Role of Platelets, Bacterial Vaginosis, Serum Interleukinsand TNF In Genital Human Papillomavirus InfectionAmong Black Female Population in Nigeria

The study was aimed at evaluating the relationship between platelets count,
bacterial vaginosis and immune response in cervical human Papillomavirus positive
women in Bayelsa State. Cervical smears were collected from apparently healthy
subjects attending clinics in Bayelsa State. Consensus primers GP5+/6+ and MY09/11
were used to detect HPV DNA in cervical smears and further subjected to multiplex PCR
for molecular typing of HPV subtypes. Nugent method was used for scoring of bacterial
vaginoses based on gram staining reaction. Interleukins were studied using sandwich
ELISA kits. The prevalence of bacterial vaginosis among HPV positive, HPV negative
and high-risk HPV was 35.9% and 18.2% and 51.7% respectively. Bacterial vaginosis
expressed statistically significant relationship with high risk HPV (OR=9.64, P=0.002,
95%CI= 1.08-86.26).Interleukin 4 (IL-4), IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, Tumor
Necrotic Factor (TNF) and Interferon Gamma (INF-γ) were equally higher in HPV positive
participants compared with negative participants and statistically significant.


Introduction
Estimated population of 47 million women aged 15 years and above are at risk of developing cervical cancer worldwide. There are reported rising incidence of cervical dysplasiain Nigeria according to the information center on HPV and cancer based on various studies done in population in Nigeria [1]. Cell growth, function and differentiation, steering immunological response and inflammatory processes are initiated by certain biological molecules known as cytokines [2]. These cytokines and chemokines are secreted by keratinocytes which are either activated or suppressed by the immune system as they participate in human Papillomavirus infection. Certain cells like the dendritic cells, Langerhans cells, natural killer cells, and natural killer T-helper cells among others are also involved in promoting immune response against HPV infection [3]. A delicate balance exists between pro-inflammatory and anti-inflammatory cytokines and their disturbances as reported in many acute disease conditions like pyrexia, rheumatic disorders and cancers. In histological sections, cervical Papillomatosis is characterized by marked keratinocyte hyperproliferation, dense inflammatory infiltrate consisting of T-helper cells and neutrophils, vascular dilatation and proliferation.
The primary defect in Papillomatosis patients is believed to be abnormal thickening of epidermal cells [4]. Genetic variations in IL-6, IL-8 or IL-10 polymorphisms is associated with the risk of cervical cancer. Also, circulating levels of cytokines IL-2, IFN-a ß, IL-2, IL-8, and IL-10 are useful in identifying women at higher risk of developing cervical invasive cancer, LSIL or HSIL, and risk of metastasis. Circulating or tissue levels of IL-6, IL-8 and IL-10 serve as additional markers in the prognosis of patients with advanced stage disease and may help the decision-making processes [2].

Platelets disorders have been reported in certain viral infection like
human immunodeficiency virus where surface glycoprotein gp120 causes an increase in megakaryocyte apoptosis in vitro due to increased TGFb and down regulation of the proliferation, inducing TNF super family member 13(TNFSF13). The envelope protein gp120 also interacts with CD4, which is expressed by immature megakaryocyte expressing CCR5 in infectious state [5]. Zapata, et al. [6] documented that in dengue virus infection, platelet production is impaired by suppression of megakaryopoiesis via infection of hematopoietic progenitor cells or indirectly via altered cytokine levels in the bone marrow due to impaired stroma cell function.
Iannacone, et al. [7] and Pozner, et al. [8] [10] and Singh, et al. [11] documented that human parvovirus do not has the ability to reproduce itself in megakaryocyte, but rather triggers a downward activation in platelet count via platelet activation. Therefore, the need to establish the effect of genital HPV on platelets count, interleukins and bacterial vaginosis among women in Bayelsa State.

Study Population/ Sampling
Fifty apparently health persons attending two hospitals in Bayelsa State participated in the study. Snowball and convenient sampling were the sampling methods adopted. Cervical-vaginal smears samples collection was done under aseptic technique; cervical smears were collected using Cytobrush and disposable speculum and swab stick respectively. Smears were made and the remaining stored in phosphate buffer used for DNA extraction for HPV detection and identification. Also, 6mls of venous blood was equally collected from each participant and dispensed into EDTA containers for blood count and 4mls into plain containers for interleukins estimation.
The process required multi-step washing, rinsing and soaking. The buffer added to each well, soaked for 2min, decanted and pat-dried followed by addition of 100uL of HRP conjugate working solution to each well, covered and incubated for 30min. at 37 0 C. The wells were decanted and washed with 350uL wash buffer, soaked for 5min decanted pat dry for 5 times. Substrate reagent (90uL) was added to each well, covered and incubated for 15min. at 37 0 C. with maximum protection from light. Thereafter, 50uL of the stop solution was added to stop the reaction. The optical density of each well was determined at once with a micro plate reader set to 450nm.

Blood Count
Blood count of the participants was assayed using automated hematology analyzer in accordance with the method of Schapkaitz and Levy [13]. The blood count analysis was based on the principle of electrical impedance. The impedance changes as the blood passed through the orifices. The change in impedance is proportional to cell volume, resulting in cell count and volume. The impedance analysis returns blood count in three parts white blood cell differentials (granulocytes, lymphocytes and monocytes) but unable to distinguish between similarly sized granular leucocytes: eosinophil, basophils and neutrophils. The procedure involves passing 2ml of venous blood collected from each participant into EDTA containers into an orifice attached to the machine. The EDTA blood container was vortexed and passed through a tiny orifice in the automated hematology analyzer and the machine sucked an aliquot of the blood sample in the container and within 30 sec.
results were displayed on the screen and were printed.

Bacterial Vaginosis
The methodologies of Nugent, et al. and Mendoza, et al. [14,15] were adopted. The principle of the reaction is based on Gram staining reaction. A score of 0 to 3 large gram-positive rods was considered normal, 4 to 6 rods was classified as abnormal vaginal flora (or intermediate) and 7 to 10 was indicative of bacterial Vaginosis. A sterile swab stick was used to collect the samples from the vagina of the participants to make a thin smear. The smears were air-dried and protected from contaminants and then fixed in absolute ethanol for 15min. The fixed smears were flooded with crystal violet stain for 1min and rapidly washed away with distilled water. They were drained and flooded again with Lugol's iodine for 1min, decolorized rapidly (for 2secs) with acetone and washed immediately with clean water and covered with safranin stain for 2min. The excess stain was washed off with water, allowed to drain and air-dried and examined microscopically first with X40 objective to check staining and then X100 oil immersion objectives to report bacteria rods, yeast cells, pus cells and epithelial cells.

Pro-inflammatory and anti-inflammatory cytokines expressions
were observed in this present study. This present study observed a statistically significant increase in the various interleukins studied, al. [28,29]. Similarly, serum circulating IL-8 level shows increased mean concentration in women with HPV infection in our present study. High levels of IL-8 have been reported in previous works related to lung, colorectal, breast, brain, liver, bone and gynecological cancers [30][31][32]. Also, Wu, et al. [2] documented that patients with cervical cancer whose cervical biopsy expresses IL-8 were likely to have lymph node metastasis. Baker, et al. [33] also documented increased plasma levels of circulating IL-8 in elderly women with persistent HPV infection. Interleukin-12 (IL-12) appears to be immuno-protective in cervical HPV in the present study. The present study reported a statistically significant reduction in the level of IL-12 expression in HPV positive participants compared with negative participants. Yang, et al. [18] reported that reduced expression of IL-12 in cervical biopsy specimens from invasive cancer cases. Souza, et al. [34] also reported higher serum level of IL-17 expression in LSIL patients as seen in the present study. Liu, et al. [35] has established an association between Tumor Necrotic Factor (TNF) polymorphism with increased risk of cervical cancer.
Interestingly TNF-a gene polymorphism can increase or decrease the susceptibility to cervical cancer depending on whether it is TNF-a-allele or TNF-a-is altered [36] wealso observeda decreased circulation of TNF among HPV positive participant. Parmar and Plantains, [37] has reported on the immunoprotective nature of interferon and that they can be defective and deficient in cervical cancer. The present study observed a defective interferon among HPV positive women as seen in (Table 3). According to Boccardo, et al. [38] interferon has been evaluated in experimental and clinical studies for immune function capabilities. IFN therapy has been used in patients with cervical intraepithelial neoplasia to cure or arrest of the cervical lesion progression [38,39]. HPV interfere with the protective action of IFNs at several levels and allow escape of HPV virus from immune degradation or clearance [3,35,40]. Gillet, et al. [41] optioned that Bacterial Vaginosis (BV) is characterized by a depletion of Lactobacillus species and a concurrent overgrowth of anaerobic bacteria and the presence of potentially pathogenic bacteria which are most frequently detected in the vaginal tract.
In our present study the prevalence of bacterial vaginosis among HPV positive, HPV negative and high-risk HPV were 35.9% and 18.2% and 51.7% as shown in Table 4 and Table 5  and ranging from absence of any association [45] to a clear positive relationship [41] in accordance with our findings.
Full blood count evaluated in our current study showed a statistically significant reduction in platelets among HPV positive subjects as well as neutrophils (Table 5) Thrombocytopenia in response to viral infections is often multifactorial.
Thrombocytopenia in viral hepatitis is caused by platelet specific glycoprotein antibodies [47] and immune complexes form bound to platelet surface to cause platelets destruction [48].
Coagulation, inflammation, and platelet activation play a role in HCV-induced decrease of platelet count. HCV also indirectly affects megakaryopoiesis [48,49]. The mechanism of platelets destruction in HPV might be linked to inflammatory response. In therefore that women exposed to high-risk HPV should be screened for bacterial vaginosis, with concomitant estimations of levels of platelets as well as inflammatory cytokines.

Conflict of interest:
There is no conflict of interest