Screening for Cervical Cancer and Its Association with Human Papilloma Virus ( HPV ) among Sudanese Women

This study investigates the presence of human papilloma virus (HPV) on cervical smear among women in Khartoum State. Four hundred specimens were taken from patients who attended different hospitals in Khartoum State during a period from July 2008 to July 2009. The specimens were processed and screened using cytological technique (Papanicolaou stain) and PCR for HPV detection. The pre-cancer cells were detected in 30/400 (7.5%) specimens among which cervical glandular intraepithelial neoplasia (CGIN) was found in one cases 1/30 (0.3%), the details were as follows: mild dyskaryosis was present in 18/30 (4.5%), moderate dyskaryosis in 5/30 (1.3%), and severe dyskaryosis 6/30 (1.5%). On the other hand, screening for HPV among the enrolled subjects revealed high ratio (36.0%). HPV was detected in all cases that had cytological changes except one (29 out of 30 cases (96.7%)). Infections other than HPV were observed during cytological assessment which include T. vaginalis 8 (2.0%), Candida spp. 11(2.8%), and Actinomyces spp. 5 (1.3%). The study concluded that the prevalence of HPV infection is high in Sudanese women (36%) who were revealed as 96.6% in pre-cancer cases and 83.3% in cervical cancer patients.


INTRODUCTION
Cancer of uterine cervix is the second leading cause of cancer death in women world wide with more than 270000 deaths reported every year.Over 80% of these deaths occur in developing countries (Muchiri et al., 2006 andKent, 2010).
Human Papilloma virus infection plays an etiological role in the development of cervical cancer, and most of cervical cancers contain HPV DNA (McMurray et al., 2001).
HPV is viewed as sexually transmitted infection (STI) as Snijders et al., (2006) andDe Sanjose et al., (2007) reported more than 50% of sexually active women have been infected with genital HPV at some time in their life.Infection prevalence of up to 82% has been reported in adolescent and young adult women in Southern Africa (Marais et al., 2000).

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HPV is a member of the papilloma virus family of viruses that are capable of infecting humans.Like all papilloma viruses, HPV establishes productive infections only in the stratified epithelium of the skin or mucous membranes.While the majority of the nearly 200 known types of HPV causes no symptoms in most people, some types can cause warts (verrucae), while others can-in a minority of cases-lead to cancers of the cervix, vulva, vagina, and anus in women or cancers of the anus and penis in men (Muñoz et al., 2003).Of these, 15 are classified as high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), 3 as probable highrisk (26,53,and 66),11,40,42,43,44,54,61,70,72,81,and CP6108) (Muñoz et al., 2003), but even those may cause cancer.Types 16 and 18 are generally acknowledged to cause about 70% of cervical cancer cases.Together with type 31, they are the prime risk factors for cervical cancer (Walboomers et al., 1999).
More than 30 to 40 types of HPV are typically transmitted through sexual contact and infect the anogenital region.Some sexually transmitted HPV types may cause genital warts.Persistent infection with "high-risk" HPV typesdifferent from the ones that cause wartsmay progress to precancerous lesions and invasive cancer (Schiffman and Wacholder, 2009).
It is quite obvious that there is a need to study the prevalence of different HPV types in different geographic areas and, particularly, in less studied regions such as Africa.In Sudan, the number of cervical cancer new cases is in dramatic increase according to Radiation and Isotope Center Khartoum records (2009).Screening for cervical cancer remains an important health and economic concern in Sudan to decline the incidence of cervical cancer and its associated mortality.

MATERIALS AND METHODS
Four hundreds patients attended different hospitals and gynecologic clinics (out patients) with different ages and gynecological symptoms in Khartoum State (Khartoum hospital, Khartoum North hospital (Bahri), and Elneelin Clinic Center) during the period from July 2008 to July 2009 were included in this study.Written consent was obtained from every patient before they were enrolled in the study, during the interview to obtain essential identification data, cervical smears were collected by using modified Salyze cyto spatula, then they were smeared immediately on clean slides and fixed while they were still moist in 95% ethyl alcohol for 15 minutes (Papanicolaou procedure) (Bancroft and Gamble, 2002) and the remaining cells on the spatula were immersed in a plastic swab tubes containing 5 mL of Tris HCl buffer (PH 8.0).Pellets from these samples were obtained by centrifugation and then resuspended in 3 mL Tris HCI buffer PH 8.0 and stored in cryo tube at -20 °C until used (Jacobs et al., 1995).
Biopsies for histopathological examination were taken only from patients suggested for cervical cancer and were stained with Papanicolaou.
Isolated DNA from 10 mL of crude cell suspensions of cervical scrapes was subjected to PCR as follows: 5 x 1.25 µL master mix (Jena Bioscience) was used for the preparation of 25 ul PCR mix containing 0.25 µL of each GP5 (sequence 5" to 3": TTT GTT ACT GTG GTA GAT ACT AC), and GP6 (sequence 5" to 3": GAA AAA TAA ACT GTA AAT CAT ATT C) primers, 5.0 µL master mix, 9.5 µL nuclease free water, and 10 µL DNA template.A 4 min denaturation step at 94 °C was followed by 40 cycles of amplification with a PCR processor (PE9600; Perkin-Elmer).Each cycle included a denaturation step at 94 °C for 1 min, a primer annealing step at 65 °C for 1.5 min, and elongation step at 72 °C for 1.5 min.The final elongation step was prolonged by 4 min to ensure a complete extension of the amplified DNA.The PCR product was visualized by UV transilluminator on 1% agarose gel according to the procedure of Sambrook et al., (2001).

DISCUSSION
The present study showed that the incidence of cervical intraepithelial neoplasia (CIN) is low by cytological investigations, which were (7.5%) cases out of the study group.This result agrees with Hassan and Khirelseed (2009) who have conducted a community-based survey in 256 Sudanese women living in Khartoum, during the period from 2003 to 2008.Indicators of cervical cancer screening participation were examined: at least one previous Pap smear and Pap testing in the last 1 year, their findings confirm low levels of cervical cancer screening among Sudanese women using pap stain.
Also, the present study showed that cervical malignancy was (1.5%), HPV infection (1.5%), and Trichomonas vaginalis infection in 2.0% cases of study subjects.These results agree with Misra et al., (2006) who studied the results of long term hospital-based cytological screening in asymptomatic women, they found that the incidence of squamous intraepithelial lesion (SIL) was 5.9% of cases studied, while cervical malignancy was seen in 0.6% of cases, HPV infection was seen in (0.4%) cases, and T. vaginalis in (2.6%) of the cases.
Revzina and Diclemente (2005) reported that the estimates of HPV prevalence vary from 14% to more than 90%.
The present study showed that the prevalence of HPV is 144 (36.0%) out of 400 subjects with age ranging from 16-83years, this prevalence is relatively high, Also the present study showed that the prevalence of HPV is 34.8% (139 out of 400 cases) among patients at age ranging 15-59 years.These results are not far away from the results reported by Aggarwal et al., (2006) which was (36.8%), they studied the prevalence of high risk human papilloma virus infections in women with benign cervical cytology (hospital based study from North India), and Dunne et al., (2007) found HPV in (26.8%) in 14-59 age group patients.
The description of HPV results by age in this study showed that, although the highest HPV prevalence was detected in patients aged 30-39 years (which is a large study group number), the incidence of HPV in age 15-19 years is higher 4 (80.0%)out of 5 cases, and also in age more than 50 it was present in 12 (54.5%)out of 22 patients.These results confirm the findings of Stamataki et al. (2010) who found that the prevalence of HPV in female aged 16 to 20 years was 57.1% and Dunne et al. (2007) who found that the age of 50 more than 80% of American women will have contracted at least one strain of genital HPV, while Zhang et al., (2010)  Screening for cervical cancer and its association with human papilloma virus (HPV) 105 Most patients attending to hospitals were at the reproductive age, so the age group from 30-39 years was more frequent in the present study.
Steben and Durate-Franco (2007) noted that the National Health and Nutrition Examination Survey determined that the prevalence of HPV infection in a representative's sample of women was highest in those aged 20-24 years (44.8%); the same result was reported by Dunne et al., (2007); who studied the prevalence of cervical cancer in relation to age.They noted that prevalence decreases with age.This may be due to HPV infection being cleared by the immune system or sinking to undetectable levels while still presents in the body.These results agree with the present result (47.4%) as HPV was present in 9 out of 19 patients.

CONCLUSION
The pre-cancer and cancer cells are low while the prevalence of HPV is high and always in association with CIN in young women and women with cervical squamous carcinoma.

Table 1 :
Description of cytological results according age group among enrolled subjects.

Table 2 :
Correlation between HPV and cytological findings among enrolled subjects.