allowed (+H, +Na, +K, +NH4) and neutral loss of H2O, compounds with more than 1000 counts were extracted, rt and m/z tolerance were 0.15 min and of 10 ppm, respectively.
Statistic analysis
All of the analyses described below were carried out using R Statistical Software version 3.6.2. The one-tailed Fisher's exact test was performed to the clinical-epidemiological variables and the FB1. The contingency tables with at least one structural zero are added 0.5 to each cell entry to measured the effect size with Cramer's V. We used the following function CramerV(Table2X2, conf.level=0.95,method = c("fisheradj")) of R package DescTools (Signorell et mult. al., 2019). A Cramer's V between 0 and 0.3 is regarded as weak, one between 0.3 and 0.70 as medium, and greater than 0.7 is considered strong.
Subsequently, a principal component analysis (PCA) was analyzed using numerical variables (age and numbers of sexual partners) and dichotomy variables (vaccine and FB1) to determine possible clusters. Next, the scores of the first principal components were clustered using a K-means clustering. PCA and K-means clustering analyses were conducted using R package FactoMineR [11], factoextra [12] and ppclust [13].