
==== Front
J Cytol
J Cytol
JCytol
J Cytol
Journal of Cytology
0970-9371
0974-5165
Wolters Kluwer - Medknow India

JCytol-41-157
10.4103/joc.joc_27_24
Original Article
Aptima HPV Genotypes in Abnormal Cervical Samples in Different Age Groups – Implication on Vaccination Strategies
Al-Awadhi Rana 1
AlRoomy Moody 1
Dashti Hawraa 2
Kapila Kusum 23
1 Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Kuwait University, Jabriyah City, State of Kuwait
2 Cytology Laboratory, Mubarak Al-Kabeer Hospital, Ministry of Health, Kuwait
3 Department of Pathology, Faculty of Medicine, Kuwait University, Jabriyah City, State of Kuwait
Address for correspondence: Dr. Rana Al-Awadhi, Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Kuwait University, PO Box: 31470, Sulaibikhat, State of Kuwait. E-mail: rana.alawadhi@ku.edu.kw
Jul-Sep 2024
18 7 2024
41 3 157161
22 2 2024
16 4 2024
03 6 2024
Copyright: © 2024 Journal of Cytology | Indian Academy of Cytologists
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Context:

Persistent infection with high-risk human papillomavirus (hr-HPV) types is associated with high-grade cervical abnormalities.

Aims:

The aim of the study was to find most hr-HPV types causing persistent infection in abnormal cytological samples using Aptima HPV testing and discuss the compatibility of the Gardasil 9 vaccine in targeting most types.

Settings:

The study was conducted in a cytology laboratory in a tertiary hospital.

Design:

This is a retrospective observational study.

Methods and Materials:

Cytology and HPV Aptima test reports were obtained for abnormal cervical samples for a 6-year period.

Statistical Analysis Used:

Pearson Chi-square test.

Results:

Reports of 2834 abnormal cervical samples were analyzed. Aptima testing was positive in 21% of samples, including 92% of squamous cell carcinoma (SCC), 76.4% of the high-grade squamous intraepithelial lesion (HSIL), 52% of low-grade squamous intraepithelial lesion (LSIL), 40% of adenocarcinoma (ADC), and 21% of atypical squamous cells that cannot exclude HSIL (ASC-H). The Aptima other hr-HPV group was most common (60%), HPV16 was 26%, HPV18/45 was 9.6%, and double HPV infection was 4.3%. HPV16 was the most common infection in HSIL+ cases. HPV infection was most common in age groups (30–39) and (40–49), and a shift to age groups (50–59) and ≥60 was seen in HSIL+ cases.

Conclusions:

This study is the first of its kind in correlating age with hr-HPV and cytology findings in the Middle East and adds to previous knowledge related to the prevalence and Aptima testing of HPV. The outcome could be used as a baseline for the Gardasil 9 vaccine and for the assessment of its effectiveness after three or five years from initiation.

Aptima HPV test
cervical samples
Gardasil 9 vaccine
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pmcINTRODUCTION

High-risk human papillomavirus (hr-HPV) testing and cytology have become the screening or triaging methods of choice in many centers worldwide. The use of both tests together or simultaneously helped triage women who were at risk of developing cervical cancer. The Aptima-Panther HPV detection system was designed to detect HPV E6/E7 messenger RNA (mRNA) using a hybridization technique for 14 hr-HPV genotypes.

This retrospective observational study was undertaken to evaluate the performance of Aptima HPV testing during a six-year period and discuss the benefit of using the Gardasil 9 vaccine in reducing the most persistent hr-HPV genotypes associated with cervical carcinoma and its precursors.

SUBJECTS AND METHODS

Samples

Information on cervical samples taken from women attending obstetrics and gynecology clinics was obtained in this study from October 2017 to November 2023. Opportunistic cervical samples were taken from women seeking clinics as a result of general gynecology complaints and are thus considered primary screening samples. ThinPrep vial (Hologic, Boxborough, MA) received at the cytology laboratory, Mubarak Al-Kabeer hospital, were prepared, Papanicolaou stained, and reported using the Bethesda System 2014 guidelines (TBS, 2014).[1] The diagnosis included atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (HSIL) (ASC-H), low-grade squamous intraepithelial lesion (LSIL), HSIL, squamous cell carcinoma (SCC), atypical glandular cells (AGCs), and adenocarcinoma (ADC). Mubarak Al-Kabeer cytology laboratory serves 15 obstetrics and gynecology clinics located in polyclinics distributed in four different health district areas.

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Health Science Centre (HSC) Ethics Committee at Kuwait University and Health and Medical Research Committee in the Ministry of Health and registered on Number: VDR/EC/3746 Date of the approval is: December 19, 2022. Informed consent was obtained from all subjects involved in the study. Each patient was interviewed and consented to enter the study. Patients were asked to sign a consent form after a verbal explanation from the physician soliciting their participation.

The age of patients, nationality, cytology diagnosis, and Aptima virology results were included in the analysis.

Aptima HPV testing

Cervical samples with an abnormal cytology diagnosis were further tested for the presence of E6/E7 viral mRNA from hr-HPV genotypes using two Aptima kits and Panther platform (Hologic, Genetic Center Drive, San Diego, CA, USA).[2] The Aptima HPV Assay Kit was used as a screening test to detect the presence from mRNA of any of the 14 hr-HPV genotypes without specifying the type of virus. The hr-HPV genotypes include the following: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The Aptima HPV GT kit was used to detect the presence of mRNA from HPV16 or HPV18/45. Virology results were analyzed as follows: HPV16, HPV18/45, HPV16/18/45, and other hr-HPV. HPV16 indicates the presence of mRNA of HPV16, HPV18/45 indicates the presence of mRNA of HPV18 and/or HPV45, and HPV16/18/45 indicates the presence of mRNA of HPV16 and/or HPV18 and/or HPV45. Other hr-HPV group indicates the presence of mRNA of one or more of the following hr-HPVs: HPV 31, 33, 35, 39, 51, 52, 56, 58, 59, 66, and 68.[2]

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software. The data were analyzed using the Statistical Package for Social Sciences (SPSS version 29.0.1.0; SPSS Chicago, IL, USA). The mean, median, range, minimum, and maximum were used as descriptive statistics. The association between two categorical variables was determined using the Pearson Chi-square test. A P value < 0.05 was considered significant.

RESULTS

This study comprised 2834 abnormal cervical samples obtained from women attending obstetrics and gynecology clinics. Table 1 shows that the mean age was 41.28 and the median was 41.0. Age was categorized into five age groups, and most of the women (68.1%) were in age groups 30–39 and 40–49. The difference between the age groups was statistically significant (P = 0.001). Cytology reports show that 36.1% of samples had an AGC diagnosis, 29.7% had ASCUS, 17.2% had ASC-H, 11.3% had LSIL, and 5.7% had HSIL, SCC, and ADC combined, (P = 0.001). Women were categorized into Kuwaiti (61.3%) and not Kuwaiti (38.7%) and were analyzed statistically based on their results in the Aptima test. The statistical test of the two groups was not significant (P = 0.164); therefore, the two groups were combined in the analysis.

Table 1 Demographic and test characteristics of 2834 cervical samples during a six-year period

Characteristic	No (%)	P*	
Age			
    Mean	41.28	-	
    Median	41.0	-	
    Minimum	18	-	
    Maximum	87	-	
Age groups			
    <20–29	351 (12.3)	0.001	
    30–39	893 (31.5)		
    40–49	1037 (36.6)		
    50–59	441 (15.6)		
    ≥60	112 (4.0)		
Nationality			
    Kuwaiti	1737 (61.3)	0.164	
    Non-Kuwaiti	1097 (38.7)		
Cytology report			
    ASCUS	839 (29.7)	0.001	
    ASC-H	490 (17.2)		
    AGC	1023 (36.1)		
    LSIL	320 (11.3)		
    HSIL	144 (5.0)		
    SCC	13 (0.5)		
    ADC	5 (0.2)		
Aptima report			
    Positive	596 (21)	0.001	
    Negative	2238 (79)		
*Pearson Chi-square test. ASCUS: Atypical squamous cells of undetermined significance, LSIL: Low-grade squamous intraepithelial lesion, ASC-H: Atypical squamous cells that cannot exclude HSIL, HSIL: High-grade squamous intraepithelial lesion, SCC: Squamous cell carcinoma, AGC: Atypical glandular cell, ADC: Adenocarcinoma

Table 1 shows that 596 out of 2834 (21%) of the abnormal samples had positive results for the presence of E6/E7 mRNA of one or more of 14 hr-HPV genotypes, and 2238 out of 2834 (79%) had negative reports (P = 0.001). The majority of 596 cases (356, 60%) were Aptima positive for other hr-HPV group including at least one type of hr-HPV 31, 33, 35, 39, 51, 52, 56, 58, 59, 66, and 68, followed by HPV16 (127, 26.1%), followed by HPV18/45 (57, 9.6%), and 26 (4.3%) had a double positive diagnosis for HPV16 and HPV18/45 [Tables 2 and 3].

Table 2 Distribution of age groups according to LSIL, HSIL, SCC, ADC, and Aptima Reports

Aptima test and cytology reports	Age groups*	χ2,†	P	
	(<20–29)	(30–39)	(40–49)	(50–59)	≥60			
LSIL (n=320)	52 (16.2)	111 (34.9)	110 (34.3)	41 (12.8)	5 (1.5)			
HPV16 (n=36, 11.2%)	6 (17)	17 (47.2)	11 (30.5)	1 (2.7)	1 (2.6)	32.083	0.042	
HPV18/45 (n=16, 5%)	4 (25)	5 (31.2)	5 (31.3)	2 (12.5)	0 (0)			
HPV16/18/45 (n=7, 2.18%)	1 (14.4)	3 (42.8)	3 (42.8)	0 (0)	0 (0)			
Other (n=108, 33.7%)	20 (18.5)	49 (45.4)	32 (29.6)	7 (6.5)	0 (0)			
Aptima negative (n=153, 47.9%)	22 (14.4)	37 (24.2)	59 (38.5)	31 (20.3)	4 (2.6)			
HSIL (n=144)	20 (13.9)	43 (29.9)	48 (33.3)	29 (20.1)	4 (2.8)			
HPV16 (n=49, 34%)	9 (18.3)	15 (30.6)	14 (28.6)	10 (20.4)	1 (2)	16.812	0.665	
HPV18/45 (n=11, 7.6%)	1 (9.1)	6 (54.5)	3 (27.3)	1 (9.1)	0 (0)			
HPV16/18/45 (n=6, 4.2%)	0 (0)	2 (33.3)	1 (16.7)	2 (33.3)	1 (16.7)			
Other (n=44, 30.6%)	7 (15.9)	12 (27.3)	17 (38.6)	8 (18.2)	0 (0)			
Aptima negative (n=34, 23.6%)	3 (8.8)	8 (23.5)	13 (38.2)	8 (23.5)	2 (6)			
SCC (n=13)	0 (0)	2 (15.3)	4 (30.8)	4 (30.8)	3 (23.1)			
HPV16 (n=6, 46.1%)	0 (0)	0 (0)	2 (33.3)	2 (33.3)	2 (33.3)	9.118	0.426	
HPV18/45 (n=4, 30.7%)	0 (0)	1 (25)	2 (50)	1 (25)	0 (0)			
HPV16/18/45 (n=0, 0%)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)			
Other (n=2, 15.3%)	0 (0)	1 (50)	0 (0)	1 (50)	0 (0)			
Aptima negative (n=1, 8%)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100)			
ADC (n=5)	0 (0)	0 (0)	1 (20)	2 (40)	2 (40)			
HPV16 (n=1, 20%)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100)	3.33	0.504	
HPV18/45 (n=0, 0%)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)			
HPV16/18/45 (n=0, 0%)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)			
Other (n=1, 20%)	0 (0)	0 (0)	0 (0)	1 (100)	0 (0)			
Aptima negative (n=3, 60%)	0 (0)	0 (0)	1 (33.3)	1 (33.3)	1 (33.3)			
*Age groups: presents as No (%). † Pearson Chi-square test. LSIL: Low-grade squamous intraepithelial lesion, HSIL: High-grade squamous intraepithelial lesion, SCC: Squamous cell carcinoma, ADC: Adenocarcinoma

Table 3 Distribution of age groups according to ASCUS, ASC-H, AGC, and Aptima Reports

Aptima test and cytology reports	Age groups*	χ 2,†	P	
	(<20–29)	(30–39)	(40–49)	(50–59)	≥60			
ASCUS (n=839)	117 (13.9)	249 (29.7)	282 (33.6)	145 (17.3)	46 (5.5)			
HPV16 (n=23, 2.7%)	6 (26.1)	7 (30.4)	7 (30.4)	3 (13.1)	0 (0)	29.906	0.071	
HPV18/45 (n=8, 0.95%)	2 (25)	5 (62.5)	0 (0)	1 (12.5)	0 (0)			
HPV16/18/45 (n=3, 0.36%)	1 (33.3)	1 (33.3)	0 (0)	1 (33.3)	0 (0)			
Other (n=95, 11.3%)	22 (23.2)	28 (29.5)	33 (34.7)	10 (10.5)	2 (2.1)			
Aptima negative (n=710, 84.6%)	86 (12.2)	208 (29.3)	242 (34)	130 (18.3)	44 (6.2)			
ASC-H (n=490)	45 (9.1)	146 (29.8)	172 (35.1)	93 (19)	34 (7)			
HPV16 (n=26, 5.3%)	6 (23)	13 (50)	4 (15.4)	2 (7.8)	1 (3.8)	57.750	0.001	
HPV18/45 (n=9, 1.9%)	5 (55.6)	2 (22.2)	0 (0)	1 (11.1)	1 (11.1)			
HPV16/18/45 (n=8, 1.6%)	1 (12.5)	6 (75)	1 (12.5)	0 (0)	0 (0)			
Other (n=60, 12.2%)	7 (11.7)	22 (36.7)	19 (31.7)	11 (18.3)	1 (1.6)			
Aptima negative (n=387, 79%)	26 (6.8)	103 (26.6)	148 (38.2)	79 (20.4)	31 (8)			
AGC (n=1023)	116 (11.3)	342 (33.4)	420 (41)	127 (12.4)	18 (1.9)			
HPV16 (n=16, 1.6%)	2 (12.5)	7 (43.7)	5 (31.3)	2 (12.5)	0 (0)	16.138	0.443	
HPV18/45 (n=9, 0.9%)	2 (22.2)	5 (55.6)	1 (11.1)	1 (11.1)	0 (0)			
HPV16/18/45 (n=2, 0.2%)	0 (0)	2 (100)	0 (0)	0 (0)	0 (0)			
Other (n=46, 4.5%)	5 (10.9)	21 (45.7)	15 (32.6)	3 (6.5)	2 (4.3)			
Aptima negative (n=950, 92.8%)	107 (11.3)	307 (32.3)	399 (42)	121 (12.7)	16 (1.7)			
*Age groups: Presents as No (%). †Pearson Chi-square test. ASCUS: Atypical squamous cells of undetermined significance, ASC-H: Atypical squamous cells that cannot exclude HSIL, AGC: Atypical glandular cell

Table 2 shows a detailed analysis of the Aptima test and cytology results and reveals that 124 (76.5%) of HSIL+ cases were Aptima positive. Cases comprised 13 (92.3%) SCC, 2 (40%) ADC, and 110 (76.4%) HSIL. Results also show that 167 (52.1%) of LSIL were Aptima-positive. Table 3 shows that 103 (21%) of ASC-H cases were Aptima positive, 129 (15.4%) of ASCUS, and 73 (7.1%) of AGC cases.

Figure 1 shows that from 124 HSIL+ cases, 56 (45%) had positive results for HPV16, followed by other hr-HPV groups (47 cases, 38%), followed by HPV18/45 (15 cases, 12%), followed by HPV16/18/45 (six cases, 5%). From 167 LSIL cases, 36 were HPV16 positive (21.6%), and 108 were positive for other hr-HPV scores (64%). From ASCUS/AGC/ASC-H cases combined (n = 305), 201 cases (65.9%) had positive results for other hr-HPV, and 65 cases (21.3%) had positive results for Aptima HPV16. Double infection was demonstrated as HPV16/18/45 was mainly seen in ASCUS, ASC-H, and AGC cases.

Figure 1 Four bar charts showing different Aptima HPV readings distributed according to age groups and cytology reports, including P value for Pearson Chi-Square test. Part (a) shows results for Aptima HPV16, (b) shows results for Aptima HPV18 and/or HPV45, (c) shows results for Aptima HPV18 and/or HPV45 and HPV16, and (d) shows results for Aptima Other hr-HPV types. HSIL+: high-grade squamous intraepithelial lesion and worse, LSIL: low-grade squamous intraepithelial lesion, ASCUS/ASCH/AGC: atypical squamous cells of undetermined significance, ASCH: atypical squamous cells that cannot exclude HSIL and AGC: Atypical glandular cells

Aptima-positive cases were mainly concentrated in age groups 30–39 (n = 230, 38.6%) and 40–49 (n = 175, 29.4%) [Figure 1], as this trend was reflected in all cytology diagnoses and Aptima HPV16, HPV16/18/45, and Aptima other hr-HPV groups, P = 0.001, P = 0.062, and P = 0.016, respectively. Two exceptions were observed, one for Aptima HPV18/45 positive cases, where 38 cases (66.7%) were concentrated in age groups (<20–29) and (30–39) and were clearly seen in ASCUS/AGC/ASC-H cases. The second exception was noted in the shift to age group (50–59), and ≥60 and 41 cases in these two groups were clearly observed in invasive carcinoma cases and were Aptima HPV16 positive.

DISCUSSION

This retrospective observational study evaluates the performance of the Aptima test in a cytology laboratory from 2017 to 2023. The study demonstrates that 21% of abnormal cytology samples and 92% of SCC cases were Aptima positive, and the least positive samples were in AGC diagnosis. It also shows that about 23.5% of HSIL+ cervical abnormalities were not HPV-infected. This finding agrees with Yang et al. 2022[3] and Zhang et al. 2022,[4] who showed that the Aptima testing identified 90–93% of invasive carcinoma cases and around 80% of HSIL.

The Aptima other hr-HPV group was the most positive HPV test in the LSIL, ASCUS, and ASC-H categories. Having other hr-HPV groups as the most detectable test is contrary to other studies that showed that Aptima HPV16 was first, HPV18/45 was second, and other hr-HPV groups were third, Zhang et al.[4] and Granados et al. 2017.[5] This outcome agrees with Mallik et al.[6] who carried out an Aptima-based study in the same center but on a smaller sample size observed over a two-year period and showed that the other hr-HPV group was the most detectable result.

The Aptima HPV16 test identified almost 50% of HSIL+ cases in this study. It agrees with Al-Awadhi et al.,[7] who studied HPV prevalence in Kuwait using polymerase chain reaction (PCR)-based sequencing and showed that the most prevalent hr-HPV genotype was HPV16, especially among HSIL+ cases.

In this study, almost 70% of women with Aptima positive outcomes were in age groups (30–39) and (40–49) and most of the women who were above the age of 50 years, and Aptima positive had HSIL+ cytology reports. This finding is contrary to other studies carried out by Granados et al.,[5] Maggino et al.[8] and Lindroth et al.[9] who showed that most Aptima-positive women were in their 20s, but the age groups in this study agree well with Al-Awadhi et al. study.[7] This could be explained by the fact that most published data were obtained from countries in Europe and the USA that have different religious and traditional backgrounds that affect the sexual lives of males and females.

Gardasil 9 HPV vaccination was introduced in Kuwait in May 2023 in six health district areas for males and females between the ages of 9 and 45 years.[1011] The nano-valent prophylactic vaccine is FDA-approved, has shown a nearly 95.7% efficacy in preventing the development of cervical cancer by targeting HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and is estimated to be effective for ten years after vaccination.[1011] The start of the nano-valent Gardasil HPV vaccine will have an effect on the studied population. It will form immunity and prevent persistent infection of six hr-HPV types (HPV 16, 18, 31, 33, 45, 52, and 58) and two low-risk (lr) HPV types (HPV 6 and 11) for about ten years after vaccination.[1011]

In this study, Gardasil will theoretically protect about 48% of women from developing HSIL+ lesions and 5.5% from developing ASCUS, AGC, and LSIL when taking into consideration results related to the Aptima HPV16, Aptima 18/45, and Aptima 16/18/45 tests. As for Aptima other hr-HPV groups, it is not known what exact HPV genotype is expressing E6/E7 mRNA; it could be one or more of the following genotypes: HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. According to Al-Awadhi et al.,[7] HPV33 was prevalent in 9.9% of abnormal samples, mainly in HSIL+ cases, HPV31 was prevalent in 3.9%, mainly in HSIL+ cases, HPV58 was prevalent in 5.3%, mainly in the ASC and LSIL cases, and there was no record for HPV52. As for the lr-HPV group, HPV11 was prevalent in 13.8% of samples, mainly in samples diagnosed as ASC and HPV6 was prevalent in 5.3% of samples, mainly concentrated in LSIL samples. This agrees with Giannella et al.,[12] who showed that the distribution of hr-HPV genotypes in women at the age of 45 and older is different from women at the age of 35 and less. They identified less common HPV genotypes from CIN3 lesions taken from women aged 45 and older who are not covered by the vaccine. Overall and based on rough estimation, Gardasil 9 could protect against 92% of HPV-related cervical abnormalities; 62% of them related to Aptima-detected hr-HPV, in addition to 30% related to HPV6, 11, and 58.

It was noted in this study that the proportion of ASCUS/ASC-H reports was 46.9% and AGC was 36.1%, which could be different than what is known worldwide, but these findings are almost in agreement with what was previously published from the same laboratory.[1314]

In conclusion, this study is the first of its kind in correlating age with Aptima hr-HPV and cytology findings in the Middle East and adds to previous knowledge related to the prevalence and Aptima testing of HPV. The outcome of this study could be used as a baseline for the Gardasil 9 vaccine and could be used to assess the effectiveness of the vaccine after three or five years. The limitations of this study are as follows: it is a single-center study and lacks PCR-based sequencing outcomes to know the exact HPV genotype. To overcome the limitations of this study the authors suggest carrying out an Aptima HPV and PCR-based sequencing comprehensive study, to include all nationwide cytology laboratories and to have the ability to sequence samples with Aptima hr-HPV readings to know the exact HPV genotype.

Key messages

Aptima hr-HPV testing detects persistent HPV infection in 76.5% of HSIL+ cases. Gardasil nano-valent vaccine provides immunity against 62% of Aptima-detected hr-HPV infections in addition to 30% low-risk (lr)-HPV in abnormal cytology samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgement

The authors would like to thank the staff of the cytology laboratory at Mubarak Al-Kabeer for their cooperation and support.
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