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Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma

Hamzan NI, Ab Rahman N, Suraiya S, Mohamad I, George Kalarakkal T, Mohamad S

Arch Oral Biol, 2021 Apr;124:105051.
PMID: 33581498 DOI: 10.1016/j.archoralbio.2021.105051

OBJECTIVE: The present study established a real-time loop-mediated isothermal amplification (qLAMP) for rapid detection of human papillomavirus subtype 16 (HPV-16) in oral squamous cell carcinoma (OSCC).
METHODS: The qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays.
RESULTS: The detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/μl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/μl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR.
CONCLUSION: This study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.

Matched MeSH terms: Human papillomavirus 16/genetics
Fulltext Evaluation of HPV 16 and HPV 18 Oncoprotein Expression as Alternative Diagnostic Tools in Cervical Lesion

Sabri NA, Shamsuddin SH, Mat Zin AA

Asian Pac J Cancer Prev, 2024 Feb 01;25(2):521-527.
PMID: 38415538 DOI: 10.31557/APJCP.2024.25.2.521

OBJECTIVE: The study aimed to evaluate E6 and E7 oncoproteins of HPV16 and HPV18 expression in formalin - fixed paraffin embedded (FFPE) tissue in different grades of the cervical lesion and evaluate the potential use of E6 and E7 oncoproteins derived from HPV 16 and 18 as diagnostic protein biomarkers for triaging cervical lesions.
METHODOLOGY: A total of 102 FFPE cervical tissues were collected from 2 tertiary hospitals and immunohistochemical reactivity staining of E6 and E7 oncoproteins of HPV16 and HPV18 were evaluated using immunoreactive scoring (IRS) system and analysed statistically.
RESULT: The result showed an increased oncoprotein expression with the progression of cervical lesions. There is a statistically significant association between histology grade and HPV16/18-E6 expression (p = 0.028). However, there are no significant association of histological grade to HPV16-E7 immunoreactivity score (p = 0.264) and HPV18-E7 (p=0.080).
CONCLUSION: The immunohistochemical expression of HPV oncoproteins is a potential alternative diagnostic tool applicable in a low-resource laboratory setting. The advantage of the histochemical evaluation is that this method is simpler to apply and less expensive in comparison to in situ mRNA hybridization. Nevertheless, our study also found that antibodies against HPV that are commercially available suffer quite substantial specificity issues such as background staining and inconsistency between different batches. Hence, the utilization of antibody-based staining warrants stringent quality control.

Matched MeSH terms: Human papillomavirus 16/genetics
Designing probe from E6 genome region of human Papillomavirus 16 for sensing applications

Parmin NA, Hashim U, Gopinath SCB

Int J Biol Macromol, 2018 Feb;107(Pt B):1738-1746.
PMID: 29030179 DOI: 10.1016/j.ijbiomac.2017.10.051

Human Papillomavirus (HPV) is a standout amongst the most commonly reported over 100 types, among them genotypes 16, 18, 31 and 45 are the high-risk HPV. Herein, we designed the oligonucleotide probe for the detection of predominant HPV type 16 for the sensing applications. Conserved amino acid sequences within E6 region of the open reading frame in the HPV genome was used as the basis to design oligonucleotide probe to detect cervical cancer. Analyses of E6 amino acid sequences from the high-risk HPVs were done to check the percentage of similarity and consensus regions that cause different cancers, including cervical cancer. Basic local alignment search tools (BLAST) have given extra statistical parameters, for example, desire values (E-values) and score bits. The probe, 'GGG GTC GGT GGA CCG GTC GAT GTA' was designed with 66.7% GC content. This oligonucleotide probe is designed with the length of 24 mer, GC percent is between 40 and 70, and the melting point (Tm) is above 50°C. The probe needed an acceptable length between 22 and 31 mer. The choice of region is identified here can be used as a probe, has implications for HPV detection techniques in biosensor especially for clinical determination of cervical cancer.

Matched MeSH terms: Human papillomavirus 16/genetics*
Detection of high-risk HPV 16 genotypes in cervical cancers using isothermal DNA amplification with electrochemical genosensor

Nakowong P, Chatchawal P, Chaibun T, Boonapatcharoen N, Promptmas C, Buajeeb W, et al.

Talanta, 2024 Mar 01;269:125495.
PMID: 38043336 DOI: 10.1016/j.talanta.2023.125495

Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/μL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.

Matched MeSH terms: Human papillomavirus 16/genetics
Self-assembled silver nanoparticle-DNA on a dielectrode microdevice for determination of gynecologic tumors

Li Z, Gopinath SCB, Lakshmipriya T, Anbu P, Perumal V, Wang X

Biomed Microdevices, 2020 Sep 17;22(4):67.
PMID: 32940771 DOI: 10.1007/s10544-020-00522-3

Nanoscale materials have been employed in the past 2 decades in applications such as biosensing, therapeutics and medical diagnostics due to their beneficial optoelectronic properties. In recent years, silver nanoparticles (AgNPs) have gained attention due to their higher plasmon excitation efficiency than gold nanoparticles, as proved by sharper and stronger plasmon resonance peaks. The current work is focused on utilizing self-assembled DNA-AgNPs on microdevices for the detection of gynecological cancers. Human papilloma virus (HPV) mostly spreads through sexual transmittance and can cause various gynecological cancers, including cervical, ovarian and endometrial cancers. In particular, oncogene E7 from the HPV strain 16 (HPV-16 E7) is responsible for causing these cancers. In this research, the target sequence of HPV-16 E7 was detected by an AgNP-conjugated capture probe on a dielectrode sensor. The detection limit was in the range between 10 and 100 aM (by 3σ estimation). The sensitivity of the AgNP-conjugated probe was 10 aM and similar to the sensitivity of gold nanoparticle conjugation sensors, and the mismatched control DNA failed to detect the target, proving selective HPV detection. Morphological assessments on the AgNPs and the sensing surfaces by high-resolution microscopy revealed the surface arrangement. This sensing platform can be expanded to develop sensors for the detection various clinically relevant targets.

Matched MeSH terms: Human papillomavirus 16/genetics
Fulltext Prevalence and viral load of oncogenic human papillomavirus (HPV) in pterygia in multi-ethnic patients in the Malay Peninsula

Chong PP, Tung CH, Rahman NA, Yajima M, Chin FW, Yeng CL, et al.

Acta Ophthalmol, 2014 Nov;92(7):e569-79.
PMID: 25043991 DOI: 10.1111/aos.12427

The aim of the study was to determine the prevalence of human papillomavirus (HPV) in primary and recurrent pterygia samples collected from different ethnic groups in the equatorial Malay Peninsula.

Matched MeSH terms: Human papillomavirus 16/genetics
Fulltext High-risk human papillomavirus viral load and persistence among heterosexual HIV-negative and HIV-positive men

Grabowski MK, Gray RH, Serwadda D, Kigozi G, Gravitt PE, Nalugoda F, et al.

Sex Transm Infect, 2014 Jun;90(4):337-43.
PMID: 24482488 DOI: 10.1136/sextrans-2013-051230

OBJECTIVES: High-risk human papillomavirus (HR-HPV) viral load is associated with HR-HPV transmission and HR-HPV persistence in women. It is unknown whether HR-HPV viral load is associated with persistence in HIV-negative or HIV-positive men.
METHODS: HR-HPV viral load and persistence were evaluated among 703 HIV-negative and 233 HIV-positive heterosexual men who participated in a male circumcision trial in Rakai, Uganda. Penile swabs were tested at baseline and 6, 12 and 24 months for HR-HPV using the Roche HPV Linear Array, which provides a semiquantitative measure of HPV shedding by hybridisation band intensity (graded: 1-4). Prevalence risk ratios (PRR) were used to estimate the association between HR-HPV viral load and persistent detection of HR-HPV.
RESULTS: HR-HPV genotypes with high viral load (grade:3-4) at baseline were more likely to persist than HR-HPV genotypes with low viral load (grade: 1-2) among HIV-negative men (month 6: adjPRR=1.83, 95% CI 1.32 to 2.52; month 12: adjPRR=2.01, 95% CI 1.42 to 3.11), and HIV-positive men (month 6: adjPRR=1.33, 95% CI 1.06 to 1.67; month 12: adjPRR=1.73, 95% CI 1.18 to 2.54). Long-term persistence of HR-HPV was more frequent among HIV-positive men compared with HIV-negative men (month 24: adjPRR=2.27, 95% CI 1.47 to 3.51). Persistence of newly detected HR-HPV at the 6-month and 12-month visits with high viral load were also more likely to persist to 24 months than HR-HPV with low viral load among HIV-negative men (adjPRR=1.67, 95% CI 0.88 to 3.16).
CONCLUSIONS: HR-HPV genotypes with high viral load are more likely to persist among HIV-negative and HIV-positive men, though persistence was more common among HIV-positive men overall. The results may explain the association between high HR-HPV viral load and HR-HPV transmission.

Matched MeSH terms: Human papillomavirus 16/genetics
Fulltext Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation

Chalertpet K, Pakdeechaidan W, Patel V, Mutirangura A, Yanatatsaneejit P

Cancer Sci, 2015 Oct;106(10):1333-40.
PMID: 26250467 DOI: 10.1111/cas.12761

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation.

Matched MeSH terms: Human papillomavirus 16/genetics
Fulltext Gold-Hybridized Zinc Oxide Nanorods as Real-Time Low-Cost NanoBiosensors for Detection of virulent DNA signature of HPV-16 in Cervical Carcinoma

Ramesh T, Foo KL, R H, Sam AJ, Solayappan M

Sci Rep, 2019 Nov 19;9(1):17039.
PMID: 31745139 DOI: 10.1038/s41598-019-53476-9

Detection of host integrated viral oncogenes are critical for early and point-of-care molecular diagnostics of virus-induced carcinoma. However, available diagnostic approaches are incapable of combining both cost-efficient medical diagnosis and high analytical performances. To circumvent this, we have developed an improved IDE-based nanobiosensor for biorecognition of HPV-16 infected cervical cancer cells through electrochemical impedance spectroscopy. The system is fabricated by coating gold (Au) doped zinc oxide (ZnO) nanorods interfaced with HPV-16 viral DNA bioreceptors on top of the Interdigitated Electrode (IDE) chips surface. Due to the concurrently improved sensitivity and biocompatibility of the designed nanohybrid film, Au decorated ZnO-Nanorod biosensors demonstrate exceptional detection of HPV-16 E6 oncogene, the cancer biomarker for HPV infected cervical cancers. This sensor displayed high levels of sensitivity by detecting as low as 1fM of viral E6 gene target. The sensor also exhibited a stable functional life span of more than 5 weeks, good reproducibility and high discriminatory properties against HPV-16. Sensor current responses are obtained from cultured cervical cancer cells which are close to clinical cancer samples. Hence, the developed sensor is an adaptable tool with high potential for clinical diagnosis especially useful for economically challenged countries/regions.

Matched MeSH terms: Human papillomavirus 16/genetics*