High Risk Human Papillomavirus Infection and Abnormal Cervical Cytology among Nepali and Bhutanese-Nepali Refugee women in Eastern Nepal

Objective: The objective of this study was to assess and compare the prevalence of high risk human papillomavirus (HR-HPV) infection and cervical abnormalities between Nepali and Bhutanese-Nepali refugee women in Jhapa District in Eastern Nepal.

Methods: Study participants were recruited from a women’s health camp organized by NFCC International in 2014. Consenting participants were administered a demographic and health questionnaire, and both self-collected and clinician collected cervico-vaginal specimens were acquired. All samples were tested for any HR-HPV infection and for HPV16 and HPV18/45 specifically using E6/E7 mRNA Genotype Assays. Cervical cytology was categorized as being either “Normal” (benign cellular changes, results within normal limits, atypical squamous cells of undetermined significance (ASCUS), or actinomycosis) or “Abnormal” (low-grade squamous intraepithelial lesion (SIL), high-grade SIL, squamous cell carcinoma, or Atypical Glandular Cells of Undetermined Significance (AGUS) .

Results: Of the 647 study participants, 15.9% were Bhutanese-Nepali women. The overall age was 38.8±8.2 years, with Nepali women being significantly older than Bhutanese-Nepali women (39.5 vs. 34.9 years; p<.0001). The overall prevalence of any HR-HPV infection was 8.9% and abnormal cervical cytology was 7.1%, with no significant difference in any HR-HPV positivity (8.5 vs. 10.9%; p=0.4423) or abnormal cervical cytology (p=0.6755) between Nepali and Bhutanese-Nepali women. Among HR-HPV positive women, 41.7% were positive for HPV 16, 18 or 45 with no significant difference between the two groups (p=0.8474).

Conclusions: HR-HPV positivity and abnormal cervical cytology were similar among Nepali and Bhutanese-Nepali women and these results are comparable to results from our previous study in Far-western Nepal.



BACKGROUND


MATERIALS & METHODS

Study setting

This study was conducted in Damak Municipality in the Terai (lowland) district of Jhapa district in eastern Nepal. The estimated population of Jhapa District in 2014 was of 855,600. It is bordered by India in the south and east and has been the site of five of the seven of the United Nations administered refugee camps for Bhutanese-Nepali since early 1990s. The district, comparatively, is one of the most developed districts in Nepal with a high level of literacy, economic development, transportation infrastructure, and healthcare facilities.

Participant Recruitment and Consent Process

The study participants for the study were recruited from a reproductive health camp organized by NFCC, International in 2014. The NFCC, International worked with a local organization to coordinate advertising and outreach activities about the health camp to women in Damak Municipality and the Bhutanese Refugee Camps located near Damak. The health camp provided women various reproductive health services including gynecologic examination, STI testing, family planning counseling, and liquid cytology testing free of charge. Eligibility criteria for participation in the study included: being older than 18 years; able to provide informed consent; not menstruating; having a cervix; currently not being pregnant. Eligible women who were interested in participating in the study were read and explained the informed consent form, and a written informed consent was obtained before enrollment. Women who were not eligible or refused to participate in the study received the same health services as those participating in the study. Reproductive health services including pelvic examinations were performed by trained clinical professionals with several years of experience. Institutional review boards from Kent State University, the University of Alabama at Birmingham, and the Nepal Health Research Council approved the study.

Demographic and Health Risk Questionnaire

The participants in the study were administered a demographic and health risk questionnaire that we have developed and used in our previous studies in Nepal. The details of the development and the pilot testing of the questionnaire have been previously described. Briefly, the survey questions were first developed in English, then translated into Nepali, and then back translated back to English independently for quality assurance, cultural sensitivity and appropriateness. The survey questionnaire included questions related to socioeconomic indicators, behavioral risk, clinical, reproductive health, and migration related factors. The questionnaire was administered by trained native Nepali speaking research staff.

Biological Specimens and Laboratory Analyses

Study protocols for biological sample collection and laboratory analyses have been described in detailed previously. Briefly, during pelvic examinations trained auxiliary nurse midwives collected cervico-vaginal specimens using the APTIMA Cervical Specimen Collection and Transport (CSCT) kit (Hologic/Gen-Probe, San Diego, CA). Cervico-vaginal samples were transported to the Hologic/Gen-Probe, Inc. laboratory in San Diego for HPV testing. Laboratory testing of HPV was first performed using a generic APTIMA HR-HPV mRNA (APTIMA HPV) probe (Hologic/Gen-Probe, San Diego, CA) to detect the presence of E6/E7 mRNA from at least one of 14 different types of HR-HPV (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Samples with a positive HR-HPV test using the generic probe were then were retested with APTIMA HPV16 18/45 Genotype Assay to detect the presence of HPV16 or HPV18/45 genotypes. Cervico-vaginal samples for cervical cytology was collected in ThinPrep PreservCyt medium (Hologic/Gen-Probe, San Diego, CA) and assessed for research purposes with results classified according to the Bethesda criteria. Women with abnormal cervical cytology were referred for further evaluation, testing and clinical follow-up to local healthcare facilities.

Demographic Characteristics, Risk Behavioral Profile, and Sexually Transmitted Infections and Cervical Cancer-related Awareness and Knowledge

Demographic characteristics and behavioral risk profile assessed in the study included age, marital status, education, nationality, number of children, previously married or not, husband previously married or not, current alcohol use, current tobacco smoking, and whether husband has migrated for work inside Nepal to other districts or outside Nepal. STI and cervical cancer-related awareness and knowledge assessed in the study included: had ever heard of STIs; had ever been told she had an STI by a healthcare provider; had ever been told by a healthcare provider that the husband had an STI; heard of cervical cancer; source of knowledge about cervical cancer; know HPV as the cause of cervical cancer; know of HPV vaccine; and would vaccinate her children against HPV, if vaccine was free.

Outcomes and Risk Factors of Interest

HR-HPV infection and abnormal cervical cytology were the two outcomes of interested assessed in this study. HR-HPV infection was defined as testing positive for at least one of the following HPV types: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Cervical cytology was categorized as being either “Normal” or “Abnormal” based on Bethesda classification system. “Normal” cervical cytology included those samples classified as benign cellular changes, results within normal limits (WNL), atypical squamous cells of undetermined significance (ASCUS), or actinomycosis. “Abnormal” cervical cytology included low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma (SCC), atypical glandular cells of undetermined Significance (AGUS), or atypical squamous cells- cannot exclude high-grade (ASC-H).

Potential risk factors for HR-HPV infection and abnormal cervical cytology assessed in this study included: age (≥45 vs. <45 years); formal education (none vs. some); married more than once or ‘previously married’ (yes vs. no); husband married more than once or ‘husband previously married’ (yes vs. no); number of children (≤1, 2, 3 or ≥4); nationality (Nepali vs. Bhutanese-Nepali); drink alcohol (yes vs. no); smoke any types of tobacco (yes vs. no); husband migrated for work (no, yes- migrated outside the district, or yes-migrated outside the country). Assessment and classification of nationality was based on answers to two questions: country of birth or whether they lived in a refugee camp. A participant was classified as Bhutanese-Nepali if they either indicated that they were born in Bhutan or currently lived in one of the U.N. administered refugee camps in Nepal; otherwise, a participant was classified was a Nepali.

Statistical Analyses

Frequency distribution and proportions were computed for categorical variables and mean (± standard deviation) were compute for continuous variable to describe the demographic and risk behavioral profile and STI and cervical cancer knowledge for the overall and sample stratified by nationality. Prevalence and 95% confidence interval (95% CI) for HR-HPV infection and cervical cytology were computed for overall and by nationality. The relationship between independent variables described above and the two main outcome variables (HR-HPV infection and abnormal cervical cytology) was assessed using univariable and multivariable logistic regression models. Crude and adjusted Odds Ratio (OR) and corresponding 95% CI and p-value are reported. Multivariable regression model included the following independent variables: age; formal education ; woman previously married; husband previously married; number of children; nationality; drink alcohol; smoke any types of tobacco; and husband migrated for work. SAS version 9.3 (SAS Institute, Cary, NC) was used to perform all statistical analyses.



RESULTS

Demographic Characteristics and Risk Behavioral Profile

Of the 679 women who provided response to some or all the demographic questionnaire, 574 (84.1%) were Nepali and 105 (15.9%) were Bhutanese-Nepali. Table 1 provides the demographic and risk behavioral factor profile of the participants in the study. The mean age of the sample was 38.8 (±8.2) years with Nepali women significantly older than Bhutanese-Nepali women [39.5 (±8.1) vs. 35.0 (±7.8) years ; p <0.001]. Almost all the women were currently married, widowed or divorced, with a mean age at marriage of 19.2 (±4.0) years. Nepali women were significantly more likely to report being currently married compared to Bhutanese-Nepali women (98.3% vs. 92.8%; p=0.002). They also reported an older age at marriage than Bhutanese-Nepali women [19.2 (±4.0) vs. 18.0 (±3.9) years; p=0.001]. Bhutanese-Nepali were significantly more likely to report being married more than once (or previously married) (8.1% vs. 1.1%; p<0.001). About 11% of the women reported that their husband had been previously married. The average number of children reported was 2.4 (±1.3). About 29% of the women reported no formal education. There was no significant difference between Nepali and Bhutanese-Nepali women in regards to husband being previously married, the number of children, or the level of formal education.

The mean number of life-time sexual partner reported was 1.0, with no significant difference between two groups. The mean age at first sexual intercourse was 19.3 (±4.1) years with Bhutanese women [18.2 (±4.1) years] reporting a younger age than Nepali women [19.6 (±4.0) years]; a difference that coincided with the difference in the mean age of marriage. About 9% of the women reported drinking alcohol and currently smoking. Bhutanese-Nepali women were significantly more like to report drinking alcohol (17.4% vs. 7.1%; p<0.001) and smoking (15.1% vs. 8.0%; p=0.025) than Nepali women. Over a quarter of the women reported that their husbands had migrated for work, with 21% reporting migration outside the country. Nepali women were almost two-times more likely to reported spousal migration for employment than Bhutanese-Nepali women (27.4% vs. 14.7%; p=0.043).

Sexually Transmitted Infections and Cervical Cancer-related Knowledge

Overall, two-thirds of the respondents reported having heard of STIs, with no significant difference between Nepali and Bhutanese-Nepali women ( p=0.944) [Table 2]. Six-percent of the women reported a healthcare provider ever telling them that they had a STI, with Bhutanese-Nepali women two-times more likely to report as such (p=0.038). Two-third of the women reported having heard of cervical cancer and Nepali women were significantly more likely to report cervical cancer awareness than Bhutanese-Nepali women (p=0.021). Friends and government clinics were reported as the source of cervical cancer information by over tw0-thirds of the women. Almost 80% of the women did not know HPV as the cause of cervical cancer and 86% were not aware of the HPV vaccine. Ninety-six percent of the women reported that they would have their children vaccinated against HPV, if the vaccine were available for free. There was no significant difference between Nepali and Bhutanese-Nepali women in regards to their knowledge of cervical cancer cause and HPV vaccine, and their willingness to have their children vaccinated against HPV.

High-risk HPV Infection and Cervical Cytology

Of the 713 had a sample available for HR-HPV testing the prevalence of HR-HPV positivity was 8.4% (95% CI: 6.4 – 10.5%). When only the women with information on nationality were considered (n=641), the prevalence of HR-positivity was 8.9% (95% CI: 6.7 – 11.1%) with no significance difference between Nepali and Bhutanese-Nepali women (p=0.399). Among HR-HPV positive women, 41.7% were positive for HPV 16, 18 or 45 with no significant difference between the two groups (p=0.8474). Among HR-HPV positive women abnormal cervical cytology was 33.3% compared to 5.0% among HR-HPV negative women (p<0.001).

Of the 688 samples available for cervical cytology testing, 7.0% (n=48) of the sample yielded unsatisfactory results. The prevalence of abnormal cervical cytology in 640 the satisfactory samples was 7.3% (95% CI: 5.3 – 9.4%). When including only women with the information on nationality (n=576), the prevalence of abnormal cervical cytology was 7.2% with no significance difference between Nepali and Bhutanese-Nepali women ( p=0.698). There was also no significant difference in cervical cytology results among women with and without the nationality information (results not shown). Table 3 provides details on HR-HPV positivity and cervical cytology data among Nepali and Bhutanese-Nepali women.

In the univariable analyses, women younger than 45 years of age and women whose husbands had migrated outside Jhapa district for employment were significantly associated in HR-HPV positivity (Table 4). In the multivariable analysis, women whose husbands had migrated outside of the districts were 4.7 times (95% CI: 1.5 – 14.3) as likely to HR-HPV positive compared to women whose husbands had not migrated for employment (Table 5). Similarly, in the multivariable analysis, women who husband had migrated outside the country for work has 2.9 times the odds of having abnormal cervical cytology (95% CI: 1.3 – 6.6) compared to women whose husband had not. Currently alcohol use was marginally significantly associated with increased odds of abnormal cytology (OR=3.3; p=0.065) (Table 5).



Table 1. Demographic Characteristics and Risk Behavioral Profile a Sample of Nepali and Bhutanese-Nepali Women in Eastern Nepal, 2014

Frequency (%)

p- value

Characteristics

Overall*

679 (100.0)

Nepali

574 (84.1)

Nepali-Bhutanese

105 (15.9)

Age, years

<.0001

≤ 30

24 (3.5)

6 (1.1)

18 (17.4)

30-34

226 (33.3)

187 (32.6)

39 (37.1)

35-39

122 (18.0)

104 (18.1)

18 (17.1)

40-44

115 (16.9)

98 (17.1)

17 (16.2)

≥45

192 (28.3)

179 (31.2)

13 (12.4)

Formal education

None

187 (28.9)

150 (27.6)

37 (35.9)

0.0868

Some

460 (71.1)

394 (72.4)

66 (64.1)

Marital Status

Currently married

602 (97.4)

512 (98.3)

90 (92.8)

0.0018

Other**

16 (2.6)

9 (1.7)

7 (7.2)

Previously married

<0.0001

Yes

14 (2.2)

6 (1.1)

8 (8.1)

No

636 (97.9)

545 (98.9)

91 (91.9)

Husband previously married

0.8569

Yes

69 (10.6)

59 (10.7)

10 (10.1)

No

581 (89.4)

492 (89.3

89 (89.9)

Number of children

<0.0001

≤1

138 (20.9)

98 (17.5)

40 (40.4)

2

259 (39.3)

240 (42.9)

19 (19.2)

3

161 (24.4)

141 (25.2)

20 (20.2)

≥4

101 (15.3)

81 (14.5)

20 (20.2)

Drink alcohol

<0.001

Yes

55 (8.7)

38 (7.1)

17 (17.4)

No

578 (91.3)

497 (92.9)

81 (82.7)

Smoke

0.0245

Yes

60 (9.1)

45 (8.0)

15 (15.10)

No

603 (90.9)

518 (92.0)

85 (85.)

Husband migrated for work

0.0432

No

398 (74.5)

328 (72.6)

70 (85.3)

Outside the district

24 (4.5)

21 (4.6)

3 (3.7)

Outside the country

112 (21.0)

103 (22.8)

9 (11.0)

*Not all values may add to 679 due to missing data; *Includes single (n=1), divorced/separated (n=6), or widowed (n=9)



Table 2. Sexually Transmitted Infections and Cervical Cancer-related Knowledge among a Sample of Nepali and Bhutanese-Nepali Women in Eastern Nepal, 2014

Frequency (%)

p- value

Characteristics

Overall*

679 (100.0)

Nepali

574 (84.1)

Nepali-Bhutanese

105 (15.9)

Have heard of sexually transmitted infections

0.9444

Yes

407 (66.3)

343 (66.3)

64 (66.0)

No

207 (33.7)

174 (33.7)

33 (34.0)

Healthcare provide ever told the participant had an STI

0.0378

Yes

28 (5.8)

20 (4.9)

8 (11.1)

No

453 (94.2)

389 (95.1)

64 (88.9)

Healthcare provide ever told the participant’s husband had an STI

0.3686

Yes

15 (2.5)

14 (2.8)

1 (1.1)

No

579 (97.5)

492 (97.2)

87 (98.9)

Have heard of cervical cancer

0.0205

Yes

426 (66.7)

370 (68.5)

56 (56.6)

No

213 (33.3)

170 (31.5)

43 (43.3)

Source of cervical cancer information

0.0171

Family

15 (3.5)

12 (3.2)

3 (5.2)

Friends

144 (33.3)

128 (34.0)

16 (27.6)

Government clinic

146 (33.6)

125 (33.2)

21 (36.2)

NGO clinic

55 (12.6)

41 (10.9)

14 (24.1)

School

49 (11.3)

48 (12.7)

1 (1.7)

Other

26 (6.0)

23 (6.1)

3 (5.2)

Know HPV as the cause of cervical cancer

0.5512

Yes

128 (20.8)

110 (21.2)

18 (18.6)

No

487 (79.2)

408 (78.7)

79 (81.4)

Know of HPV vaccine

0.186

Yes

83 (13.6)

74 (14.2)

9 (9.4)

No

526 (86.4)

439 (85.6)

87 (90.6)

Would give children HPV vaccine if free

0.3335

Yes

18 (95.5)

14 (4.1)

4 (6.9)

No

385 (95.5)

331 (95.9)

54 (93.1)

*Not all values may add to 679 due to missing data;



Table 3. High-risk HPV infection and abnormal cervical cytology among Nepali and Nepali-Bhutanese women in Eastern Nepal

Characteristics

n (%)

p- value

Overall

Nepali

Nepali-Bhutanese

High-risk HPV Infection (n=641)

0.3993

No

584 (91.1)

496 (91.5)

88 (88.9)

Yes

57 (8.9)

46 (8.5)

11 (11.1)

Cervical Cytology Ɨ (n=576)

0.6982

Normal

535 (92.9)

458 (92.7)

77 (93.9)

Abnormal

41 (7.2)

36 (7.3)

5 (6.1)

Cytology results in detail

0.5387

Normal

WNL

473 (82.1)

406 (82.4)

66 (80.5)

ASCUS

62 (10.8)

51 (10.3)

11 (13.4)

Abnormal

ASC-H

7 (1.2)

5 (1.0)

2 (2.4)

AGUS

10 (1.7)

10 (2.0)

0 (0.0)

LSIL

20 (3.5)

18 (3.6)

2 (2.4)

HSIL

4 (0.7)

3 (0.6)

1 (1.2)

Ɨ Cervical Cytology Classification

“Normal” includes: benign cellular changes, WNL (results Within Normal Limits); ASCUS (Atypical Squamous Cells of Undetermined Significance, or actinomycosis

“Abnormal” includes: ASC-H (Atypical Squamous Cells- cannot exclude High-grade); LSIL (Low-grade Squamous Intraepithelial Lesion; HSIL (High-grade Squamous Intraepithelial Lesion); AGUS (Atypical Glandular Cells of Undetermined Significance); and Squamous Cell Carcinoma (none in this sample)



Table 4. Univariable Analysis of Risk Factors for High-risk HPV Infection among a Sample of Nepali and Bhutanese-Nepali Women in Eastern Nepal

Characteristics

High-risk HPV

n (%)

Odds Ratio

(95% Cl)

p- value

Positive

Negative

Age, years

≥45

9 (5.1)

166 (94.9)

1.00

<45

48 (10.3)

416 (89.7)

2.13 (1.02 – 4.44)

0.0438

Formal education

None

15 (8.1)

170 (91.9)

1.00

Some

42 (9.2)

414 (90.8)

1.15 (0.62 – 2.13)

0.6570

Current marital Status

Other**

0 (0.0)

15 (100.0)

Married

54 (9.1)

543 (88.9)

3.1 (0.18 – 52.7)

0.2229

Previously married

No

54 (9.0)

549 (91.0)

1.00

0.8146

Yes

1 (7.1)

13 (92.9)

0.78 (0.10 – 6.09)

Husband previously married

No

49 (8.9)

501 (91.10

1.00

Yes

6 (9.0)

61 (91.0)

1.01 (0.41 – 2.45)

0.9900

Number of children

≤1

13 (10.0)

117 (90.0)

1.00

2

22 (9.1)

220 (90.9)

0.90 (0.44 – 1.85)

0.7746

3

11 (7.1)

143 (92.9)

0.69 (0.30 – 1.60)

0.3907

≥4

9 (9.3)

88 (90.7)

0.92 (0.38 – 2.25)

0.8557

Drink alcohol

No

49 (8.6)

524 (91.4)

1.00

Yes

6 (11.1)

48 (88.9

1.34 (0.55 – 3.28)

0.5258

Currently smoke

No

53 (9.3)

519 (90.7)

1.00

Yes

2 (3.6)

54 (96.4)

0.36 (0.09 – 1.53)

0.1674

Husband migrated for work

No

30 (7.6)

363 (92.4)

1.00

Outside the district

5 (21.7)

18 (78.3)

3.36 (1.17 – 9.69)

0.0248

Outside the country

10 (9.0)

101 (91.0)

1.20 (0.57 – 2.53)

0.6363

Nationality

Nepali

46 (8.5)

496 (91.5)

1.00

Bhutanese-Nepali

11 (11.1)

88 (88.9)

1.35 (0.67 – 2.70)

0.4000



Table 5. Multivariable Analysis of Risk Factors for High-risk HPV and Abnormal Cervical Cytology among a Sample of Nepali and Bhutanese-Nepali Women in Eastern Nepal

Characteristics

High-risk HPV

(n=496)

Abnormal Cervical Cytology (n=447)

Odds Ratio

(95% CI)

p- value

Odds Ratio

(95% CI)

p- value

Age, years

≥45

1.00

1.00

<45

2.39 (0.87 – 6.56)

0.0899

0.84 (0.29 – 2.34)

0.7361

Formal education

None

1.00

1.00

Some

0.88 (0.39 – 1.97)

0.7502

0.92 (0.36 – 2.38)

0.8632

Previously married

No

1.00

0.4956

1.00

Yes

0.46 (0.05 – 4.31)

1.12 (0.11 – 11.65)

0.9271

Husband previously married

No

1.00

Yes

1.39 (0.49 – 3.92)

0.5341

0.91 (0.24 – 3.49)

0.8946

Number of children

≤1

1.00

1.00

2

1.19 (0.50 – 2.85)

0.6913

0.91 (0.35 – 2.41)

0.8526

3

0.99 (0.35 – 2.78)

0.9774

0.74 (0.23 – 2.39)

0.6136

≥4

2.26 (0.72 – 7.07)

0.1608

1.09 (0.29 – 4.09)

0.9022

Drink alcohol

No

1.00

Yes

2.49 (0.78 – 8.00)

0.1257

3.25 (0.93 – 11.47)

0.0650

Currently smoke

No

1.00

Yes

0.23 (0.04 – 1.27)

0.0919

0.35 (0.06 – 2.07)

0.2478

Husband migrated for work

No

1.00

Outside the district

4.65 (1.51 – 14.35)

0.0075

1.39 (0.17 – 11.37)

0.7613

Outside the country

1.25 (0.56 – 2.78)

0.5816

2.88 (1.26 – 6.55)

0.0118

Nationality

Nepali

1.00

1.00

Bhutanese-Nepali

1.35 (0.55 – 3.28)

0.5121

0.97 (0.30 – 3.10)

0.9612