Original Article

A single group study to evaluate the perinatal transmission of Human papilloma virus in high prevalence area

¹Raunak R Mankodi, ¹Royana Singh, ²Rashmi Jaiswal, ²Ruhi Dixit, ³Baldev D Bhatia, ⁴Anuradha Khanna, ²Manoj Pandey

• ¹Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India.
• ²Department of Surgical Oncology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India.
• ³Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India.
• ⁴Department of Gynaecology and Obstretics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India.

• Submitted: August 7, 2012.
• Accepted September 21, 2012 .
• Published: September 23, 2012.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In areas of high prevalence of HPV in the population, it may be transmitted to the oral cavity of the child from the mother. We collected endocervical swabs from 84 mother and oral swabs from the neonate. DNA was extracted and amplified and checked for HPV-16 and HPV-18 DNA. It was found that 2% of the neonates of the HPV positive mothers tested positive showing for HPV suggesting a low frequency of the perinatal transmission of HPV.

Introduction

Human papillomavirus (HPV) is known to cause laryngeal papillomatosis [1], oral and genital warts [2]. HPV types are classified as low-risk, nononcogenic, types, associated with anogenital warts and laryngeal papillomatosis or high-risk, oncogenic, HPV types [3] associated with cancers of the cervix, anogenital areas, and head and neck [4]. The most prevalent HPV types associated with genital and oral cancers are HPV-16, 18 [5]. It is estimated that nearly 7% of US population harbours HPV in oral cavity [6], the prevalence in other part of the world including India is even higher [7].

Although the predominant mode of viral transmission occurs through sexual contact [8,9,10,11]; HPV has been found in young and virginal women prior to first coitus [3,12]. However, in a conservative society like that in India, high-risk behaviours like multiple sexual partners and oral sex practises are expected to be negligible. The prevalence of HPV in the oral cavity in Varanasi is reported to be about 64.5% in oral cancer patients and in 20% of normal subjects [7]. The prevalence in the cervix of normal women is reported to be 12% which rises to 49% in cases of chronic cervicitis. It is proposed that there may be some other mode of transmission of HPV, other than classical genito-oral route.

Studies suggest that the virus can be transmitted from mother to infant before or during childbirth [13,14,15,16,17]. Some have found that the risk of vertical transmission of HPV DNA to the oral or genital mucosa of new-borns to be rare, 1–5% [13,16,17,18]. In contrast, other studies suggest the vertical transmission is common, 40%–80% [13,19,20,21,22].

In our study it was not possible to distinguish true infection from mere inoculation due to lack of post-partum follow-up. However several studies on persistent HPV DNA, a method for distinguishing inoculation from true infection, have reported maternal/new-born concordance after birth to be maintained between 37%–83% at 6 weeks to 6 months after birth [19,23] whereas another study has shown a lower 10% prevalence in infants at 24 months of follow-up [22].

Transmission of human papillomavirus during sexual activity is the most publicized, but this is not the only way it can be spread. HPV infection can also occur during birth as proposed by Cason et al, 1995 [19] and Rombaldi et al, 2009 [15]. Rice et al, 1999 [21] presented evidence for vertical transmission from at least 30% HPV positive mothers to their infants, resulting in persistent infection in children. That the mother is the source of infant infection was confirmed by DNA sequencing. Though most of these infections clear subsequently, however those who persist can lead to substantial morbidity [24]. Xavier Castellsagué et al, 2009 [18] also confirmed that the risk of vertical transmission of HPV genotypes is relatively low. HPV persistence in infants is a rare event.

Few larger studies have evaluated concordance based on a broad spectrum of human papillomavirus (HPV) types in oral and genital specimens of mothers and their recently born infants. This information is important in determining whether HPV vaccines administered prior to pregnancy may be useful for preventing vertical transmission. Hence we propose the perinatal mode of transmission of HPV in high-risk areas.

Aim and Objective

It is hypothesized that in areas of high prevalence of HPV in the population, The HPV may be transmitted to the oral cavity of the child from the mother (vertical or perinatal transmission).

Figure 1: showing HPV 16/18 DNA positivity in mother and neonate after UV exposure of eectrophoresed gel .

Materials and Methods

After permission from Institute Ethics Committee, the subjects were selected by convenient sampling. The study included pregnant mothers admitted for delivery at department of Gynaecology & Obstetrics over a period of three months. After obtaining the written informed consent a cervical scrap of mother and a scrap from buccal mucosa of newborn were taken. DNA was extracted from these paired samples and PCR was done for detection of HPV 16 and 18 genome. Associations between potential determinants of HPV infection in pregnant women and of HPV positivity in infants were also explored by logistic regression modelling.

Inclusions criteria

1. Pregnant mothers willing to give consent at the time of child bearing.
2. Above the age of 18 years.

Exclusion criteria

1. Women with concomitant medical illness.
2. Women with excess post-partum bleeding.
3. Women on anti-viral therapy.

DNA analysis

DNA extraction was performed using 200 μL of cervical or oral cells suspensions and 400 μL from epithelial cells using the chloroform /phenol method in accordance with the manufacturer's instructions. Amplification was performed using 5 μL extracted DNA with the consensus primers defined by following sequences: MY9 [5’-CGTCMARRGGAWACTGATC-3’], MY11 [5’-GCMCAGGGWCTATAAYAATGG-3’], GP-5 [5’-TTTGTTACTGTGGTAGATACYAC-3’], GP-6 [5’-GAAAAATAAACTGTAAATCATATTC-3’]. The mixture was cycled following an initial incubation at 95 °C for 5 min through repetitive denaturation at 94 °C for 45s, annealing at 47.5 °C for 1 min, and extension at 72 °C for 30s to a final incubation at 72 °C for 6 min , which amplify a fragment from the L1 HPV gene. PCR was performed in 25μL of reaction mixture containing 0.8 μL of dNTPs, 1 μL BSA, 2.5 μL of Taq buffer, 50 μL of each primer and 1μL of Taq polymerase (Roche Diagnostics S.L.). The resultant PCR product was then subjected to Gel Electrophoresis for UV visualization.

Figure 2: showing HPV 16/18 negativity in neonates of HPV positive mothers(not shown in the figure).

Results

Among the 84 women, there were 74 (88%) vaginal deliveries and 10 (12%) caesarean sections. Of these the HPV DNA was positive in 50 subjects. Among the 50 DNA positive mothers, the proportions of vaginal and caesarean section deliveries were also 87% and 13%, respectively. Only 1 of the babies born by vaginal delivery was positive for HPV DNA, although 60% of the mothers demonstrated HPV DNA positivity from endocervix.

The overall prevalence of HPV DNA in newborns was seen in 2% and there was no significant difference by time of sample collection between the HPV positive and HPV negative newborns (40 hrs. versus 41.5 hrs).

The mean HPV specimen collection interval between enrolment at the third trimester and delivery was 71.4 days. There was no significant association between the interval between the third trimester and delivery specimen collection period and subsequent risk of HPV transmission. The overall mean interval time (based on both serology and DNA) was not significantly related to the transmission rate.

Discussion

This study evaluated HPV concordance between mothers and newborns by examining viral frequency and identified risk factors for vertical transmission. Previous studies [13,28,22,17], have evaluated HPV vertical transmission by examining the prevalence of HPV DNA positivity in mothers and newborns, and less often, in investigations with sufficient DNA of oral/genital specimens have determined HPV types and type-specific concordance between mother and newborn[19,29,23,17,27]. Only half of the HPV DNA positive mothers were detected with these same HPV.

There was no association between type of delivery and rate of vertical transmission; thus vaginal delivery does not appear to convey a greater risk of HPV from mother to newborn even where there was concordance in HPV type.

Among mother/baby pairs infected with HPV, Smith et al, 2004 [25] found low type-specific concordance, 17% (1/6), in a study of 574 mother/baby pairs. In contrast, Tseng et al., 1998 [26], detected 100% of HPV-16/18 concordance in oral/genital specimens of 301 mother/baby pairs (27/27) as did Tenti,1999 [27] (11/11: 4 HPV-16/18, 7 other unspecified types matched), but the latter study finding was based on newborn nasopharyngeal aspirates taken at birth and likely reflects maternal contamination to the newborn. These two studies limited assessment to HPV-16 and -18 which accounted for only half of the HPV DNA types detected. Previous studies by Watts et al, 1998, [17]. Puranen et al, 1996, [13,28]; Smith et al, 2004, [25]; Rintala et al, 2005 [22], have evaluated HPV vertical transmission by examining the prevalence of HPV DNA positivity in mothers and newborns, and less often, in investigations with sufficient DNA of oral/genital specimens. Several other authors have determined HPV types and type-specific concordance between mother and newborn [19,29,23,17]. In past studies, they found that the maternal prevalence rate of HPV DNA from oral/genital specimens was 31% whereas it was 2% in the infant.

According to the study of Medeiros et al, 2005 [30]. Infants born through vaginal delivery were at higher risk of exposure to HPV. However Smith et al, 2010 [31], pointed out that there was no association between the type of the delivery and the rate of the vertical transmission.

Although others suggest that the frequency of vertical transmission is much higher [19,29,23,22], these findings are most likely due to small sample sizes that produce unstable frequency rates, and inoculation rather than infection from specimens taken immediately after birth especially those from nasopharyngeal aspirates, or PCR contamination. We prepared and maintained collected specimens, and performed PCR preparation in a separate building from that where the PCR assay was conducted to reduce contamination. Our study failed to show any evidence for vertical transmission or higher incidence in vaginal deliveries.

Conclusion

Vertical transmission is low between mother/newborn oral and genital sites around 2%, even though the HPV infection rate is higher in mothers.

Authors Contribution

RM: Prepared the draft manuscript
RS: Conceived and designed the study and helped in preparation of manuscript
RJ: Conducted the PCR and did the literature search
RD: Helped in preparation of manuscript, editing
BDD: Designed the study in neonates, preparation of manuscript
AK: Design of study in Gynaecology and preparation of manuscript
MP: Concept and design, preparation of final manuscript and editing.

Funding

None

Acknowledgement

Dr. Raunak Mankodi was awarded the short term research studentship by ICMR for conducting this study

Ethical considerations

The study was approved by Institute Ethics committee

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