Study On The Detection Of HPV In Phase 3B Cervical Cancer Patients

Cervical Malignancy and HPV

Cervical malignancy is a malignant tumor that develops in the uterine wall, which joins the womb to the vaginal canal. The human papillomavirus -HPV, a chronic infection, functions in most cervical cancer cases. Cervical cancer develops on the cervix's membrane. It occurs when a cancer body in a woman's cervix starts to transform into abnormal cells (Srivastava et al.,2018). Although not all pre-malignant cells will develop malignant, detecting them and managing them until they become malignant is critical to avoiding cervical cancer. Cervical cancer is divided into hepatocellular carcinoma and adenocarcinoma (Hull et al.,2020). Basal cell carcinoma accounts for 80 to 90 percent of cervical malignancies, while carcinoma tumors account for 10 to 20 percent. Approximately 14,000 individuals in the United Kingdom are diagnosed with cervical cancer every year. Cervical cancer is most commonly detected in people aged 35 and 44 (Ou et al.,2020). At the point of diagnosis, the typical person is 50 years old. Cervical cancer claims the lives of about 4,000 people each year. This percentage decreases because of inspections and the HPV vaccine (Bergman et al., 2019).

The female patient in this investigation was identified with phase 3B cervical cancer regarding a colposcopy protocol. Since the latest smear test revealed high-quality dyskaryosis in the uterus, she was initially diagnosed with polycystic ovary disorder at early onset. She had chosen not to have the HPV vaccine. Enhanced cytological alterations in the cervical could signal the presence of phase 3B carcinoma. HPV-16 and 18 identification by PCR technique, on the other hand, can help detect dyskaryosis in the cervical (Mahmoodi et al.,2019). The two primary gene mutations, E6 and E7, which are induced continuously and contribute to carcinogenesis, are responsible for this kind of cancer's initial formation and advancement. As a result, manipulating these genes has been the most effective method of treating cervical cancer.

The goal of this study is to see if cervical cancer is connected to Human papillomavirus utilizing the PCR method to identify the occurrence of  HPV-L1 genotype, western blot to evaluate the articulation of E6 protein, including a protease immunosorbent assay -ELISA to approximate the renal function of the sickly person in correlation to a good health patient. The relevance of testing requirements and the improvement of western blot also will be addressed.

The immunodetection-positive E6 peptide at 18kDa for the ill individual was seen on a western blot using the SDS-PAGE electrophoresis methodology, even though there was no band for the ordinary person. The results also revealed that the desirable and undesirable samples were, respectively, E6 positively and negatively.

The PCR study reveals that the ill individual was significant for the HPV-L1 genome at 450bp; however, the typical individual was negative. The study also indicated that the pleasant and unpleasant samples were positive and negative (Jalil et al.,2021).

After computing the creatinine calibration graph, the findings show an increased creatinine concentration level for the sick individual -2.12mg/dl. The creatinine measurement of -0.71 mg/dl recorded for the verified normal patient, on the other hand, was within the stated acceptable limit of -0.5-1.1 mg/dl. Conversely, during the ELISA method on day 7, mild QC, moderate QC, and severe QC were run simultaneously on the test specimens and yielded 0.36mg/dl, 0.766mg/dl, including 2.06mg/dl, correspondingly. Computed average scores for the whole seven days time, 0.39mg/dl, 0.80mg/dl, and 2.43mg/dl for weak, moderate, and strong QC, correspondingly, were compared to pre-determined goal levels of 0.41mg/dl, 0.81mg/dl, and 2.6mg/dl, respectively, for scientific rigour at P >.06. Following confirming regularity with the Shapiro-Wilk test S-W, a single sample t-test was done to verify for Fairly low and Moderate.

Detection of Phase 3B Cervical Cancer using PCR, Western Blot and ELISA

After passing the normality test at p >.05, the Wilcoxon rank analysis was employed to determine analytical validity for High-QC. For the weak QC, medium QC, and great QC, there were non-essential differences (t(df) =-1.26, p =.26), (t(df) = -.870, p=.420), and (t(df) = -1.750, p =.080), respectively. 

Figure 1:Western blot immunodetection of E6 oncoprotein

The specimen configurations are DNA size -L, Negative specimen for control -N, Positive control specimen -P, Normal individual -H, and Sickly patient -D. SDS-PAGE electrophoresis split protein samples, followed by western blotting using a mouse monoclonal antibody suitable for HPV16 E6 immunodetection.   

Serum creatinine content (mg/dl) 

Serum creatinine content  in mg/dl

Illustration 2:ELISA calibration graph for creatinine

A standard diagram depicts the average absorbance of creatine standards at various contents, with error margins denoting the standard deviation.   

The quantitative variables for the significant variation between the weekly mean contents and the defined target scores of the minimal, moderate, and elevated controls are shown in the quality assurance data  for ELISA.  

HPV is now known to be the most prevalent trigger of carcinoma. All HPV strains have been linked to severe cervical cancer. The majority of research demonstrates that having more constant partners and starting sexual relations at an early age increases the risk of cervical malignancy (Sadri Nahand et al., 2020). The presence or absence of Human papillomavirus genotype, E6 enzyme transcription, and creatinine level in a female patient having phase three cancer were investigated in this study. The etiology of carcinoma was established using a terminal polymerase chain reaction approach. It was also confirmed by a western blot to indicate the existence and deletion of HPV-L1 transcript and E6 oncoprotein in the studied cases. The afflicted participant's creatinine levels were also assessed using ELISA, yielding a result of 2.13mg/dl.

Conversely, the PCR approach applied in this research focused on a 450-bp area of the HPV-L1 transcript that correlates to adjacent nucleotide locations 6584/7035 on Human papillomavirus 16 of the L1 gene prevalent trigger of cervical cancer. Even though the selection of MY09/MY11 synthesized primer variety resulted in unsatisfactory efficiency and unpredictable fluctuation, the results of this investigation, like earlier studies, demonstrated that the Polymerase chain reaction method could be efficient in recognizing HPV Types. Furthermore, the report's use of control samples revealed that the experimental findings were trustworthy (Wait, 2021).

As a result, detecting the HPV sub-type for the afflicted individual at the 450bp here on Plasmid DNA revealed that HPV16 illness was the etiology of cervical cancer.

 The western blot revealed the existence of an E6 oncogene at 18kda of the sick individual in complement to the PCR outcome by using a pre-stained peptide ladder by utilizing a pre-stained peptide ladder. There are some ideas that the western blot There really are doubts that using western blot analysis may not be precise and can be restricted by a few elements. Western blotting's major downside is that technology can only be performed if a target protein against the target protein is obtainable (He et al., 2018). Primary antibodies that target phosphorylated sites are utilized to identify post-translational changes like phosphorylation of target molecules.

Creatinine Measurement

Nevertheless, E6 oncoprotein never was discovered in the average individual in this same experiment, and the favorable and unfavorable standards both were negative and positive, indicating that the technique was reliable. Squamous cell carcinoma induced by HPV-16 Genetic code exhibits the 18-kDa E6 oncoprotein. That is inherent in the premise that E6 is created by HPV16 and might even lead to cervix tumor formation, as reported in a prior study (Zhu et al., 2021). Unlike colposcopy, such studies require a confirmed diagnosis via HPV sequencing and protein characterization to enact medical decisions.

Nevertheless, stage 3B cervical cancer is defined as lymph node involvement in the uterus wall and the existence of hydro-nephrosis, according to current cervical cancer categorization (Salina Zhang et al.,2019). Conversely, serum creatinine is perhaps the most commonly utilized biomarker to diagnose and track the course of hydro-nephrosis. Serum creatinine was assessed in both the sick and normal patients using the ELISA technique, with minimal, moderate, and elevated standards used to control the methodological approach to diagnosing hydro-nephrosis. The ill participant's projected creatinine level was 2.13 mg/dl, higher than the base value -of 0.5-1.1 mg/dl, whereas the healthy participant's level was 0.71 mg/dl. Following correlating controls to the specified target levels -table 2, no substantial change (p>.05) was found, indicating that the findings are reliable. According to a comparable study, the high creatinine result of -2.13 mg/dl acquired that used the ELISA analysis methodology for the afflicted individual indicates hydro-nephrosis. Generally speaking, the conclusions from the multiple inquests and the colposcopy outcomes affirmed that the sickly female patient seemed to have phase 3B cervical cancer due to a high HPV16 or E6 with hydro-nephrosis, which she could have avoided if she had expressed a preference to get the HPV vaccine at a younger age (Oh et al., 2021).

The recognition of the HPV E6 enzyme employing a western blotting methodology separated by dimension -18kDa required multiple steps, including specimen preparation, and gel readiness, using appropriate direct and indirect antibodies to view the specific protein. In the meantime, some optimal control initiatives were designed to decrease the occurrence of unpredictable outcomes such as no spot, sluggish or lightheaded bands, and poor backstory. They are generally linked with the western blot methodology and have accounted for about 30% of tampered outcomes in other study findings (Pong et al., 2020). It's important to mention that the selectivity and impetuosity of the selection immune response, and the quantity, incubating, cleaning, and overall blocking or latency conditions enforced all play a role in its high efficiency.

Despite preventative steps such as suitable scheduling, buffer content, and good overall methods used in the study, the result of the western blot analysis technique in this investigation was many backgrounds and unanticipated bands on the affected and unaffected patients pathways. In the meantime, insufficient rinsing and antibody usage could be blamed for the numerous detected bands. It's important to remember that reduced avidity could be linked to the antisera utilized, contributing to cross-reactivity and a false-positive consequence, as shown in statistic 2. Appropriate usage of antisera may alleviate similar cases by limiting the mistake of off-target indication by principal specific antibodies and the potential of numerous or erroneous spots also on western blot test for the assessment of E6 molecule in a subsequent study (Huang et al., 2021).

Results of the Study

Suitable antibody concentrations have been linked to high-quality ambient results and better goal adherence. In contrast, using the wrong amount of antibodies in a western blot test can result in negative consequences. For instance, on a western blot, far too much target molecule can conceal the specific protein by rising off-target adhesion and cross-reactive bands and "burnt-out" bands brought about by rapid potting medium exhaustion, which outcomes in bars with no transmitter in the middle. At the same time, too little can affect faint transmissions. As a result, the pipetting mistake could produce either cross-reactivity or numerous band development found in this investigation. Regardless, a sequential dot blot titrimetric assessment which was before specific antibodies appropriate concentrations, generating the most exemplary blot notification ratio as the reference standard, can be used in a prospective study to find advisable direct and indirect antibody accumulation for western blot context enhancement (Ren et al.,2019). Poor interference in western blot assays has been attributed to overabundance and inappropriate antibody response.

Illustration 2, on the other hand, had a low backdrop sharpness. The backdrop sharpness may have been enhanced if the cleaning procedure had been changed. As opposed to typical buffers application, significant water rinsing is among the adjustments shown to eliminate different antibodies effectively. Due to water's lower pH., it allows it to accept other solvent and solute bio-molecules. Existing methodologies have anticipated that using water would induce protein devastation (Hameed et al.,2021).

Nevertheless, studies have shown that water does not cause protein loss or bi-layer damage. Finally, the use of  Tween buffered fluid for rinsing affects the best antibody reactivity and has been linked to better results in stains in the background. Even though the 0.04 percent Tween buffered solutions employed in this analysis of E6 oncoprotein through western blot had poor knowledge, an investigation is employing the use of a cleaning solution containing 0.50 percent v/v Tween 20 suggests that weak  Tween 20 concentration;0.03–0.007 percent v/v, as per the standard methodology.

It's possible that 0.1 percent -v/v is only required for antibody development and not for other purposes,  cleaning solutions, which may necessitate a high proportion for maximum efficiency. In a nutshell, illustration 2's numerous bands and weak background could be improved by using appropriate antisera and extended cleaning.

Data quality control is implementing methods or techniques to ensure that data meets overall performance goals and defined quality requirements for critical properties. Quality control, which maintains the correctness of patient test data, is one of the essential components of laboratory investigations. The consistency of analyzed specimens is necessary for maintaining overall performance and meeting competency evaluation standards. QC issues such as chemical derivatization and validation mismatch of evaluation and approval should be resolved To determine possible inconsistencies with clinical information (Xiao et al.,2018). To ensure that patient inspection is carried out accurately and adequately, rigorous and regular quality assurance checks of experimental lab verification are required.

If quality standards are followed correctly, they can uncover and remedy flaws in a hospital's statistical methodology before issuing misleading patient results. The failure of QC evaluation could be due to "clerical, procedural, practical, PT component durability, and interference." Using quality assurance approaches, a lab can self-control its analysis and achieve the intended it provides that are precise. Medical labs manage documentation and use a strategic action plan to improve the quality assurance process.

Relevance of Testing Requirements

Quality control specimens are supposed to look like clinical specimens and be assessed the same way. A repeated quality assurance study aims to ensure that the patient experimental verification results are exact and reliable. The Levey-Jennings chart; L-J chart is a popular tool for monitoring scientific quality assurance specimens. During quality control, and L-J chart and the Westgard Guidelines are frequently used to examine patterns, prejudices, and flaws. The standard error is calculated using the Westgard Rules, checking for the projected standard curve. Rules violations can be identified by adding Westgard rules into an L-J chart, depending on the specimen tester's specification limit (Ma et al.,2022).On 14- or 30-day assessments of QC assessment, several laboratories use L-J charts. Whereas daily QC departures from normal parameters maintain specimen test accuracy, comprehensive reviews are much more effective for regularly diagnosing trends and prejudices in tests that might be undetected. Patients can also be used as their controls when using the L-J chart lacking quality control samples. A laboratorian can detect deviation or issues with analyzer performance that are not seen by quality control assessment by following the rolling means of the patient data.

In this investigation, a quantitative study was utilized to check the analytical performance and consistency of the patient outcomes by evaluating the weekly average value of the QCs vs. a specified value employing SPSS -Appendix C. To verify the efficacy of the complete ELISA analysis procedure for seven days and decide precisely the patient's plasma creatinine values, minimal, moderate, and elevated controls were used for both diseased and healthy patients. The selection of these high concentrations was crucial in determining the quantitative performance's responsiveness threshold. On day 7, the low-QC collection had an average content of 0.35 mg/dl, and for the entire 7-day run, it had a mean range of 0.38 mg/dl. As a result, the Shapiro-Wilk method was used to evaluate the rationality of dispersion within the seven days' content levels to correlate the mean amount during the seven days with the set target value of 0.41mg/dl, and it revealed that disclosed information was evenly distributed throughout the seven days.

As a result, just one t-test, another of the descriptive procedures employed throughout analysis for normally distributed variables, was performed to examine the statistical relevance difference between the weekly average value and the given goal value. When matched to the preset goal value, the average value for the week before the minimal collection showed a non-significant variation, suggesting that the minimal outcomes met the acceptance criterion. Similarly, following the Shapiro-Wilk test, the Medium-QC data, such as the Low-QC sample, were distributed evenly (Appendix F). The week's Moderate score was 0.79 mg/dl, with 0.75 mg/dl on day 7. The significance of the difference between the average value during the week and the preset goal value of 0.80mg/dl was assessed using the T-test. In addition, there was no substantial variation -p =.422.

Moreover, in contrast to the other two Quality control levels, the Elevated-QC missed the normality dispersed check shortly after the S-W assessment revealed a statically vital variation set within the seven days;p =.001. Rather than using the t-test, the Wilcoxon signed-rank experiment, a non-parametric analysis technique, was used to check if the High-Quality Control mean value of 2.43mg/dl was considerably different from the preset objective value. Nonetheless, there was no significant variance across the analyzed means because the outcome was not substantially different -P =.078 as from the intended ideal weight. Generally, the 3 QC test specimens refuted the alternate theory while retaining the null hypothesis, indicating that the findings for both afflicted and healthy individuals were considered credible.

Conclusion and Future Implications

Conclusion

Finally, the female patient's phase 3B carcinoma was attributed to the high HPV 16/E6 illness earlier identified by colposcopy. Terminal PCR is a reasonably precise approach for detecting the HPV subtype that causes cervical cancer at the molecular scale. Furthermore, while the overall quality of a western blot analysis might be difficult to achieve, if properly tuned, it could be used to evaluate the transcription of an oncoprotein -E6 in vitro.   

References

Bergman, H., Buckley, B. S., Villanueva, G., Petkovic, J., Garritty, C., Lutje, V., ... & Henschke, N. (2019). Comparison of different human papillomavirus (HPV) vaccine types and dose schedules for prevention of HPV?related disease in females and males. Cochrane database of systematic reviews, (11).

Hameed, A., Farooq, T., Hameed, A., & Sheikh, M. A. (2021). Sodium nitroprusside mediated priming memory invokes water-deficit stress acclimation in wheat plants through physio-biochemical alterations. Plant Physiology and Biochemistry, 160, 329-340.

He, Y., Yao, X., Taylor, N., Bai, Y., Lovenberg, T., & Bhattacharya, A. (2018). RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells. Journal of neuroinflammation, 15(1), 1-13.

Huang, B., Dai, L., Wang, H., Hu, Z., Yang, X., Tan, W., & Gao, G. F. (2021). Neutralization of SARS-CoV-2 VOC 501Y. V2 by human antisera elicited by both inactivated BBIBP-CorV and recombinant dimeric RBD ZF2001 vaccines. BioRxiv.

Hull, R., Mbele, M., Makhafola, T., Hicks, C., Wang, S. M., Reis, R. M., ... & Dlamini, Z. (2020). Cervical cancer in low and middle?income countries. Oncology Letters, 20(3), 2058-2074.

Jalil, A. T., Al-Khafaji, A. H. D., Karevskiy, A., Dilfy, S. H., & Hanan, Z. K. (2021). Polymerase chain reaction technique for molecular detection of HPV16 infections among women with cervical cancer in Dhi-Qar Province. Materials Today: Proceedings.

Ma, H., Cao, J., & Li, M. (2022). Application of PDCA Process Management in Day Operation Ward and the Influence of Nursing Quality and Safety. Computational and Mathematical Methods in Medicine, 2022.

Mahmoodi, P., Fani, M., Rezayi, M., Avan, A., Pasdar, Z., Karimi, E., ... & Ghayour?Mobarhan, M. (2019). Early detection of cervical cancer based on high?risk HPV DNA?based nanosensors: A systematic review. Biofactors, 45(2), 101-117.

Oh, N. L., Biddell, C. B., Rhodes, B. E., & Brewer, N. T. (2021). Provider communication and HPV vaccine uptake: a meta-analysis and systematic review. Preventive Medicine, 148, 106554.

Ou, R., Lv, J., Zhang, Q., Lin, F., Zhu, L., Huang, F., ... & Xu, Y. (2020). circAMOTL1 motivates AMOTL1 expression to facilitate cervical cancer growth. Molecular Therapy-Nucleic Acids, 19, 50-60.

Pong, Y. H., Su, Y. R., Lo, H. W., Ho, C. K., Hsieh, C. C., Chu, C. T., ... & Wu, G. J. (2020). METCAM/MUC18 is a new early diagnostic biomarker for the malignant potential of prostate cancer: Validation with Western blot method, enzyme-linked immunosorbent assay and lateral flow immunoassay. Cancer Biomarkers, 27(3), 377-387.

Ren, W., Mohammed, S. I., Wereley, S., & Irudayaraj, J. (2019). Magnetic focus lateral flow sensor for detection of cervical cancer biomarkers. Analytical Chemistry, 91(4), 2876-2884.

Sadri Nahant, J., Moghoofei, M., Salmaninejad, A., Bahmanpour, Z., Karimzadeh, M., Nasiri, M., ... & Hamblin, M. R. (2020). Pathogenic role of exosomes and microRNAs in HPV?mediated inflammation and cervical cancer: a review. International journal of cancer, 146(2), 305-320.

Salina Zhang, B. S., Batur, P., & Ncmp, C. (2019). Human papillomavirus in 2019: An update on cervical cancer prevention and screening guidelines. Cleveland Clinic journal of medicine, 86(3), 173.

Srivastava, A. N., Misra, J. S., Srivastava, S., Das, B. C., & Gupta, S. (2018). Cervical cancer screening in rural India: Status & current concepts. The Indian journal of medical research, 148(6), 687.

Wait, A. D. (2021). The Importance of Data Reliability and Usability When Assessing Impacts of Marine Mineral Oil Spills. Toxics, 9(11), 302.

Xiao, S., Xiao, M. Z., Zhao, Q. H., & Xie, L. L. (2018). Construction of nursing quality control information system in large Hospitals. In Nursing Informatics 2018 (pp. 193-194). IOS Press.

Zhu, J., Chen, L., Zheng, Q., Han, R., Chen, X., Zhou, Q., & Cheng, H. (2021). Immunohistochemical Detection of Human Papillomavirus 16 E7 Oncoprotein in Cervical Lesions. Journal of Cancer, 12(23), 7130.