Abstract
Objective: The distribution of human papillomavirus (HPV) genotypes varies by countries and regions. Data regarding HPV genotype distribution for women with cervical intraepithelial neoplasia 2/3 (CIN2/3) are still limited. This study aimed to investigate the distribution of HPV genotypes for women with CIN2/3 in Shanxi province, China.
Methods: Prior HPV testing/genotyping resulted from women with histologic diagnosis of CIN2/3 were retrospectively collected between August 2011 and August 2016.
Results: A total of 1387 women with a diagnosis of CIN2/3 were included in this study. The average age of the women was 45.7 years and the average time period from HPV testing to histological diagnosis was 0.4 months. The overall high-risk HPV (hrHPV) positive rate was 91.6%. In all tested women, the most commonly detected hrHPV genotype was HPV16 (59.3%) followed by HPV58 (14.4%) and HPV33 (10.0%), while HPV18 was in the 6th position (6.0%).
Conclusions: This is one of the largest studies of HPV genotyping for women with CIN2/3 in China. The prevalence and distribution of HPV genotypes in these Chinese women differed from those in women from Western countries. Our results provide important information for developing specific HPV vaccines for Chinese women.
Keywords: HPV genotyping, Neoplasia 2/3, Prevalence
Introduction
Cervical cancer is the fourth most common cancer among women worldwide in terms of morbidity and mortality. More than 85% of cervical cancers occur in the developing countries.1 China accounts for 14% of the world's annual incidence of cervical cancer, and hence it remains an important public health problem in China. 2, 3 The incidence of cervical cancer in Shanxi Province, China is strikingly high.4 Persistent infection of high-risk Human Papillomavirus (hrHPV) infection is critical for the development of almost all cervical cancers and precancerous lesions.5 A meta-analysis of 48 studies showed that HPV16 and HPV18 are the most prevalent genotypes worldwide.6 It has been shown that the distribution of HPV genotypes in cervical cancers varies by countries and regions.7 China is a large country with varied socioeconomic conditions among different regions, and HPV infections differ accordingly.8 A large study from China, which included 51,345 women, has demonstrated three most common hrHPV types were HPV52, 16, and 58, while HPV18 was only the 6thmost common type.9
HPV vaccination is one of the most important measures to prevent HPV infection.10 However, all currently available vaccines were developed based on the epidemiological data from Western countries, and as a result, target HPV16, HPV18 and several other commonly identified subtypes in Western countries.11, 12, 13 Implementation of these vaccines in China may not be able to achieve the desired preventive efficacy. Therefore, in order to develop HPV vaccines specific for Chinese women, it is necessary to investigate HPV genotype distributions in China.
Materials and Methods
Study participants
The cases with histological diagnosis of CIN2/3 rendered between August 2011 and August 2016 were retrospectively searched in the patient information system at Second Hospital of Shanxi Medical University (SHSMU). Only the cases with prior HPV genotyping results in the preceding 6 months were included in the study and analyzed. Other clinical information, including age and clinical history were also collected. Histopathologic diagnoses were reported on samples including endocervical curettage specimens, cervical biopsies, loop electrosurgical excision procedures or cold-knife conization. The Second Hospital of Shanxi Medical University is a large, integrated, public health system located in the capital of Shanxi, which accommodates patients from all over Shanxi province.
HPV Geno-array test
HPV detection and genotyping was tested using HybriMax HPV Geno-Array kit (Hybribio Biotechnology Limited Corp., Chaozhou, China) at the Second Hospital of Shanxi Medical University laboratory. The HPV Geno-Array can determine 21 HPV types, including 15 hrHPV types(16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and 6 low risk HPV (lrHPV) types (6, 11, 41, 42, 44, and CP8304).Test was performed according to the manufacturer's instructions. 14 Briefly, PCR was performed in a 25 μL reaction mixture containing 5 μL extracted DNA, 0.75 μL DNA Taq polymerase and 19.25 μL PCR-mix solution containing MY09/11 primer system. The PCR protocol was: denaturing at 95 °C for 9 min, followed by 40 cycles of 20s at 95 °C, 30s at 55 °C, and 30s at 72 °C, at last a finally extension at 72 °C for 5 min. A positive control and a negative control were run in each PCR analysis process to control for possible contamination and accuracy. The kit can simultaneously identify 21 HPV genotypes: adding NBT/BCIP solution to display the results, a positive result was indicated by a clearly visible indigo dot. The HPV-genotype result was determined by the position of the HPV-genotype probes on the microarray chip. The kit can simultaneously identify 21 HPV genotypes: adding NBT/BCIP solution to display the results, a positive result was indicated by a clearly visible indigo dot. The HPV-genotype result was determined by the position of the HPV-genotype probes on the microarray chip. Multiple dots indicated multiple infections. 15
Colposcopy and cervical histopathologic diagnosis
Colposcopy exams were performed by specialists according to a standardized protocol. During the exam, all visually abnormal areas were biopsied. The squamocolumnar junction was biopsied (“random biopsy”) if no visible lesion identified. Endocervical curettage (ECC) was then performed according to the aforementioned indications.
Statistical analysis
The Pearson chi-square test was used for statistical analysis, which was conducted with SAS 9.1 software (SAS Institute, Cary, North Carolina). A P value less than. 05 was considered statistically significant.
Results
Overall HPV prevalence
A total of 1,387 CIN2/3 cases with HPV genotyping results within 6 months prior to histological diagnosis were included in the study. The overall hrHPV positive rate was 91.9% (1274/1387). The average time period from HPV testing to histological diagnosis was 0.4 months (range: 0.1-6 months) and the average age was 45.7 years (range: 21-83 years).
Distribution of single and multiple hrHPV infections
Single hrHPV infection was identified in 65.0% of all women (902/1,387), accounting for 71.0% (902/1,271) of hrHPV positive cases. Infection with multiple hrHPV types was identified in 26.6% of all women, accounting for 29.0% of hrHPV positive cases. Among the women infected with multiple hrHPV types, infection of two genotypes was most commonly observed with an overall prevalence rate of 17.5%. Infection with three and more hrHPV types were found in 99 women with an overall prevalence rate of 7.1% (Table 1).
Table 1.
1387 cases with genotyping results 6 months before CIN2/3 diagnosis
| HPV infection | Positive Case | % |
|---|---|---|
| 1 HrHPV only | 902 | 65.0 |
| 2 HrHPV only | 244 | 17.5 |
| ≥3 HrHPV only | 96 | 6.9 |
| Any mixed Hr and LrHPV | 32 | 2.3 |
| LrHPV only | 10 | 0.7 |
| Negative (no both Hr and Lr HPV | 103 | 7.4 |
| Total | 1387 | 100% |
Abbreviations: HrHPV, high-risk human papillomavirus; LrHPV, low-risk human papillomavirus number; CIN2/3, cervical intraepithelial neoplasia 2/3.
HrHPV genotype distribution
The most commonly detected hrHPV genotype was HPV16 (59.3%), followed by HPV58 (14.4%) and HPV33 (10.0%), while HPV18 was in the 6th position (6.0%) (Table 2).
Table 2.
HPV genotypes screening test results 6 months before CIN2/3 diagnosis (1387 cases)
| HPV type | Case No | Percentage |
|---|---|---|
| HrHPV | ||
| 16 | 823 | 59.3 |
| 18 | 79 | 6.00 |
| 31 | 105 | 7.6 |
| 33 | 139 | 10.0 |
| 35 | 31 | 2.2 |
| 39 | 47 | 3.4 |
| 45 | 25 | 1.8 |
| 51 | 67 | 4.8 |
| 52 | 98 | 7.1 |
| 53 | 60 | 4.3 |
| 56 | 35 | 2.5 |
| 58 | 200 | 14.4 |
| 59 | 27 | 1.9 |
| 66 | 22 | 1.6 |
| 68 | 0 | 0.00 |
| LrHPV | ||
| 11 | 23 | 1.7 |
| 42 | 12 | 0.9 |
| 81 | 14 | 1.0 |
| 6 | 7 | 0.5 |
| 41 | 3 | 0.2 |
| 44 | 4 | 0.3 |
Abbreviations: HrHPV, high-risk human papillomavirus; LrHPV, low-risk human papillomavirus number; CIN2/3, cervical intraepithelial neoplasia 2/3.No, number.
HrHPV infection among women of different Ages
All the 1,387 cases were divided into three age groups (<40 years, 40-49 years, and ≥50 years). The overall prevalence of hrHPV was 93.2% in women<40 years, 90.0% in women 40-49 years, and 92.2% in women ≥50 years. HrHPV positive rates showed no significant difference among these three age groups (p=0.1978). Additionally, the positive rates of HPV16 in the three groups were also similar and were 59.2% in women <40 years, 58.2% in women 40-49 years, and 60.6% in women ≥50 years respectively (p=0.7273).
Discussion
Epidemiological studies have suggested a substantial difference in the prevalence and genotype distribution of hrHPV infection among women from different countries. 16,17,18,19,20 Several large studies from China have demonstrated hrHPV prevalence rates in general population range from 9.9% to 21.1%. 9, 21, 22 However, studies of hrHPV infection in Chinese women with CIN2/3 were lacking. A study has reported that approximately 82.4% of cervical precancerous lesions and 92% of cervical invasive carcinoma are HPV-positive23. Zhao et al. reported that all 12 cases of cervical cancer and 31 cases of CIN3, and 95.3% of CIN2 (43 cases) were hrHPV positive.24 To our knowledge, the current study is one of the largest studies to investigate hrHPV prevalence in Chinese women with CIN2/3, demonstrating that 91.6% of these women were hrHPV positive.
In consistent with previous reports,25, 26 single HPV type infection is the most common infection in the current study, followed by the HPV infection with two types. Infection with 3 or more HPV types is rare. It remains exclusive whether HPV infection with multiple types is linked to occurrence or progression of cervical cancerous/precancerous lesions. One study suggested HPV infection with multiple types has increased risk for cervical cancerous/precancerous lesions27, while other studies did not show any increased risk for HPV infection with multiple types. 28, 29 Further studies are still warranted to elucidate the role of infection with multiple HPV types in the development of cervical cancer.
HPV genotyping results for Chinese women with CIN2/3 are limited. The current study demonstrated the most commonly detected hrHPV genotype was HPV16 (59.3%), followed by HPV58 (14.4%) and HPV33 (10.0%), while HPV18 was only in the 6th position (6.0%). HPV16 was the predominant type among women with CIN2/3 in this study, consistent with previous studies from Western countries. 30,31 However, the 2nd and 3rd most commonly detected types in current study were HPV58 and HPV33, which are different from the results observed in women from Western countries. 32, 33, 34, 35HPV 16, HPV 33, and HPV 52 have been reported to be the most prevalent HPV types in Chinese women, however, the distribution of HPV genotype has been shown to be different from regions to regions in China. 9, 36, 37, 38A study of 6723 subjects in four large cities (Beijing, Shanghai, Shenzhen, and Shenyang) and four rural areas (Shanxi: Xiangyuan, Yangcheng, Henan Xinmi, and Xinjiang: Hotan) in China demonstrated HPV16 is the most common type, followed by HPV58 and HPV18. 39 However, all these results were derived from general Chinese population, none of the studies specifically investigated hrHPV genotyping in women with cervical precancerous/cancerous lesions. Our current study is the one of the largest to investigate hrHPV genotype distribution among women with CIN2/3, showing the top three most common types were HPV16 (59.3%), HPV58 (14.4%) and HPV33 (10.0%), while HPV18 was in the 6th position (6.0%). A recent study indicated that the prevalence of HPV58 was 22.1% and 21.1% among HIV-1 positive women with CIN2 in Burkina Faso and South Africa respectively, and 23.1% and 15.1% among women with CIN3 and above lesions.40
In China, the development and application of HPV vaccines still face many challenges. Our study demonstrated that, even though HPV16 was the predominant type of HPV among Chinese women with CIN2/3, the following most common HPV types were not exactly the same as those found in other countries or regions. These data may provide a basis for formulating cervical cancer prevention strategies in China.
On April 24, 2014, the US Food and Drug Administration (FDA) approved a proposal by Roche Molecular Systems, (HPV) test as an alternate primary cervical cancer screening method for women aged 25 to 65 years. Current study showed 8% CIN2/3 patients had a negative HPV testing result before the histological diagnosis of high grade dysplasia, indicating the limitation of HPV testing as primary cervical cancer screening.
In summary, current study is the one of the largest case studies of HPV genotyping in Chinese women with histological diagnosis of CIN2/3. The knowledge of variable hrHPV genotypes in different countries and regiones may be important for the development and application of HPV vaccines.
Acknowledgments
The study was supported by the grant from the Special Public Welfare Industry Research of National Health and Family Planning Commission (Beijing, China) (201402010).
References
- 1.Torre LA, Bray F, Siegel RL. et al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. doi: 10.3322/caac.21262. [DOI] [PubMed] [Google Scholar]
- 2.Bray F, Ren JS, Masuyer E, Ferlay J. Global estimates of cancer prevalence for sites in the adult population in 2008. Int J Cancer. 2013;132:1133–1145. doi: 10.1002/ijc.27711. [DOI] [PubMed] [Google Scholar]
- 3.Shi JF, Qiao YL, Smith JS. et al. Epidemiology and prevention of human papillomavirus and cervical cancer in China and Mongolia. Vaccine. 2008;26(Suppl 12):M53–59. doi: 10.1016/j.vaccine.2008.05.009. [DOI] [PubMed] [Google Scholar]
- 4.Wang X, Liu K, Yuan F. et al. Analysis of both high incidence of esophageal and cervical cancer in Yangcheng County, Shanxi Province. China Cancer. 2011;(4):259–261. [Google Scholar]
- 5.Walboomers JM, Jacobs MV, Manos MM. et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J of Pathol. 1999;189:12–19. doi: 10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
- 6.de Sanjose S, Diaz M, Castellsague X. et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. The Lancet Infectious diseases. 2007;7:453–459. doi: 10.1016/S1473-3099(07)70158-5. [DOI] [PubMed] [Google Scholar]
- 7.Clifford GM, Gallus S, Herrero R. et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet. 2005;366:991–998. doi: 10.1016/S0140-6736(05)67069-9. [DOI] [PubMed] [Google Scholar]
- 8.Zhang R, Velicer C, Chen W. et al. Human papillomavirus genotype distribution in cervical intraepithelial neoplasia grades 1 or worse among 4215 Chinese women in population-based study. Cancer Epidemiol. 2013;37(6):939–45. doi: 10.1016/j.canep.2013.10.005. [DOI] [PubMed] [Google Scholar]
- 9.Zeng Z, Yang H, Li Z. et al. Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory. J Cancer. 2016;7(9):1037–43. doi: 10.7150/jca.14971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Giuliano AR, Kreimer AR, de Sanjose S. The beginning of the end: vaccine prevention of HPV-driven cancers. J Natl Cancer Inst. 2015;107(6 ):djv128. doi: 10.1093/jnci/djv128. [DOI] [PubMed] [Google Scholar]
- 11.Burchell AN, Winer RL, de Sanjosé S. et al. Chapter 6: Epidemiology and transmission dynamics of genital HPV infection. Vaccine. 2006;24:S3. doi: 10.1016/j.vaccine.2006.05.031. /52-61. [DOI] [PubMed] [Google Scholar]
- 12.Sankaranarayanan R. HPV vaccination: The most pragmatic cervical cancer primary prevention strategy. Int J Gynaecol Obstet. 2015;131(Suppl 1):S33–35. doi: 10.1016/j.ijgo.2015.02.014. [DOI] [PubMed] [Google Scholar]
- 13.Skinner SR, Apter D, De Carvalho N, Human Expert review of vaccines. 2016; 15 (3):367-87. [DOI] [PubMed]
- 14.Tao P, Zheng W, Wang Y, Bian M. Sensitive HPV genotyping based on the flow-through hybridization and gene chip. J Biomed Biotechnol. 2012 doi: 10.1155/2012/938780. doi:10.1155/2012/938780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Xu XX, Zhou JS, Yuan SH. et al. Distribution of HPV genotype in invasive cervical carcinoma and cervical intraepithelial neoplasia in Zhejiang Province, Southeast China: establishing the baseline for surveillance. Int J Environ Res Public Health. 2015;12(9):10794–10805. doi: 10.3390/ijerph120910794. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Li N, Franceschi S, Howell-Jones R. et al. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication. Int J Cancer. 2011;128:927–935. doi: 10.1002/ijc.25396. [DOI] [PubMed] [Google Scholar]
- 17.Human papillomaviruses. In: IARC, Humans WHitEcoURt. Biological Agents. Volume 100 B. A review of human carcinogens. IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer. 2012;100:1–441. [Google Scholar]
- 18.Bruni L, Diaz M, Castellsague X. et al. Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis. 2010;202:1789–1799. doi: 10.1086/657321. [DOI] [PubMed] [Google Scholar]
- 19.Castellsagué X, de Sanjosé S, Aguado KS. WHO/ICO Information Centre on HPV and Cervical Cancer. HPV and cervical cancer in the 2007 report. Vaccine. 2007;25(Suppl 3):C1–230. doi: 10.1016/S0264-410X(07)01183-8. [DOI] [PubMed] [Google Scholar]
- 20.Jing L, Zhong X, Zhong Z. et al. Prevalence of human papillomavirus infection in Guangdong province, China: a population-based survey of 78,355 women. Sex Transm Dis. 2014;41:732–738. doi: 10.1097/OLQ.0000000000000201. [DOI] [PubMed] [Google Scholar]
- 21.Li C, Wu M, Wang J. et al. A population-based study on the risks of cervical lesion and human papillomavirus infection among women in Beijing, People's Republic of China. Cancer Epidemiol Biomarkers Prev. 2010;19:2655–64. doi: 10.1158/1055-9965.EPI-10-0212. [DOI] [PubMed] [Google Scholar]
- 22.Cairenzhuoma Zhang J, Wei C. Human papillomavirus infection in 26,622 women in Qinghai. Chin J Fam Plann. 2013;21(12):812–816. [Google Scholar]
- 23.Fernandes JV, Meissner RV, Carvalho MG. et al. Prevalence of human papillomavirus in archival samples obtained from patients with cervical pre-malignant and malignant lesions from Northeast Brazil. BMC Res Notes. 2010;3(1):96. doi: 10.1186/1756-0500-3-96. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Zhao F, Li Na, Ma J. et al. Study of the association between human papillomavirus infection and cervical cancer in Xiangyuan County, Shanxi province. Chin J Epidemiol. 2001;22(5):375–378. [PubMed] [Google Scholar]
- 25.Wang L, Li J, Chen L. Prevalence of human papillomavirus and its genotype among 1336 invasive cervical cancer patients in Hunan province, central south China. J Med Virol. 2015;87:516–521. doi: 10.1002/jmv.24094. [DOI] [PubMed] [Google Scholar]
- 26.Dickson EL, Vogel RI, Bliss RL. et al. Multiple-type human papillomavirus (HPV) infections: across-sectional analysis of the prevalence of specific types in 309,000 women referred for HPV testing at the time of cervical cytology. Int J Gynecol Cancer. 2013;23(7):1295–1302. doi: 10.1097/IGC.0b013e31829e9fb4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Fife KH, Cramer HM, Schroeder JM. et al. Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia. J Med Virol. 2001;64(4):550–559. doi: 10.1002/jmv.1085. [DOI] [PubMed] [Google Scholar]
- 28.Munoz N, Bosch FX, de-Sanjose S. et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348(6):518–527. doi: 10.1056/NEJMoa021641. [DOI] [PubMed] [Google Scholar]
- 29.An HJ, Cho NH, Lee SY. et al. Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method. Cancer. 2003;97( 7):1672–1680. doi: 10.1002/cncr.11235. [DOI] [PubMed] [Google Scholar]
- 30.Wright TC Jr, Stoler MH, Behrens CM, Sharma A, Sharma K, Apple R. Interlaboratory variation in the performance of liquid-based cytology: insights from the ATHENA trial. Int J Cancer. 2014;134(8):1835–1843. doi: 10.1002/ijc.28514. [DOI] [PubMed] [Google Scholar]
- 31.Kjær SK, Munk C, Junge J, Iftner T. Carcinogenic HPV prevalence and age-specific type distribution in40,382 women with normal cervical cytology, ASCUS/LSIL, HSIL, or cervical cancer: what is the potential for prevention? Cancer Causes Control. 2014;25(2):179–189. doi: 10.1007/s10552-013-0320-z. [DOI] [PubMed] [Google Scholar]
- 32.Peng Guan, Rebecca Howell-Jones, Ni Li. et al. Human papillomavirus types in 115,789 HPV-positive women: A meta-analysis from cervical infection to cancer. Int J Cancer. 2012;131:2349–2359. doi: 10.1002/ijc.27485. [DOI] [PubMed] [Google Scholar]
- 33.Human papillomaviruses. In: IARC, Humans WGotEoCRt: Biological agents. Volume 100 B. A review of human carcinogens. IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer. 2012;100:1–441. [Google Scholar]
- 34.Castellsagué X, de Sanjosé S, Aguado KS, HPV WIICo, Cervical C. HPV and cervical cancer in the 2007report. Vaccine. 2007;25(Suppl 3):C1–230. doi: 10.1016/S0264-410X(07)01183-8. [DOI] [PubMed] [Google Scholar]
- 35.Bosch FX, Burchell AN, Schiffman M. et al. Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia. Vaccine. 2008;26(Suppl10):K1–16. doi: 10.1016/j.vaccine.2008.05.064. [DOI] [PubMed] [Google Scholar]
- 36.G M Clifford, S Gallus, R Herrero. et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled Analysis. Lancet. 2005;366:991–998. doi: 10.1016/S0140-6736(05)67069-9. [DOI] [PubMed] [Google Scholar]
- 37.Ding X, Liu Z, Su J. et al. Human papillomavirus type-specific prevalence in women referred for colposcopic examination in Beijing. J Med Virol. 2014;86:1937–43. doi: 10.1002/jmv.24044. [DOI] [PubMed] [Google Scholar]
- 38.Chih HJ, Lee AH, Colville L. et al. A review of dietary prevention of human papillomavirus-related infection of the cervix and cervical intraepithelial neoplasia. Nutr Cancer. 2013;65(3):317–28. doi: 10.1080/01635581.2013.757630. [DOI] [PubMed] [Google Scholar]
- 39.Zhao Y, Zhao F, Hu S. et al. Multi-center cross-sectional study on type-specific human papillomavirus infection among Chinese women. Chin J Epidemiol. 2015;36(12):1351–1356. [PubMed] [Google Scholar]
- 40.Kelly HA, Ngou J, Chikandiwa A. et al. Associations of Human Papillomavirus (HPV) genotypes with high-grade cervical neoplasia (CIN2+) in a cohort of women living with HIV in Burkina Faso and South Africa. PLoS One. 2017;12(3):e0174117. doi: 10.1371/journal.pone.0174117. [DOI] [PMC free article] [PubMed] [Google Scholar]