Self-collected versus clinician-collected samples for HSV-2 and HSV-2/HPV screening in HIV-infected and -uninfected women in the Tapajós region,Amazon,Brazil

Abstract

The aim of this study was to evaluate the prevalence of herpes simplex virus 2 (HSV-2) and HSV-2/human papillomavirus (HPV) co-infection by self-collected samples compared to clinician-collected samples in human immunodeficiency virus 1 (HIV-1)-infected and -uninfected women from the Tapajós region, Amazon, Brazil. A cross-sectional study was conducted with 439 anal and cervical scrapings and cervico-vaginal self-collected samples obtained from 153 eligible HIV-infected and -uninfected women. Real-time PCR for HSV-2 and nested PCR for HPV detection were performed. A multivariate analysis identified risk factors for HSV-2/HPV co-infection. The anogenital prevalence of HSV-2 was 9.2% (14/153), HPV was 67.3% (103/153) and HSV-2/HPV co-infection was 6.5% (10/153). There was a significant overall agreement (95.5%, 11/133, kappa 0.64, 95% CI 0.38–0.90, p < 0.0001) for HSV-2 detection by the self-collected and clinician-collected samples. HSV-2 genital infection was more prevalent than anal infection in all participants. HIV-infected women had a higher prevalence of HSV-2 and HSV-2/HPV. No woman with a cervical squamous intraepithelial lesion had HSV-2/HPV co-infection. Risk factors for HSV-2/HPV were age ≤25 years (aOR = 10.07) and being single (aOR = 3.79). In general, young and single women are at greater risk for HSV-2/HPV infection. Self-collection can be a useful strategy for the screening of HSV-2 and HPV in limited-resource settings.

Keywords

HSV-2 HPV HIV self-collected epidemiology

Introduction

The herpes simplex virus 2 (HSV-2), more recently referred to as human alphaherpesvirus 2 (HHV-2),¹ human immunodeficiency virus 1 (HIV-1) and human papillomavirus (HPV), among other sexually transmitted infections (STIs), represents a major burden for global health.² Herpesviruses establish life-long persistent infections and latency.³ HPV is the main etiological agent of cervical cancer, one of the most frequent occurring cancers in women worldwide. HPVs are DNA viruses with at least 200 different genotypes, of which 40 can infect the human anogenital tract. HPV and HSV-2 infections are more severe in HIV-1-infected individuals, and both viruses share similar routes and sites of infection.³ Several studies observed that women with HSV-2 have a higher risk of developing cervical carcinoma, and HSV-2 can be considered as a cofactor.^3,4 Evidence shows a possible synergism between HSV-2 and HPV in cervical cancer etiology.^5,6 However, some studies report from their findings that it is necessary to continue investigating whether HSV-2 may be a cofactor of cervical cancer.^7,8 HIV is a risk factor in HPV-associated cancers; HPV infection has also been suggested to be a risk factor for HIV infection.^3,9,10 Although Northern Brazil has the highest rates of cervical cancer, epidemiological data on STIs are still limited in that region, making it difficult to develop more broad and efficient strategies of prevention and control. In a previous study, the cervico-vaginal self-collection acceptability and HPV prevalence were evaluated in HIV-infected and -uninfected women from the Tapajós region, Amazon, Brazil.¹¹ Considering the role of HSV-2 infection in cervical cancer, the purpose of the study was to estimate the prevalence of HSV-2 and HSV-2/HPV co-infection by cervico-vaginal self-collection compared to clinician-collected samples in this population.

Methods

Study population

A cross-sectional study comprised of 153 non-indigenous HIV-infected and -uninfected women living in the Tapajós region, Amazon, Brazil. The Tapajós region has a territory of 281,082 mi² of the state of Pará in Northern Brazil and with almost two million inhabitants.¹¹ The main socioeconomic center and the best infrastructure in this region is Santarém, which supports health services for 22 neighboring municipalities.¹³ Women living in this region were recruited between August 2015 and August 2016 at nine public health units in Santarém, including a counseling and testing center, the only referral center for the care and follow-up of people living with HIV in this region.

Ethical approval and consent to participate

The study was approved by the Ethics and Research Committees of the Universidade do Estado do Pará (protocol permit number 1.099.852) and Instituto Oswaldo Cruz/Fiocruz (protocol permit number 1.059.253). All women agreed to participate in the study and signed an informed consent form in compliance with the Brazilian human ethical guidelines.

Data collection

This study was performed in a cohort of women who participated in a previous study, where the acceptability of the cervico-vaginal self-collection and HIV and HPV tests was evaluated.¹¹ All women signed a written informed consent form after understanding the study objectives explained by the physician. Participation in this study included the collection of sociodemographic and clinical data using a standardized form followed by the collection of specimens.

Specimen collection and DNA extraction

In the outpatient public health units, there were medical doctors, nurses, and nursing technicians who were denominated as health care providers. They were the ones who performed the collection of cervical and anal scrapings denominated, in this study, “clinician-collected samples”. Anal scrapings were collected with a sterile brush with protected tip (Kolplast, São Paulo, Brazil) for the collection of anal cells, and were immediately stored in ThinPrep (Hologic, Inc., San Diego, CA, USA). The term anal scraping refers to cells obtained by brushing in the anal canal and squamo-columnar junction. Cervical scrapings were collected with a sterile cervical brush with a protected tip (Kolplast, São Paulo, Brazil) for the collection of endocervical cells during the Pap smear. Clinical evaluation of clinician-collected samples using Pap smear cytological analysis was performed according to the routine procedures of the public health clinical centers participating in the study. Soon after, the women were invited to perform the cervico-vaginal self-collection, and those who agreed received an individual collection kit and specimen collection instructions with the aid of an illustrated, self-explanatory pamphlet, with step-by-step instructions. Cervico-vaginal self-collection was done immediately after the Pap smear. The procedure was done in a separate office within the health care provider’s office, where the patient performed the collection on their own. After the procedure, those women handed the cervico-vaginal self-collected sample immediately to the health care provider for identification and storage.^11,14,15 The collected specimens were identified and stored at 5°C and shipped to the Universidade Federal do Oeste do Pará, in Santarém, Pará, for storage until they were sent to the Laboratório de Virologia Molecular at the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, in Rio de Janeiro, Brazil. DNA extraction was performed with the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol.

HSV-2 detection

The HSV-2 DNA was detected by quantitative PCR (qPCR) using the primers, probes and synthetic curve previously described.¹⁶ Each reaction was performed in duplicate and contained 12.5 µL of TaqMan® Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 2 µM of each primer, 1.5 µM of probe, and DNase- and RNase-free H₂O. The cycling conditions were as follows: 95°C for 10 min (initial denaturation and polymerase activation) followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 60 s.

HPV detection and genotyping

A nested PCR was performed to increase the specificity of HPV DNA detection and genotyping. In the first round, PGMY09/11 primers were used and in the second round, GP5+ and GP6+ primers were used as previously described.¹⁷ HPV genotyping was done according to a previous study.¹¹

Statistical analysis

Descriptive statistics of the qualitative variables were determined by frequency distribution and quantitative variables by medians and interquartile ranges (IQRs) or mean and standard deviation (SD). The Chi square test was used for categorical variables and Mann–Whitney U test for continuous variables, both at 95% confidence intervals (CIs) and p ≤ 0.05. For the identification of risk factors, initially, the univariate analysis was used and odds ratio (OR) was calculated; variables with p ≤ 0.20 were used in the multivariate analysis. The agreement rate between cervico-vaginal self-collection and cervical scraping for HSV-2 and HSV-2/HPV detection was performed using the kappa test with 95% CI and p ≤ 0.05. The statistical analyses were carried out using SPSS version 19.0 (IBM Corp., Armonk, NY, USA).

Results

Anal and cervical scrapings and cervico-vaginal self-collected samples were obtained from 153 eligible women who agreed to participate in the study. Participants were divided into HIV-infected women (N = 41) and HIV-uninfected women (N = 112). Twenty women did not accept to perform the cervico-vaginal self-collection. The overall prevalence found was 9.2% (14/153) for HSV-2 infection, 67.3% (103/153) for HPV infection and 6.5% (10/153) for HSV-2/HPV co-infection, considering positivity in at least one of the three clinical sample types (Table 1).

Table 1.

Prevalence of HSV-2, HPV and HSV-2/HPV co-infection in HIV-infected and HIV-uninfected women.

Sample type	Anal scraping		Cervical scraping		Cervico-vaginal self-collection		At least one of the three clinical sample types
Sample type	HIV+(N = 41)n/N (%)	HIV−(N = 112)n/N (%)	HIV+(N = 41)n/N (%)	HIV−(N = 112)n/N (%)	HIV+(N = 39)n/N (%)	HIV−(N = 94)n/N (%)	HIV+(N = 41)n/N (%)	HIV−(N = 112)n/N (%)
HSV-2	1/41(2.4%)	1/112(0.9%)	2/41(4.9%)	7/112(6.3%)	4/39(10.3%)	7/94(7.4%)	4/41(9.8%)	10/112(8.9%)
HPV	40/41(97.6%)	35/112(31.3%)	39/41(95.1%)	37/112(33.0%)	38/39(97.4%)	37/94(39.4%)	41/41(100.0%)	62/112(55.4%)
HSV-2/HPV	1/41(2.4%)	0/112(0.0%)	2/41(4.9%)	3/112(2.7%)	4/39(10.3%)	5/94(5.3%)	4/41(9.8%)	6/112(5.4%)

HIV+: HIV-infected women; HIV−: HIV-uninfected women.

The prevalence among groups of women and by clinical sample type is detailed in Table 1. Genital infection was more prevalent than anal infection in all participants, and HIV-infected women had a higher prevalence of HSV-2, HPV and HSV-2/HPV co-infection (Table 1), but there was no statistically significant difference between the groups (Figure 1).

Figure 1.

Number of HSV-2-infected and HSV-2/HPV co-infected women in the HIV-uninfected and HIV-infected groups. Chi square test was performed for HSV-2/HPV co-infection between the groups.

There was a high and significant overall agreement rate for HSV-2 detection of 95.5%, 11/133, kappa 0.64 (95% CI 0.38–0.90), p < 0.0001 and for HSV-2/HPV co-infection detection of 96.2%, 9/133, kappa 0.59 (95% CI 0.28–0.91), p < 0.0001 by the cervico-vaginal self-collected and clinician-collected samples (cervical scraping) for all participants, respectively. Additionally, HIV-infected and HIV-uninfected women were analyzed separately, and a similar agreement rate was found for HSV-2 detection of 94.9%, 4/39, kappa 0.64 (95% CI 0.19–1.00), p < 0.0001 and 95.7%, 7/94, kappa 0.65 (95% CI 0.32–0.96), p < 0.0001, respectively; for HSV-2/HPV co-infection of 94.9%, 4/39, kappa 0.64 (95% CI 0.19–1.00), p < 0.0001 and 96.87%, 5/94, kappa 0.56 (95% CI 0.18–0.99), p < 0.0001, respectively.

All HIV-uninfected women had negative cytology. Of the HIV-infected women, 12.2% (5/41) had cytological abnormalities with three cases (7.3%) of low-grade squamous intraepithelial lesion (LSIL), and two cases (4.8%) of high-grade squamous intraepithelial lesion (HSIL). Women diagnosed with HSIL were referred for colposcopy and biopsy for confirmation and medical care follow-up. No woman had clinical signs of genital or anal ulcers. All HIV-infected women with abnormal cytology had high-risk HPV, but were HSV-2/HPV co-infection negative, so the statistic correlation between cervical disease and HSV-2/HPV could not be performed (Table 2).

Table 2.

Cytological results, CD4+ T-cell counts and viral load in HIV-infected women who tested negative and positive for HSV-2/HPV co-infection considering positivity in at least one of the three clinical sample types.

Categorical variables	HSV-2/HPV negative co-infection n/N (%)	HSV-2/HPV positive co-infection n/N (%)	p-value^a
Cytological results (N=153)^b			–*
Negative	138/143 (96.5%)	10/10 (100.0%)
LSIL	03/143 (2.1%)	0 (0.0)
HSIL	02/143 (1.4%)	0 (0.0)
HIV viral load (N=41)^c			0.245
<40 copies/mL	29/37 (78.4%)	02/04 (50.0%)
≥40 copies/mL	08/37 (21.6%)	02/04 (50.0%)
CD4+ T-cell counts (N=41)^c			–*
<200 cells/mm³	08/37 (21.6%)	00/04 (0.0%)
200–500 cells/mm³	17/37 (45.9%)	03/04 (75.0%)
>500 cells/mm³	12/37 (32.4%)	01/04 (25.0%)
Continuous variables	Median (IQR)	Median (IQR)	p-value^d
HIV viral load (N=41)^c	39 (0)	52.2 (8135.25)	0.457
CD4+ T-cell counts (N=41)^c	365 (336)	469.5 (579.25)	0.382

LSIL: low grade squamous intraepithelial lesion; HSIL: high grade squamous intraepithelial lesion; IQR: interquartile ranges.

^aPearson Chi square test was used for categorical variables.

^bOverall women (HIV-infected women and HIV-uninfected women).

^cOnly HIV-infected women.

^dMann–Whitney U test was used for continuous variables by medians and IQR, 95% CI.

*A statistical test could not be performed, because there are houses with zero value.

All HIV-infected women were on combination antiretroviral therapy (cART), and no significant association was found between HIV viral load categories or median and HSV-2/HPV co-infection (p = 0.245; p = 0.457); and also, no significant association was found between median CD4+ T-cell count and HSV-2/HPV co-infection (p = 0.382) (Table 2).

Univariate analysis showed the following associated variables (p ≤ 0.20) significantly increasing the risk of having HSV-2/HPV co-infection (Table 3): age ≤25 years (OR = 7.02, 95% CI = 1.84–26.72), being employed or student (OR = 3.05, 95% CI = 0.76–12.27), single marital status (OR = 3.32, CI 95% = 0.80–13.84), education 18 years or more (OR = 2.61, CI 95% = 0.62–10.95) and no pregnancy (OR = 4.39, CI 95% = 0.99–19.53). The multivariate analysis identified the following risk factors for HSV-2-HPV co-infection in the final model (p ≤ 0.05): age ≤25 years (aOR = 10.07; 95% CI 2.28–44.59) and single marital status (aOR = 3.79; 95% IC 0.84–17.01). All the variables for uni- and multivariate analyses included in this study are depicted in Table 3.

Table 3.

Univariate and multivariate analyses of risk factors for HSV-2/HPV-coinfected women considering positivity in at least one of the three clinical sample types.

	HSV-2/HPV co-infection
Variables	Total(153)	Non (%)	Yesn (%)	p-value^a	Bivariate models OR (95% CI)	Multivariable modelaOR (95% CI)
HIV status				0.459
HIV-uninfected	112	106 (94.6)	6 (5.4)		1.00	–
HIV-infected	41	37 (90.2)	4 (9.8)		1.91 (0.51–7.15)	–
Age (years)				0.005^b
≤25	31	25 (80.6)	6 (19.4)		7.02 (1.84–26.72)^b	10.07 (2.28–44.59)
26+	121	117 (96.7)	4 (3.3)		1.00	1.00
Employment status				0.186^b
Unemployed	84	81 (96.4)	3 (3.6)		1.00	–
Employed or student	69	62 (89.9)	7 (10.1)		3.05 (0.76–12.27)^b	–
Marital status				0.155^b
Single	59	53 (89.8)	6 (10.2)		3.32 (0.80–13.84)^b	3.79 (0.84–17.01)
Married/living together	91	88 (96.7)	3 (3.3)		1.00	1.00
Education (years)				0.177^b
Up to 17	129	122 (94.6)	7 (5.4)		1.00	–
18+	23	20 (87.0)	3 (13.0)		2.61 (0.62–10.95)^b	–
Pregnancies				0.071^b
None	17	14 (82.4)	3 (17.6)		4.39 (0.99–19.53)^b	–
1+	129	123 (95.3)	6 (4.7)		1.00	–
Hormonal contraception				0.770
No	67	63 (94.0)	4 (6.0)		1.00	–
Yes	46	42 (91.3)	4 (8.7)		1.5 (0.36–6.33)	–
No information	40	38 (95.0)	2 (5.0)		0.83 (0.15–4.74)	–
Alcohol use				0.724
No	106	98 (92.5)	8 (7.5)		1.80 (0.37–8.81)	–
Yes	46	44 (95.7)	2 (4.3)		1.00	–
Tobacco use				1.000
No	130	121 (93.1)	9 (6.9)		1.56 (0.18–12.9)	–
Yes	22	21 (95.5)	1 (4.5)		1.00	–
Age of first sexual intercourse (years)				–^*
Up to 17	104	94 (90.4)	10 (9.6)		–	–
18+	48	48 (100.0)	0 (0.0)		–	–
Lifetime number sex partners				0.756
1–3	68	63 (92.6)	5 (7.4)		1.22 (0.34–4.41)	–
4+	82	77 (93.9)	5 (6.1)		1.00	–
Condom use				1.000
No	127	118 (92.9)	9 (7.1)		1.83 (0.22–15.13)	–
Yes	25	24 (96.0)	1 (4.0)		1.00	–
History of STI				0.724
No	105	97 (92.4)	8 (7.6)		1.81 (0.37–8.89)	–
Yes	46	44 (95.7)	2 (4.3)		1.00	–

Note: Complete case analysis; final model fit by finding the model with the lowest AIC and OR adjusted by the variables that continued in the final model.

aOR: adjusted odds ratio; OR: odds ratio.

^aPearson Chi square test.

^bp-value <0.20 in bivariate models.

*A statistical test could not be performed.

Discussion

This is the first study on HSV-2 infection and HSV-2/HPV co-infection detected by molecular testing in cervico-vaginal self-collected samples compared to clinician-collected samples in women living in the Tapajós region, Amazon, Brazil. In the present study, the HSV-2 DNA was detected in 9.2% of the participants without any clinical signs of genital or anal ulcers. The overall prevalence of HSV-2 found in this study was slightly lower than the worldwide prevalence of HSV-2 in women (14.8%).¹⁸ Few studies are available about the prevalence of HSV-2 in Brazil; the data are divided into serological or molecular diagnostic testing in specific populations, which makes it difficult to completely understand the impact of HSV-2 in the country. A prevalence of 10.9% of HSV-2 DNA was identified in asymptomatic HIV-infected women, whereas 5.7% of HSV-2 DNA was detected in HIV-infected pregnant women from Southern and Southeastern Brazil, respectively,^19,20 in contrast to 65.2% of HSV-2 DNA detected in women with genital ulcer disease in the Brazilian Amazon, Northern Brazil.²¹ Moreover, a seroprevalence of 72.1% of HSV-2 was identified in women living at the borderlands in the Brazilian Amazon.²² HSV-2 infection is also an important infection in its own right, because it can cause recurrent genital ulcer disease, and is potentially fatal when transmitted to neonates, and likely contributes substantially to the spread of HIV infection.¹⁸

The overall prevalence of HSV-2/HPV co-infection was 6.5%. Some authors described that HSV-2-infected and HSV-2/HPV-co-infected women can have an increased risk of developing cervical cancer and cervical intraepithelial neoplasia (CIN).^3,23,24 Moreover, HSV-2 co-infection with the high-risk HPV types 16 and 18 has been observed in approximately 25–30% of women with CIN, and 13–25% of invasive cervical squamous cell carcinoma and adenocarcinomas compared to 0–4% of normal cervical tissues.²⁴ Several biological and environmental cofactors have been implicated in the development of HPV-associated cancer that include immune status, hormonal changes, parity, dietary habits, tobacco usage, and co-infection with other sexually transmissible agents, like HIV and HSV-2 infection. In fact, epidemiological studies suggested that other concomitant STIs also contribute to an increased risk of cervical and anal cancer development and that non-HPV STIs exert a synergistic action with high-risk HPV types and act as co-factors in the cancer progression.^5,25 Although some studies have found no evidence of the association between HSV-2 and the development of cervical cancer, the scientific community recommends the need to continue investigating whether infection with this virus can be a cofactor in the development of cervical and anal cancer.^7,8

This study demonstrated that HIV-infected women had a higher prevalence of HSV-2, HPV, and HSV-2/HPV co-infection compared to HIV-uninfected women. This finding was similar to what has been observed in a clinic-based population of African-Caribbean women in Toronto where HSV-2 and HPV infection were markedly more frequent in HIV-positive than HIV-negative women.²⁶ Another study observed a higher prevalence of HSV-2 in HIV-infected patients suggesting that the HSV-2 can be a gateway to HIV, and conversely, HIV can be a risk factor for the acquisition of herpes.²⁷

The cervico-vaginal self-collection had a high and significant overall agreement rate for HSV-2 DNA detection and HSV-2/HPV co-infection detection when compared to clinician-collected samples (cervical scraping). This finding corroborates other studies, where self-collected samples that could be collected easily at home were a sensitive and efficient method to molecularly detect virus.^28–31 Such inclusion of genital sites other than the cervix can be important.

It is well established in the literature that young women may be more vulnerable to sexual activity in unstable relationships, and who are at higher risk of HSV-2 infection and other STIs.^19,32,33 Therefore, the risk factors identified in this study are in agreement with the other scientific reports. This study reinforces the importance of such an approach in adolescents and young women in controlling STIs.

Study limitation

This study had limitations to be addressed. The cross-sectional epidemiological design does not allow us to establish a temporal relationship between the exposure and causal effect, as well as the small sample size of the HIV-infected women enrolled in this study, which may have been influenced by the difficulty of accessing those women in health units in Santarém. However, the lack of data in the literature on the prevalence of HIV and HPV infection for this population of women is one of the strengths of the study. Accurate estimates are crucial for guiding broader STI prevention and control efforts, primarily in limited-resource settings at higher risk and with very few scientific data available. New cases of HIV infection in young people have increased in recent years in Brazil;³⁴ moreover, in the Northern of the country, where cervical cancer is the most incident and of higher mortality.³⁵

Conclusion

Cervico-vaginal self-collection can be a new technology for diagnostics implementation in prevention and control programs for STIs. The unavailability of laboratory STI testing and inaccessible medical appointments are real barriers for the access of early diagnosis of STI in the Tapajós region. Women from this region of Brazil are in urgent need of a method such as cervico-vaginal self-collection, that can simultaneously detect HSV-2, HPV, and other STIs, since early diagnosis may decrease the risk of HIV acquisition and its transmission, decrease HSV-2 mother-to-child transmission (MTCT) risk especially among HIV-infected women, besides being an important tool for the screening and prevention of cervical cancer.

Footnotes

Acknowledgments

The authors thank all the patients participated in the study, the Post-graduation in Tropical Medicine at the Oswaldo Cruz Institute and the Universidade Federal do Oeste do Pará for all the support, the Secretaria Municipal de Saúde for having authorized the recruitment of the women in the health units, Dr Charlotte A Gaydos for the mentorship and Justin Hardick for all support during training and experiments of the Luana LS Rodrigues as a doctoral student at Johns Hopkins University, Division of Infectious Diseases.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; Programa Estratégico de Apoio a Pesquisa em Saúde (PAPES VII), Fundação Oswaldo Cruz (FIOCRUZ)/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 401810/2015–1]; and National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH) [grant number U54EB007958]. CAPES Foundation, PAPES VII, FIOCRUZ/CNPq, and NIBIB, NIH were not involved in the study design, in data collection and interpretation, in the design and writing of this manuscript, or in the decision to submit the manuscript for publication.

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