Optimization of preservation solutions to execute testing on cervical smear sample

Background: The common methods to preserve cell for protein analyses are in cold condition or treated with freeze solution and packaging in dry ice for shipping. Solution which can preserve cervical cells at room temperature is preferable and cost consuming for laboratory testing. Aims and Objective: Research and optimized the storage and transport solution for cervical sample which can preserve cells at room temperature for laboratory testing. Materials and Methods: In this study, cervical specimens were collected in 3 different preservation solutions. Storage and transport of samples was at ambient or refrigerated temperature. The effect of preservation solution and temperature was check by cell visualization under microscope and protein measurement. Results: Presence of cells were detected in all three solutions. Among those, HEPES solution can preserve the highest number of cells and at room temperature. Conclusion: HEPES solution appeared suitable to preserve cervical cytology specimens at ambient temperature for further laboratory testing at protein and DNA level.

Soluble production of a full-length human papillomavirus type 16 L1 protein by Escherichia coli

Persistent infection with human papillomavirus type 16 (HPV16) causes the development of cervical cancer. Escherichia coli is a cost-effective host successfully used to develop a second-generation vaccine against HPV, based on the purification of soluble truncated L1 protein variants. Previous attempts to produce soluble full-length HPV16-L1 protein by E. coli have failed. This study was aimed at cloning a Cuban HPV16-L1 gene in E. coli and assessing its expression as a soluble full-length L1 protein by manipulating culture conditions. The L1 gene was amplified from a Cuban patient’s cervical sample and cloned into pET28a and pBAD/Myc-HisA vectors. Production and solubility of L1 protein were evaluated in E. coli TOP10 harboring pBADHPV16-L1 plasmid and E. coli BL21-(DE3), Rosetta-(DE3)/pLysS, and SHuffle® T7 Express lysY strains harboring pETHPV16-L1 plasmid, grown under arabinose (0.2%)- or isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 µM)-induction or Super Broth-based auto-induction for 24 and 48 h. The recombinant plasmids pETHPV16-L1 and pBADHPV16-L1 were constructed. The HPV16-L1 protein was produced insoluble to high levels in conventionally IPTG-induced E. coli-pETHPV16-L1 cells. However, under auto-induction, soluble full-length HPV16-L1 protein was successfully produced at similar levels by E. coli BL21 (DE3), Rosetta (DE3) pLysS and SHuffle® T7 Express lysY cells, reaching up to 7.2 ± 0.5% and 14.3 ± 1.6% of the total proteins in the soluble fraction after growing for 24 and 48 h, respectively. It is concluded that the auto-induction procedure at 18 °C with 30 µM IPTG and 100 rev/min promotes soluble full-length HPV16-L1 protein production by E. coli.

Triaging HPV-positive, cytology-negative cervical cancer screening results with extended HPV genotyping and p16INK4a immunostaining in China

Background Self-sampling for human papillomavirus (HPV) testing is a feasible option to improve the cervical screening coverage. However, an ideal triage method for HPV-positive self-samples does not yet exist. The aim of this study was to explore the utility of HPV genotyping and p16INK4a immunostaining (p16) in triaging HPV-positive self-samples, focusing on HPV-positive, cytology-negative (HPCN) women. Methods A total of 73,699 women were screened in a cervical screening project in China via SeqHPV assay on self-samples. HPV-positive women were called-back and collected cervical sample for p16 immunostaining and liquid-based cytology, those who met any result of HPV16/18+ or visual inspection with acetic acid (VIA) + or p16+ were referred for colposcopy, and HPCN women with adequate data on p16 and pathology were analyzed. A triage strategy was considered acceptable if the negative predictive value (NPV) for cervical intraepithelial neoplasia 3 or worse (CIN3+) was 98% or more, combined with an improvement of sensitivity and specificity for CIN2+/CIN3+ in reference to the comparator, being HPV16/18 + . Results A total of 2731 HPCN women aged 30–64 years were enrolled, 136 (5.0%) CIN2+ and 53 (1.9%) CIN3+ were detected. Five triage strategies met the criteria: p16+; HPV16/33+; ‘HPV16+ or HPV33/58/31/35+&p16+’; ‘HPV16/33+ or HPV58/31/35+&p16+’; HPV16/18/31/33/45/52/58 + & p16+. These strategies required less or similar colposcopy referrals, and less colposcopies to detected one case of CIN2+/CIN3+, achieving favorable false positive (negative) rates to the comparator. Among them, p16 staining detected 83.1% (79.2%) of underlying CIN2 + (CIN3+) in HPCN women. Moreover, three triage strategies were favorable in sensitivity and/or specificity to the ‘HPV16/33+’ strategy: p16+; ‘HPV16+ or HPV33/58/31/35 + &p16+’; HPV16/18/31/33/45/52/58 + &p16 + . Conclusions Genotyping for HPV16/33 could be utilized to optimize the management of HPCN women. Moreover, p16 immunostaining, either alone or combined with extended genotypes, is more effective than HPV genotypes alone in the triage of HPCN women.

Acquisition, prevalence and clearance of type-specific human papillomavirus infections in young sexually active Indian women: A community-based multicentric cohort study

In context of the ongoing multi-centric HPV vaccine study in India, unvaccinated married women (N = 1484) aged 18–23 years were recruited in 2012–2015 as age-matched controls to the vaccinated women and followed up yearly. We assess type-specific prevalence, natural history and potential determinants of human papillomavirus (HPV) infection in these unvaccinated women. Cervical samples were collected yearly for at least four consecutive years. A Multiplex Type-Specific E7-Based polymerase chain reaction assay was used to detect 21 HPV types. HPV prevalence was 36.4% during 6 years. Most common HPV types were 16 (6.5%) and 31 (6.1%). Highest persistence were observed for HPV 35 (62.5%) and 52 (25%). New HPV acquisition rate was 5.6/1000 person-months of observation (PMO), highest for HPV 16 (1.1/1000 PMO). Type-specific clearance rates ranged between 2.9–5.5/100 PMO. HPV 16 and/or 18 infections were 41% (95% CI 4–63%) lower among women with 2-<3 years between marriage and first cervical sample collection compared to those with <2 years. HPV prevalence and acquisition rates in young Indian women were lower than their Western counterparts. HPV 16 infections being most common shows the importance and potential impact of HPV vaccination in India. Women with 2–3 years exposure had reduced risk possibly due to higher infections clearance.

1710. Comparison of herpes simplex prevalence in serum and semen-cervical sample of infertility patients: A systematic review and meta-analysis

Background Herpes simplex virus (HSV) is a common pathogen of sexually transmitted infections, however the role it plays in the development of infertility is unknown. In animal studies, inoculating murine rete testis with HSV-1 revealed irreversible atrophy of the germinal epithelium. Another study found that human herpesvirus 1 thymidine kinase (HHV-1 TK) protein disrupts spermatogenesis by creating immature sperm and accelerating apoptotic cell death in rodent. Although it is well established that herpes virus affects fertility in male animal models, the question remains as to the effect of HSV in human infertility. Routine testing of serum HSV IgG/IgM/DNA or HSV PCR in semen-cervical sample is not commonly done in clinical practice, and there are no set guidelines as when to screen. We aim to review the available literature and compare the prevalence of HSV in serum versus semen-cervical samples, focusing on the infertile patient population. Methods We searched PubMed, Embase, Cochrane Library, and ClinicalTrials.gov from inception to December 2019. Our search terminology included: “Herpes, Human herpesvirus, infertility.” Inclusion criteria required testing to be done on either serum, sperm, menstrual fluid, or endocervical sample in infertile patients. PRISMA Flow Diagram for study selection. Results 17 retrospective studies were included in this review. In the male-infertility cohort, a total of 11 studies were compared. The random-effects pooled prevalence was 12.7% in semen sample, and 16.8% in serum sample. In the female-infertility cohort, a total of 6 studies were compared. The random-effects pooled prevalence was 12.1% in menstrual fluid /endocervical sample, and 17.8% in serum sample. Figure 1. Studies enroll in this meta-analysis, Male Figure 2. Studies enroll in this meta-analysis, Female Conclusion The prevalence of HSV in semen-cervical sample was about 12%, compared to HSV in serum sample is about 17%. Therefore, HSV contribution to infertility will be overestimated when we use serum sample for diagnosis. It is noteworthy to mention that the seroprevalence of HSV IgG is much higher in general population, previously reported at 35% to 50%. In addition, given that current antiviral treatment for HSV has side effects that could cause infertility on its own, as seen in animal studies. More studies are needed to evaluate the role HSV plays in causation of infertility. Disclosures All Authors: No reported disclosures

Prevalence and Incidence of Human Papillomavirus (HPV) Infection Before and After Pregnancy: Pooled Analysis of the Control Arms of Efficacy Trials of HPV-16/18 AS04-Adjuvanted Vaccine

Objective Data on human papillomavirus (HPV) prevalence around pregnancy were inconsistent. We assessed HPV prevalence before and after pregnancy, HPV incidence after pregnancy, and risk factors for HPV infection. Method Data from 15 754 women in control arms of 5 AS04-HPV-16/18 vaccine efficacy trials were analyzed, including 3001 women with at least 1 pregnancy. Results of HPV deoxyribonucleic acid testing on cervical samples were available. We analyzed risk factors, including age, region, pregnancy and its outcomes, duration from pregnancy resolution to collection of first postresolution cervical sample, previous HPV infection, cigarette smoking, and number of sexual partners with Cox regression. Results Prevalence of high-risk oncogenic (hr)-HPV types was similar before and after pregnancy (20.8% vs 19.8%). Incidence of hr-HPV was 40.1 per 1000 person-years (95% confidence interval [CI], 23.4–64.2) at 0–3 months, 266.7 (95% CI, 217.4–323.7) at 3–6 months, and 95.7 (95% CI, 83.9–108.7) at &gt;6 months after pregnancy. Risk factors associated with HPV infection after pregnancy are previous HPV infection, elective abortion, and younger age at pregnancy resolution. Conclusions Pregnancy could not be confirmed as a risk factor for HPV infection in this population despite an increased incidence detected 3–6 months after pregnancy resolution. Most women remained HPV negative after pregnancy. Clinical Trial Registration NCT001226810 (HPV-008 trial), NCT00294047 (HPV-015 trial), NCT00316693 and NCT00929526 (HPV-032/063 trials), and NCT00779766 (HPV-039 trial).

Acceptability of Human Papillomavirus Self-Sampling for Cervical Cancer Screening in an Indigenous Community in Guatemala

Purpose Cervical cancer rates in Latin America are higher than those in developed countries, likely because of the lower prevalence of screening. Specifically, less than 40% of women in Guatemala are regularly screened and even fewer women are screened in indigenous communities. Current screening strategies—Pap smears and visual inspection with acetic acid—might not be the most effective methods for controlling cancer in these settings. We thus investigated the potential of self-collection of cervical samples with testing for human papillomavirus (HPV) to help prevent cervical cancer in an indigenous community in Guatemala. Patients and Methods A community representative random sample of 202 indigenous women age 18 to 60 years residing in Santiago Atitlan, Guatemala, were surveyed to assess knowledge of and risk factors for HPV and cervical cancer. Women were then invited to self-collect a cervical sample using HerSwab collection kits to assess the prevalence of HPV and the acceptability of self-sampling. Results Of 202 women who completed the survey, 178 (89%) provided a self-sample. In all, 79% of these women found the test comfortable, 91% found the test easy to use, and 100% reported they were willing to perform the test periodically as a screening method. Thirty-one samples (17%) were positive for at least one of 13 high-risk HPV types, and eight (4.5%) were positive for HPV 16/18. Conclusion HPV testing by using self-collected samples was well accepted, suggesting that it is a plausible modality for cervical cancer screening in indigenous communities. Further studies are needed to assess rates of follow-up after a positive test and to determine whether these findings extend to other indigenous and nonindigenous communities in Guatemala and Latin America.