PCR-RFLP and FTIR-based detection of high-risk human papilloma virus for cervical cancer screening and prevention.

BACKGROUND: Approximately 70% of cervical carcinoma cases show the presence of high-risk Human Papilloma Virus (HPV), especially HPV-16 and HPV-18, and can be used to stratify high risk patients from low risk and healthy. Currently, molecular biology techniques such as polymerase chain reaction (PCR) are used to identify the presence of virus in patient samples. While the methodology is highly sensitive, it is labor intensive and time-consuming. Alternative techniques, such as vibrational spectroscopy, has been suggested as a possible rapid alternative. Therefore, in this study, we evaluate the efficiency of cervical fluid Fourier Transform Infrared spectroscopy (FTIR) in patient risk stratification informed by PCR. METHODS: Cervical fluid samples (n = 91) were obtained from patients who have undergone routine Papanicolaou (Pap) test. Viral genome was identified and classified as high/low-risk by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). FTIR spectra were acquired from samples identified by PCR-RFLP as No-HPV (n = 10), high-risk HPV (n = 7), and low-risk HPV (n = 7). RESULTS: Of the 91 samples, was detected the viral genome by PCR in 36 samples. Of these 36 samples, nine samples were identified to contain high-risk HPV (HR-HPV) and nine samples were found to have low-risk HPV (LR-HPV). The FTIR spectra acquired from No-HPV, LR-HPV, and HR-HPV showed differences in 1069, 1437, 1555, 1647, 2840, 2919, and 3287 cm-1 bands. Principal Component Analysis (PCA) showed distinct clusters for No-HPV and HR-HPV and No-HPV and LR-HPV, but there was significant overlap in the clusters of HR-HPV and LR-HPV. PCA-Linear Discriminant Analysis (PC-LDA) after Leave One Out Cross Validation (LOOCV) classified No-HPV from HR-HPV and No-HPV from LR-HPV with 100% efficiency in the 1400-1800 cm-1 spectral range. LOOCV classifications for LR-HPV and HR-HPV from each other were 71 and 75%, respectively, in the 2800-3400 cm-1 spectral range. CONCLUSIONS: The results highlight the high sensitivity of PCR-RFLP in HPV identification and show that FTIR can classify samples identified as healthy, low, and high-risk samples by PCR-RFLP. GENERAL SIGNIFICANCE: We show the possibility of using FTIR for initial cervical cancer risk stratification followed by detailed PCR-RFLP investigations for suspect cases.

ATR-FTIR spectroscopy and CDKN1C gene expression in the prediction of lymph nodes metastases in papillary thyroid carcinoma.

Thyroid cancer has become in recent years the most common endocrine malignancy. Among its different types, papillary thyroid carcinoma (PTC) has the highest incidence. PTC is slow growing, but shows a high rate of lymph node metastasis. Tissue biochemical characterization and identification of molecular markers can facilitate stratification of patients into those requiring surgical assessment of lymph nodes and patients for whom this surgical procedure is unnecessary; thus, leading to a more accurate prognosis. To this end, the study aimed to predict lymph node metastasis by Attenuated Total Reflectance - Fourier transform infrared (ATR-FTIR) spectroscopy of primary PTC tumors. Another objective of the study was to determine whether CCNA1, CDKN1C, FOS, HSPA5, JUN, KSR1, MAP2K6, MAPK8IP2 and SFN gene expression in primary PTC tumors could be used as predictive markers of lymph node metastasis. Three PTC with lymph node involvement (PTC+), six PTC without lymph node involvement (PTC-), and five normal (N) thyroid tissues were used for FTIR spectroscopy analysis; while 18 PTC+, 17 PTC-, and 6 N samples were used for molecular analysis by real-time quantitative PCR (RT-qPCR). FTIR spectral analysis revealed changes in phosphate groups possibly associated with nucleic acid (1236 cm-1), and protein/lipids (1452, 2924, 3821 cm-1) in PTC + compared to PTC-, and multivariate analysis could distinguish the two groups. Molecular analysis showed significant increase in CDKN1C gene expression in PTC + compared to PTC-. Being a cell growth regulator, increased CDKN1C provides some supporting evidence to the FTIR spectroscopy based finding of increased nucleic acids in PTC+. Thus, the study suggests the possibility of using FTIR spectroscopy and CDKN1C expression for predicting metastasis using primary tumor alone.

Molecular detection of HPV and FT-IR spectroscopy analysis in women with normal cervical cytology.

BACKGROUND: The increase in the incidence of Cervical Cancer in the female population worldwide has been an issue that deserves further attention from the scientific community. Several studies have already proven the relationship of its development with the molecular mechanisms that Human Papillomavirus (HPV) induces in cervical cells. The gene amplification provided by molecular biology techniques has been used as the gold standard diagnostic method of this virus because of its high specificity and sensitivity.However, the high investments associated with the acquisition of reagents, equipment and labor demonstrate the need for the development of more accessible techniques that present the same accuracy. FT-IR spectroscopy has been studied as an inexpensive and easily accessible technology that can provide the differentiation of malignant and benign cells. This study aimed to demonstrate the effectiveness and sensitivity of molecular analysis by PCR in relation to cytological analysis and to evaluate the sensitivity of FT-IR spectroscopy in the diagnosis of HPV for cervical cancer prevention. METHODS: Cervical fluid samples obtained from 50 patients with absence of cellular lesion by cytological analysis were analyzed by molecular and spectroscopic analyzes. Oncotic colpocitology analysis was performed by the Papanicolaou staining, amplification of the L1 viral gene by PCR was performed using primers MY09 and MY11 and biochemical analysis of the fluids by FT-IR was performed using the Spectrum 400 system equipped with a microscope. RESULTS: Of the 50 patients without evident morphological alteration of the cells, seven were diagnosed by molecular analysis as positive for presence of HPV. Principal component analysis of spectroscopy was not able to separate the negative samples from the HPV positive samples and, therefore, did not present as an effective diagnostic technique. CONCLUSIONS: We highlight the efficacy, sensitivity and specificity of molecular biology by PCR in the identification of the virus and we emphasize that more studies should be used for the application of FT-IR spectroscopy in the diagnosis of this infection and its application in the prevention of cervical cancer.

The Xenon Test Chamber Q-SUN® for testing realistic tolerances of fungi exposed to simulated full spectrum solar radiation.

The low survival of insect-pathogenic fungi when used for insect control in agriculture is mainly due to the deleterious effects of ultraviolet radiation and heat from solar irradiation. In this study, conidia of 15 species of entomopathogenic fungi were exposed to simulated full-spectrum solar radiation emitted by a Xenon Test Chamber Q-SUN XE-3-HC 340S (Q-LAB® Corporation, Westlake, OH, USA), which very closely simulates full-spectrum solar radiation. A dendrogram obtained from cluster analyses, based on lethal time 50 % and 90 % calculated by Probit analyses, separated the fungi into three clusters: cluster 3 contains species with highest tolerance to simulated full-spectrum solar radiation, included Metarhizium acridum, Cladosporium herbarum, and Trichothecium roseum with LT50 > 200 min irradiation. Cluster 2 contains eight species with moderate UV tolerance: Aschersonia aleyrodis, Isaria fumosorosea, Mariannaea pruinosa, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, and Torrubiella homopterorum with LT50 between 120 and 150 min irradiation. The four species in cluster 1 had the lowest UV tolerance: Lecanicillium aphanocladii, Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium inflatum with LT50 < 120 min irradiation. The QSUN Xenon Test Chamber XE3 is often used by the pharmaceutical and automotive industry to test light stability and weathering, respectively, but it was never used to evaluate fungal tolerance to full-spectrum solar radiation before. We conclude that the equipment provided an excellent tool for testing realistic tolerances of fungi to full-spectrum solar radiation of microbial agents for insect biological control in agriculture.