We used both HPV probes and site-specific probes: 3p14 (FHIT), 8q24 (MYC), 13q22 (KLF5/KLF12), 3q28 (TP63), and 5p15 (TERT). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence  Cervical cancer remains the leading cause of gynecological tumor-related mortality worldwide and the second most common malignancy in women, with 570,000 women diagnosed with cervical cancer and 311,000 dying from the disease each year .Most HPV infections are cleared by the immune system, but in some cases, the infection persists. Persistent infection with high-risk HPV leads to cervical intraepithelial neoplasia (CIN), which occasionally develops into cervical cancer . High-riRecent studies have suggested that the integration of HPV in the human genome is an important cause of cervical cancer. Integration can preserve the upstream regulatory region (URR) of the virus. This HPV replication initiation region in the human genome is not stable but activates the cell’s DNA replication and other systems, which is an important “trigger” for cancer . The intIncreasing research on HPV integration has led to the recognition of HPV integration status as a potential biomarker for the prediction of diagnosis, progression, and survival and even as a biomarker for cancer screening . With thFormalin-fixed and paraffin wax-embedded cervical cancer samples were selected from the Department of Pathology, Tongji Hospital, Wuhan, China. All the samples were reviewed and confirmed independently by two pathologists, and cases with discrepancies were discussed until a consensus was reached. Samples with poor morphology or too many lymphocytes were excluded.Clinical characteristics were obtained from patient charts. The TNM staging system, which is based on surgical and pathological reports, was used in this study to evaluate cervical cancer patients. We collected a total of 96 samples: 93 squamous cell carcinoma (SCC) samples, 3 adenocarcinoma (AD) samples. The study was approved by the hospital’s ethics committee.Bacterial artificial chromosome (BAC) plasmids for 8q24 (RP11-1145O20), 3p14 (RP11-191B8), 3q28 (RP11-373I6), 5p15 (RP11-326E20) and 13q22 (RP11-179I20) were purchased from Life Technologies . The whole-genome plasmid of HPV types 16 and 18 was a gift from Haraud zur Hausen. The probes were labeled by standard nick translation with biotin- or digoxigenin-dUTPs. The biotin-labeled HPV probes and digoxigenin-labeled BAC probes were then coprecipitated with human Cot-1 DNA and salmon sperm DNA. Pelleted probes were then dissolved in a hybridization buffer composed of 50% formamide, 2×SSC, and 10% dextran sulfate. The detailed protocol and parameters of HPV-BAC dual-color probes are provided in the 2O2 for 10 min at room temperature. The slides were incubated in 1 M NaSCN for 20 min at 80°C, followed by digestion with 4 mg/ml pepsin  in 0.02 M HCl for 12 min at 37°C. The slides were rinsed 2 times in 2× SSC, postfixed in 4% formaldehyde for 10 min at room temperature, and dehydrated in an ascending ethanol series.Four-micrometer thick paraffin wax tissue sections were dewaxed, dehydrated with 100% ethanol for 3 min twice before air drying, and pretreated with 3% HProbes and target DNA were denatured simultaneously for 7 min at 90°C before hybridization overnight at 37°C. After hybridization, the preparations were washed stringently in 50% formamide (3 × 5 min) and 2× SSC at 43°C.The biotin- and digoxigenin-labeled probes were detected consecutively using the dual-color tyramide signal amplification (TSA) procedure. The biotin-labeled probe was detected by streptavidin-HRP . Then, the first amplification reaction was carried out under a coverslip by applying 50 μl of Cy3-tyramide  for 20 min at room temperature. Thereafter, the slides were soaked in blocking reagent (Perkin Elmer) for 15 min at room temperature to block the remaining peroxidase activity. Subsequently, the digoxigenin-labeled probe was detected by anti-digoxigenin-HRP , followed by TSA amplification using FITC-tyramide . Finally, the slides were washed in 4× SSC, dehydrated in an ascending ethanol series, and mounted in Vectashield (Vector Laboratories).Images were recorded with a fluorescence microscope (Olympus BX53) equipped with FITC, TRITC, and DAPI bandpass filters. A minimum of 100 nuclei in each sample were observed for HPV integration site evaluation. Among the cells containing HPV signals, the ratio of cells with colocalized HPV probe signals and BAC probe signals was calculated. Samples with a ratio greater than 60% were evaluated for HPV integration in a specific site.HPV and BAC probe hybridization on HPV-positive cell lines  was used as a control for HPV integration site detection. On sample tissue sections, BAC probes were also used as a control for effective hybridization.HPV16/18 probes were hybridized in SiHa and CaSki cells, two smaller HPV signals were observed in SiHa cells with only two copies of HPV16  signal were colocalized, as expected  in cultured cells and paraffin sections are completely different. Through a review of the literature and experiments , we concluded that HPV was integrated at this site , 15, we gression   in cancer cells within cervical cancer. For example, in sample No. 1321, two groups of cancer cells in the same cervical cancer tissue showed no difference in cell and nucleus morphology after HE staining and were pathologically identified as the same type of cells  There was no HPV integration in this sample. (2) As 80% of cervical cancers are infected by HPV16/18, we selected HPV16/18 probe. However, a few samples may not be HPV16/18 integrated but integrated by other subtypes. (3) Considering the intra-tumoral heterogeneity of cervical cancer tissues, some tumor cell clones in the same tissue have HPV integration but some clones do not. The tumor cell population at the paraffin sections we examined may happened to be with no HPV integration.We used a threshold value for identifying an HPV integration site: in cells containing HPV signals, the ratio of cells with colocalized HPV signals and chromosomal site-specific signals is more than 60%, which is interpreted as HPV integration at a specific site. The application of FISH in fusion gene research could be used as a reference, since fusion gene and HPV integration have similarities. Tomlins used FISH to evaluate the TMPRSS2:ETV1 fusion gene in paraffin sections of prostate cancer and found that, fusion signals were observed in an average of 31% of the 100 cancer cells in the positive case  . TherefoFHIT is a tumor suppressor at 3p14, which is significant copy number loss in cervical cancer, as loss of its activity results in replication stress and DNA damage. MYC is an oncogene, and it has been suggested that MYC activation is associated with HPV integration at 8q24. KLF5 is a transcriptional activator at 13q22 and may play a role in cell proliferation. Telomerase expression plays a role in cellular senescence, and deregulation of telomerase expression is found in somatic cells. 3q28 is significant copy number gain in cervical cancer, amplification and overexpression of TP63 at 3q28 is a biomarker of progression from CIN to cervical cancer. In our study, 3p14 and 8q24 were two high-frequency sites of HPV integration. Future studies may be able to reclassify cervical cancer at the molecular level from the perspective of HPV integration.Our selection of five candidate chromosomal sites is based on a review of HPV integration in cervical cancer research , 15. FHIMoreover, the integrated HPV signal can be used as a guide for the discovery of cervical cancer cell subgroups with different integrated viral loads. These subgroups may reveal the origin, metastasis, and recurrence of cervical cancer. This finding suggests that previous PCR and high-throughput sequencing methods for detecting HPV integration sites in entire cervical cancer tissue cannot accurately reveal the characteristics of different subgroups of cancer cells. This limitation may be why previous studies often report many integration sites, but they cannot determine whether these integration sites are the initiating factors before carcinogenesis or the consequence of genome instability after carcinogenesis.In clinical applications, high-throughput sequencing technologies such as WGS and HIVID can be combined with FISH to detect HPV integration after cervical cancer tissues are obtained by surgery. On the one hand, FISH can be used to verify sequencing results. Since the determination of HPV integration sites by sequencing is based on the similarity of base sequences, sequencing reads with low specificity or containing repetitive sequences will lead to sequence alignment errors, which can be verified by FISH. On the other hand, FISH can be used to detect whether the cancer cells containing a specific HPV integration site are the majority or the minority, which can be indirectly shown by the abundance of the human-virus fusion sequence in the sequencing results, while it can be intuitively observed by FISH. If cancer cells containing a specific HPV integration site are the majority in the tissue, HPV integration at this site may be the key factor promoting cervical cancer. If they are the minority, HPV integration at this site may be a consequence of genome instability in cancer cells.In conclusion, our study provides a method for the detection of HPV integration sites in paraffin-embedded cervical cancer samples using dual-color FISH and reports the heterogeneity within cervical cancer from the perspective of HPV integration. Our study provides new methods and ideas for research on HPV integration in cervical carcinogenesis.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Tongji Hospital. The patients/participants provided their written informed consent to participate in this study.HW and DZ designed and supervised the research together with JX. DZ and HS provided technical support. DZ, JX, CR, LW, and WD performed the experiments. CG, TZ, and XL obtained specimens. JC collected clinical data. HW, DZ, JX, and HS provided analysis and interpretation of data. The manuscript was drafted by DZ and JX. All authors contributed to the article and approved the submitted version.This work was supported by funds from National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P = 0.036). The HR-HPV infection rate was 72.73% in G. vaginalis women, which was significantly higher than that of women with lactobacillus as the dominant microbiota (44.72%) (P = 0.04). The positive rate of sialidase (SNA) was higher in women with HR-HPV infection (P = 0.004) and women diagnosed with cervical intraepithelial neoplasia (CIN) (P = 0.041). In HPV (+) women, the α-diversity was significantly higher than that in HPV (−) women. The 16S rRNA gene-based amplicon sequencing results showed that Lactobacillus was the dominant bacteria in the normal vaginal microbiota. However, the proportion of Gardnerella and Prevotella were markedly increased in HPV (+) patients. Gardnerella and Prevotella are the most high-risk combination for the development of HPV (+) women. The SNA secreted by Gardnerella and Prevotella may play a significant role in HPV infection progress to cervical lesions.This study aimed to explore the changes of the vaginal microbiota and enzymes in the women with high-risk human papillomavirus (HR-HPV) infection and cervical lesions. A total of 448 participants were carried out HPV genotyping, cytology tests, and microecology tests, and 28 participants were treated as sub-samples, in which vaginal samples were characterized by sequencing the bacterial 16S V4 ribosomal RNA (rRNA) gene region. The study found the prevalence of HR-HPV was higher in patients with BV ( Most women will have been infected with HPV by intercourse during their lives, most HPV infections fade away by themselves in a few months, a few HPV infections persist and cause lesions2. Although most instantaneous HPV infections are cleared by the immune system, persistent infections can cause viral gene integration into the host genome and lead to HPV-related cancer3. Previous studies have found that local microbiota, epithelial surface integrity, immune regulation were synergistic factors in the progression of HPV to cancer. Nevertheless, little is known about the functional composition of the local microbiota and how it varies by cervicovaginal syndromes, infections, and diseases4.Human papillomavirus (HPV) infections gave rise to over 600,000 cases of cancer in a yearLactobacillus spp. was the dominant microbiota in CST I, II, III, and V. And the CSTs have different Lactobacillus species types, such as Lactobacillus-crispatus, Lactobacillus-iners, Lactobacillus-jensenii, Lactobacillus-gasseri, and so on5. Notably, The features of CST IV are higher vaginal pH (> 4.5), lack of Lactobacillus, and abundance of Gardnerella. These features are also features of bacterial vaginosis (BV)6. BV is a kind of mixed infection characterized by the reduction of Lactobacillus and the multiplication of pathogen, mainly Gardnerella vaginalis , accompanied by increased vaginal pH. It has been reported that BV infection in the Chinese population ranges from 10.5 to 51.6%7. Previous studies indicate that there is a close correlation between BV infection and HR-HPV persistence. A study by Gillet8 showed that patients with BV are more prone to HR-HPV infections, while Guo9 further found that a BV infection prolongs the duration and the regression time of HR-HPV infections.The vaginal microenvironment can be categorized into five kinds of community state types (CSTs), Gardnerella was significantly increased in HR-HPV-positive women10. In a 2-year longitudinal prospective study, the presence of specific anaerobic groups, including Gardnerella, was associated with the persistence and slow degradation of CIN211. Meanwhile, different dominant bacterial communities produce different metabolomes. Gardnerella secretes SNA while elevated SNA concentration was associated with increased risk for cervical lesion12. The vaginal metabolome of HPV (−) women differed from HPV (+) women in terms of several metabolites, including biogenic amines, glutathione, and lipid-related metabolites13. L. crispatus producing hydrogen peroxide (H2O2) show the strongest associations with vaginal health and are depleted in dysbiosis14. With the development of high-throughput sequencing, there is still a lack of systematic and comprehensive studies to investigate the types and enzymes of vaginal microbiota and their relationship with HPV infection and cervical lesions.As the main pathogen of BV, the detection rate of To solve the above problems, this study started from clinical data, with the help of high throughput sequencing, aimed to explore the changes of the vaginal microbiota and enzymes in the HR-HPV infection progress to cervical lesions and provide ideas for further exploration of the interaction mechanism between vaginal microbiota and HPV infection.Characteristics of 448 participants were analyzed. Participants with normal cervical pathology or negative cytology and HPV (−) were defined as the normal group. Our study indicated that there were no significant differences in terms of age, nationality, marital status, and reproductive history among the groups  and 72.55% (37/51), and the difference was statistically significant (P < 0.001). Among all participants, the most susceptible HPV types were HPV52 (6.7%), HPV58 (4.7%), and HPV16 (4.2%). Among the women diagnosed with NILM, the most susceptible HPV types were HPV52 (5.5%), HPV58 (2.9%), and HPV51 (2.9%). Among women diagnosed with CIN I, the most susceptible HPV genotypes were HPV52 (27.3%), HPV16 (18.2%), and HPV18 (18.2%). The most susceptible HPV types were HPV58 (20.7%), HPV16 (17.2%), and HPV18 (10.3%) in patients diagnosed with CIN II and above. The detailed results are shown in Table G. vaginalis women, which was significantly higher than that of women with lactobacillus as the dominant microbiota (44.72%) (P = 0.04). However, no obvious correlation with HR-HPV infection was found between other dominant microbiota. We also found that the positive rate of SNA was higher in HR-HPV infection women (P = 0.004). The results are shown in Table In this study, the infection rate of BV was the highest, accounting for 41.96% (188/448), followed by vulvovaginal candidiasis (VVC), aerobic vaginitis (AV), cytolytic vaginosis (CV), and trichomonal vaginitis (TV), accounting for 12.05% (54/448), 5.13% (23/448) and 1.78% (8/448) and 0.89% (4/448). BV and HR-HPV infections were associated with each other. The difference was statistically significant (P = 0.036). The HR-HPV infection rate was 72.73% in Among the 448 participants in this study, 18 were excluded because they did not undergo cytological or pathological examination. The remaining participants were in a ratio of approximately 1:2 according to HR-HPV infection or not. The pathological results of NILM and ≥ CIN I patients were matched, and they were divided into a case group (≥ CIN I) and a control group. Ultimately, a total of 177 patients were included in the second part of this study, including 51 cases and 126 controls. In women diagnosed with CIN, the positive rate of SNA increased . However, catalase or leukocyte esterase (LE) were not significantly associated with cervical lesions  and five HPV (−) samples were collected for the study and the control groups, respectively. There was no significant difference in age between HPV (+) and HPV (−) groups (P = 0.666). The curve plateaued Fig. a,b when Firmicutes, Actinobacteria, Bacteroidetes, Fusobacteria, Proteobacteria  women. However, the proportion of Firmicutes decreased (68.26%), and the proportions of Actinobacteria and Bacteroides increased  in the HPV (+) women. The composition and differences of vaginal microbiota between the two groups were further analyzed at the genus level , with a small amount of Gardnerella (1.39%), Atopobium (0.03%) and other genera. In the HPV (+) women, the composition of the vaginal bacterial community structure had significant changes, mainly reflected in the decrease of Lactobacillus (62.91%), and the increase of Gardnerella (12.22%) and Prevotella (6.78%).The distribution of phylum, class, order and genus of vaginal microbiota in HPV infected women were shown in Fig. ria Fig. a. Firmiceased 68.6%, and tThe diversity indicators of the two group samples were shown in Fig. Lactobacillus was the main dominant bacteria in the HPV (−) women, and Prevotella was a distinct microbiota that plays a significant role in the HPV (+) women according to the Linear discriminant analysis (LDA) scores.To discover the changes of vaginal microbiota in HPV infection women, LEfSe analysis was performed on the two groups Fig. e,f. The Gardnerella and Prevotella and the decrease of Lactobacillus are closely associated with HPV infection.The emerging studies suggest that vaginal microenvironment plays an essential role in women’s health, specifically in sexually transmitted diseases. This is a cross-sectional study to explore the changes of the vaginal microbiota and enzymes in the HPV infection and cervical lesions. Overall, a significantly higher microbiota diversity was observed in HPV (+) women than that in HPV (−) women. The increase of Lactobacillus is the dominant bacteria, which play important role in protecting the health of women’s lower reproductive tract15. The vaginal microbiota is primarily dominated by one of the four most common Lactobacillus species: Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii5. Lee et al.16 found the proportion of Lactobacillus was lower in HPV (+) patients. On the one hand, Lactobacillus maintains the weak acid environment of the vagina through its own lactic acid. On the other hand, a large quantity of Lactobacillus can reduce and inhibit the planting and growth of some opportunistic pathogenic bacteria to protect the lower reproductive tract from infection18. The presence of the signature “abnormal vaginal microbiota” in CIN was found by a laboratory culture in 1992 and confirmed in subsequent studies19. When the vagina’s protective microbiota was destroyed, the defense against pathogen infection was weakened.Previous studies indicated 20. Vaginal microenvironment disturbance was associated with increased inflammatory cytokines, mucosal injury and chronic inflammation. In order to investigate the mechanism of BV and HPV infection, Rodriguez-Cerdeira et al. suggested that G. vaginalis is one of the common microbiota in HPV (+) women21, and this has been reported in other researches that using next-generation sequencing23. Women with BV had higher levels of the cytokine interleukin (IL)-1β and lower levels of IL-1725.In this study, We have identified vaginal microenvironment disorder was bound up with HPV infection. Compared with BV (−) women, the HR-HPV infection rate in BV (+) patients increased. BV is associated with an increased risk of detection of HPV, and HPV infections are associated with an increased risk of BV2O2 produced by Lactobacillus can catalyze peroxidase and further produce hypochlorite. It is a process that can prevent HPV from invading into cervical epithelial cells and prevent cervical lesions26. LE is an intracellular enzyme. When vaginal inflammation occurs, a large number of white blood cells can gather to engulf pathogens, resulting in the destruction of white cell membrane, and thus LE can be detected. However, no studies have linked LE to any type of vaginal inflammation. Similarly, this study also showed that there was no statistical significance between LE and HPV infection or cervical intraepithelial neoplasia. Gardnerella adheres tightly to the surface of vaginal epithelial cells, forms a dense biofilm, and can release vaginal cytolysin, which may inhibit the effect of vaginal mucosal barrier immunoglobulin A. Gardnerella can produce SNA, which can degrade mucosal protective factors (such as mucin) and causes vaginal epithelial cells to dissolve and expel27. SNA is an enzyme that cleaves terminal sialic acid residues and is associated with tissue destruction, immune response evasion, bacterial invasion, and access to bacterial-associated nutrients28. In addition to Gardnerella bacteria, such as Prevotella bacteria, Bacteroides bacteria and Mobiluncus bacteria also produce SNA29. SNA usually occupies terminal positions attached to mucosal defense factors, such as secretory IgA, secretory components, lactoferrin, and secretory leukocyte protease inhibitors30.As an important component of vaginal microenvironment, metabolomes play an important role in the pathogenicity of microbiota. The H31 the expression level of Toll-like receptor—(TLR) 4 mRNA in vaginal and cervical epithelial cells was significantly increased in patients with G. vaginalis-infected BV. Previous studies showed that TLR9 recognizes HPV infection and initiates immune response. Experiments at the transcriptional level confirmed that E6 and E7 oncoproteins directly downregulate TLR932. However, whether Gardnerella activates the TLRs-related pathways through SNA to cause HPV infection is still unknown and needs to be assessed. All of these mucosal, bacterial, and immune activations associated with BV and G. vaginalis may lead to progression of HPV infection to cervical cancer.In order to investigate the mechanism of cervical lesions caused by vaginal microbiota and metabolomes, Zariffard et al. found thatGardnerella and Prevotella contributes to HPV causing cervical lesions. Secondly, our study only focused on the distribution of different microbiota, ignoring the identification of Lactobacillus species. Recent researches suggest that Lactobacillus iners is a transitional species that colonize after the vaginal environment is disturbed and leads to BV, sexually transmitted infections, and adverse pregnancy outcomes33. Further studies are necessary to identify the exact role of different Lactobacillus species in larger samples.This study also has some limitations. First, this is a cross-sectional study to explore the changes of the vaginal microbiota and enzymes in women with HPV infection and cervical lesions, further experiments are needed to confirm that SNA secreted by Gardnerella and Prevodella are the most high-risk combination for the development of HPV (+) women. The SNA secreted by Gardnerella and Prevodella may play a significant role in HPV infection progress to cervical lesions. This finding provides ideas for further exploration of the interaction mechanism between vaginal microbiota and HPV infection.Our research demonstrates vaginal microenvironment, especially BV was closely related to HPV infection. So more attention should be paid to the prevention, discovery and proper management of BV in HPV infection women. Particularly, compared to HPV (−) women, The study participants were selected from the Fujian Cervical Lesions Screening Cohorts (FCLSCs), China. A total of 448 participants have carried out microecology tests, HPV genotyping and cytology tests, and 28 participants were treated as sub-samples, in which vaginal microecological samples were characterized by sequencing the region of bacterial 16S V4 ribosomal RNA (rRNA) gene . The cotton swabs were inserted into a tube  carefully avoiding skin contamination. The samples were stored at − 20 °C as soon as possible after collection for the vaginal microbiota analysis.The researchers used plastic brushes to collect cervical cells from all participants’ cervix, elutioned in ThinPrep PreservCyt Solution , and stored specimens at 4 °C in laboratory immediately.2O2, LE and SNA in secretions were detected by bPR-2014A vaginitis automatic detector and supporting detection kit . Vaginal PH value was determined by color strips. If the PH value was no more than 4.5 (pH ≤ 4.5), the result was defined as normal. On the contrary, pH > 4.5 was defined as abnormal. Vagina cleanness was diagnosed in accordance with the standard of National Clinical Laboratory Practice Guideline 18: I–II were defined as normal vagina cleanness, and vagina cleanness III–IV defined as abnormal. AV, BV, CV, TV and VVC were all negative or positive. The Nugent scoring method was used to diagnose BV. The Nugent score was calculated by assessing the numbers of Lactobacillus morphotypes (scored as 0–4), G. vaginalis morphotypes (scored as 0–4), and Mobiluncus morphotypes (scored as 0–2). A Nugent score of 7–10 was interpreted as consistent with BV and a score of 4–6 as intermediate, while a score of 0–3 was interpreted as negative for BV. SNA colorless is normal (−), red or purple is positive (+). LE colorless is normal (−), and green or blue is positive (+). H2O2 > 2 mmol/L is red or purple, negative (−), H2O2 < 2 mmol/L is positive (+), blue. All laboratory procedures were conducted according to the manufacturer’s instructions.The Vaginal secretions were obtained on 1/3 of the vaginal sidewall. Check whether there is trichomonad, mycelium, clue cells under the microscope after daubing on clean slide. H34. This method and kit have been approved by China Food and Drug Administration . The procedures were conducted according to the manufacturer’s instructions.The HPV genotypes  were detected by Polymerase chain reaction-reverse dot blot (PCR-RDB) HPV genotyping kit The cytological samples were blinded and independently evaluated by two experienced cytopathologists and re-evaluated until reach a consensus when the diagnoses were different. Samples were classified as NILM, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), atypical squamous cells, and it was not possible to exclude high-grade squamous intraepithelial lesions (ASC-H), squamous cervical cancer (SCC) and atypical glandular cells (AGC).According to the cervical cancer screening procedure, women with HR-HPV infection or abnormal cytology results may be referred for colposcopy or biopsy. When biopsy diagnosis results in ≥ HSIL, patients underwent a loop electrosurgical excision procedure cone or conization by cold knife to biopsy. Formalin (10%) was used to fix specimens, which were routinely processed for paraffin embedding. Then, 4 µm thick histological sections were cut and stained with hematoxylin and eosin using standard methods. Cervical biopsy specimens were examined and diagnosed according to the CIN system. If the review reading is inconsistent, conduct a second histological review. If two-thirds of the diagnoses are the same, the result is considered the final result.Genomic DNA of vaginal secretions samples was extracted by E.Z.N.A Mag-Bind Soil DNA Kit  according to the manufacturer’s protocol. DNA samples were quantified by the Qubit 3.0 DNA Kit  and transferred to Sangon Biotech Testing Center  for high-throughput sequencing.Bacterial DNA was amplified by 16S V4F primers (5′ CCTACGGGNGGCWGCAG 3′) and 16S V4R primers (5′ GACTACHVGGGTATCTAATCC 3′) complementary to the V4 region of 16S rRNA gene. This variable region has been verified can accurately amplify and resolve DNAs of vaginal microbiome. PCR reaction system consisted of 9–12 µL of nuclease-free water, 15 µL of 2 × Hieff Robust PCR Master Mix, 5 µM of each primer and 20–30 ng of genomic DNA. The cycling conditions include: initial denaturation at 95 °C for 3 min, then 94 °C for 30 s, 45 °C for 20 s, 65 °C for 30 s, then 94 °C for 20 s, 55 °C for 20 s, 72 °C for 5 min. The samples were analyzed using Roche LightCycler 480 PCR system .Purified PCR products were accurately quantified by Qubit 3.0 Fluorometric High-Sensitivity dsDNA Assay  and then constructed library by KAPA LTP Library Kit . High-throughput sequencing of 2 × 300 paired-end reads was performed on an Illumina MiSeq platform  at Sangon Biotech . Raw FASTQ files were obtained and merged.http://www.mothur.org/wiki/Chao; http://www.mothur.org/wiki/Ace) indices, for calculating the abundance of community distribution were used. Furthermore, the Shannon and Simpson  indices were used to calculate the diversity of community distribution. Biomarker discovery analysis was carried out by the LEfSe tool and LDA scores higher than 2.0 were considered statistically significant.The version of mother was v1.30.1. SILVA release 132 was used as database. R v3.6.3 was used for statistical analyses. The similarity truncation rate of the operational taxonomic units cluster was 97%. The diversity analysis of the sample (α or β diversity) could indicate the diversity or abundance of microbial communities, including the Chao and ACE  in this study. The significance level was set at a two-tailed p-value < 0.05.The study was approved approved by the Ethics Committee of Fujian Maternity and Child Health Hospital (2020KY015).


Serum antibodies against human papillomavirus 16 (HPV16) proteins are associated with HPV-driven oropharyngeal cancer (HPV-OPC). The HPV status of OPC cases is clinically relevant because patients with HPV-OPC show improved survival and treatment response compared to tobacco- or alcohol-induced OPC. In clinical settings, molecular HPV tumor status is usually determined by tissue-based methods detecting molecular markers, such as viral nucleic acids or p16 overexpression. Antibodies against HPV16 in peripheral blood were shown to be very accurate in determining the molecular HPV tumor status in multiple studies. In this work, we reviewed and summarized the available literature on the performance of HPV16 serology for E2, E6 and E7 antibodies to determine molecular HPV tumor status in OPC cases in comparison with tissue-based reference methods. We calculated summary estimates across different studies for sensitivity and specificity, and we investigated factors influencing test performance.n = 23 studies meeting all eligibility criteria and included these in the meta-analysis. E6 serology showed the best performance with pooled sensitivity and specificity estimates of 83.1%  72.5–90.2%) and 94.6% (95% CI 89.0–97.4%), respectively, while E2 and E7 serological assays were highly specific (E2: 92.5% (95% CI 79.1–97.6%); E7: 88.5% (95% CI 77.9–94.4%)) but moderately sensitive (E2: 67.8% (95% CI 58.9–75.6%); E7: 67.0% (95% CI 63.2–70.6%)). Subgroup analyses revealed increased pooled sensitivity for bacterially (89.9% (95% CI 84.5–93.6%)) vs. in vitro expressed E6 antigen (55.3% (95% CI 41.0–68.7%)), while both showed high specificity (95.2% (95% CI 93.0–96.7%) and 91.1% (95% CI 46.6–99.2%), respectively). Pooled specificity estimates for HPV16 E2, E6 and E7 serology were significantly lower in studies utilizing HPV DNA PCR as the only molecular reference method compared to those using a combination of any two reference methods , p16 immunohistochemistry (IHC)), or histopathological reference methods (ISH or p16 IHC) as stand-alone marker. In conclusion, HPV16 E6 seropositivity is a highly sensitive and specific biomarker for HPV-OPC. However, its performance differs between serological assays and depends on molecular reference methods.Antibodies against HPV16 early proteins have been shown to be promising biomarkers for the identification of HPV-driven oropharyngeal cancer (HPV-OPC) among OPC cases in multiple studies. A systematic literature search was performed to identify original research articles comparing HPV early antigen serology with established reference methods to determine molecular HPV tumor status. Random-effects models were used to calculate summary estimates for sensitivity and specificity of HPV16 E2, E6 and E7 serology for HPV-OPC. Subgroup analyses were performed to explore heterogeneity across studies and describe variables associated with test performance. We identified  The incidence of oropharyngeal cancer (OPC) has been rising over the last decades in many Western countries due to an increase in incident OPC cases attributable to infection with oncogenic human papillomaviruses (HPV) . The HPVHPV-OPC on the one hand, and tobacco- and/or alcohol-induced HPV-negative OPC on the other are considered distinct disease entities as they differ in their epidemiology, molecular characteristics and clinical behavior . HPV staink4 overexpression in cancerous cells caused by the E7 oncoprotein )A recent review and meta-analysis by Balachandra et al. investigated blood-based biomarkers for the identification of HPV-driven cancers including HPV16 E6 serology for oropharyngeal cancer . After eThis systematic literature review presents a comprehensive overview of the published literature on HPV16 serology performance for several early antigens to determine molecular HPV tumor status among OPC patients in comparison with tissue-based reference methods. However, there are also some limitations. First, there was substantial heterogeneity between the included studies, potentially limiting the precision of the derived sensitivity and specificity estimates and the power of the performed analysis. However, this was expected as a common occurrence in diagnostic test accuracy studies. Second, studies which reported serology results and their agreement with reference methods only as validation subsets often featured very small sample sizes which can increase uncertainty. However, our sensitivity analysis did not identify small sample size as a source of bias. Third, although the detection of HPV mRNA is nowadays considered the laboratory gold standard for the diagnosis of HPV-OPC, only very few studies utilized this reference method; most relied on less reliable reference methods such as HPV DNA PCR, p16 IHC and DNA ISH. Systematic misclassification of samples by less sensitive and/or specific reference methods may have resulted in higher type I and type II error rates, and may have caused over- or under-estimation of serology performance. Indeed, stratification of studies according to the used reference method suggested higher error rates caused by HPV DNA PCR than any other reference method. Lastly, the differences in study designs restrict the generalizability of our specificity estimates and suggest independent analyses in cancer-free controls in the future.In this systematic literature review, we provided summary estimates and further identified the expression system and reference methods as significant moderators for HPV16 serology performance. This shows that the performance of HPV16 early protein serology has to be evaluated in the context of the characteristics of the reference method and the setting in which it was applied, e.g., a region/study with high or low HPV-AF. The preparation of this systematic review further revealed the lack of available information on the performance of HPV serology for other oncogenic HPV types, and serological HPV(16) biomarker combinations. The identification of parameters associated with performance of HPV early antigen serology may facilitate further method optimization in the future.In this systematic review and meta-analysis, we provide robust evidence that antibodies against HPV16 early proteins, especially HPV16 E6, are tumor-tissue-independent and highly sensitive and specific biomarkers for the detection of HPV-driven OPC at diagnosis.


This study focused on the prevalence of high-risk human papilloma virus (HR-HPV) infection types, other than HPV-16 and -18, in women throughout Kazakhstan due to the high rates of cervical cancer in Kazakhstani women. Approximately one quarter of the of the participants were infected with HR-HPV types other than HPV-16 and -18; 72% of these women were infected with one HR-HPV type with HPV-31 being the most prevalent, and the remaining 28% of these infected women were infected with multiple HR-HPVs with HPV-68 being the most prevalent type in these women. Introducing HR-HPV testing for all 14 cancerogenic types into cervical cancer screening program could help to reduce the rates of cervical cancer in Kazakhstan.Cervical cancer represents a great burden to public health of women. This study aimed to obtain a nationwide genotyping survey and analysis of high risk-HPV including those that are caused by HPV types other than HPV-16 and HPV-18, among women in Kazakhstan. This study was conducted based on the collection of survey and cervical swabs of 1645 women across the country. The samples were genotyped for high-risk HPV types based on real-time PCR methods. Collected data was analyzed with the focus on high-risk HPV types other than HPV-16 and -18. Infection was present in 22% of women who participated in the study. The most prevalent types were HPV-31 among single infections and HPV-68 among multiple infections. Conclusively, despite the lack of attention high-risk HPV types beyond HPV-16 and -18 get in attempts of cervical cancer prevention in Kazakhstan, their prevalence is high and plays a large role in cervical cancer epidemiological situation.  Cervical cancer is the leading cause of cancer-related deaths in developing countries. Moreover, cervical cancer is estimated to be the fourth most common cancer in women worldwide . MajoritDespite the presence of the national cervical cancer screening program in Kazakhstan, the disease incidence has increased during the recent decade. The annual rate of cervical cancer incidence in the country increased from 16.3 ± 0.4 per 100,000 female population in 2009 to 19.5 ± 0.5 in 2018 [Previous HR-HPV genotyping studies conducted in Kazakhstan include both HPV-16 and HPV-18 and HR-HPV types other than 16 and 18 ,6. HowevA prospective cross-sectional study among Kazakhstani women was conducted from May 2019 until December 2020. Sample collection was conducted in five major cities from different regions of Kazakhstan .After obtaining informed consent, only patients who agreed to participate were included in the study. The following inclusion criteria were used to choose women for study participation: (1) age from 18 to 70; (2) attends a gynecological center located in one of chosen cities for the study; and (3) able to fill questionnaire in Kazakh, Russian or English language. Each woman participating in the study had a Papanicolaou (Pap) smear test performed as gynecologists were taking cervical swabs. Gynecologists also recorded the age of the patients along with other demographic data  that identifies 14 high-risk HPV types  on the CFX 96 Real-Time PCR machine (Bio-Rad Laboratories). Genotyping was performed following the instructions of the manufacturer, including positive and negative controls, samples concentration and thresholds for positivity of the samples. The samples were collected into 1.5 mL Eppendorf tubes and were kept frozen until further use. WizardThe data collected was analyzed using STATA 16 software . PrevaleThe study was approved by the Institutional Research Ethics Committee of Nazarbayev University (NU IREC) on 23 April 2019 (IREC number: 146/4042019). All study participants were given information on the risks, benefits, goals and methods of the study. Confidentiality of the collected data was protected.In total 1645 women participated in the study. The mean age of the participants was 35.98 ± 9.97, and 40.49% of women were aged between 26 and 35. The minimum age was 18 years and the maximum age was 70 years. More than three quarters (76.25%) of the participants identified themselves as ethnic Kazakh. There was almost equal regional distribution of women who participated in the study (around 20% from each region of the country) with the exception of the north region (around 16%). Almost half (47.17%) of the participants had graduate-level education and more than one quarter (34.77%) of women had an undergraduate-level diploma. A majority of women (82.07%) were married or in a committed relationship. More than three quarters of the participating women (79.45%) had one or more children n = 370) were infected with HR-HPV other than HPV-16 or 18, which amounts to 71.62% (n = 265) of HPV positive women or 16% of all study participants. These women had a single infection with only one HR-HPV type. Among HR-HPV types, excluding HPV-16 and 18, HPV-31 (18%), HPV-51 (14%), HPV-68 (11%) and HPV-52 (9%) were the most prevalent single HR-HPV infection (Out of the total number of participants, 22.49% (nfection . Infectinfection . In terms of age distribution, the HR-HPV genotypes were most frequently detected in the 26–35 age group, followed by 36–45 age group . In bothThe geographical distribution of HR-HPV types other than HPV-16 and HPV-18 is presented in To our knowledge, this is the first study that focuses on the distribution and prevalence of HR-HPV genotypes other than HPV-16 and HPV-18 in the different regions of Kazakhstan. Moreover, this study looked at the prevalence of single and multiple HR-HPV infections among the participants. The results of this investigation showed that 22% of women included in the study were positive for one or more HR-HPV types other than HPV-16 and HPV-18, whereby 16% had a single HR-HPV infection and 6% had multiple HR-HPV infection. When comparing to the results of previous study [We found it difficult to compare HR-HPV prevalence with previous studies conducted in Kazakhstan, as these studies have included HPV-16 and HPV-18 ,6 and prThere are both similarities and differences in the distribution of HR-HPV genotypes other than HPV-16 and HPV-18 in the studies conducted in Kazakhstan: HPV-31, HPV-39, HPV-51 and HPV-52 were found to be the most prevalent. However, unlike previous reports, this study found a high prevalence of HPV-35, HPV-59, HPV-58 and HPV-68. Therefore, even within one country HR-HPV genotype distribution can vary significantly among regions. Our study also found that women at the reproductive age had the highest prevalence of HR-HPV infection. Women aged between 26 and 35 had the highest HR-HPV prevalence, followed by the age group between 36–45 years. This finding is similar to the previous studies, which found that HPV prevalence was highest in the young age, and HPV prevalence was lower among women aged older than 60 ,11. In cThe strengths of this study include large sample size, wide-geographical distribution of collected data including western, northern, eastern and southern regions, and differentiation between single and multiple HR-HPV infection, as well as a focus on detecting HR-HPV genotypes other than HPV-16 and HPV-18. However, the study results should be interpreted and generalized cautiously because the participants were non-randomly selected, using the convenience sampling technique.Currently, the cervical cancer prevention program in Kazakhstan does not include screening for HPV infection or HPV vaccination as a primary prevention. Moreover, as HPV testing is not covered by the government or insurance, and investigation of only HPV-16 and 18 is a cheaper option, many gynecologists do not require their patients to test for other HR-HPV types with the financial burden in mind. More research needs to be conducted to study prevalence of all HR-HPV types including different cervical intra-epithelial neoplasia stages. Nationwide HPV genotyping among the general population in Kazakhstan including both male and female patients is required to have a full picture of HR-HPV burden. Additionally, the national cervical cancer prevention program should be expanded beyond regular Pap smear tests.To conclude, this study focused on genotyping analysis of HR-HPV other than HPV-16 and HPV-18 among women attending gynecologists’ offices in Kazakhstan. Despite the fact that other HR-HPV genotypes are responsible for less numbers of cervical cancer, they still play an important role in the epidemiology of the disease. Introducing compulsory testing for HR-HPV types other than HPV-16 and HPV-18 and more attention on the problem from gynecology and oncology specialists could help to reduce the increasing rates of cervical cancer in Kazakhstan.


Natl. Acad. Sci. USA 87:463–467), several studies have suggested a possible link between the pathogenesis of breast cancer and viral infection. Infection with oncogenic HPV has been one of the viruses implicated in breast cancer cases worldwide.Since the work of Band To investigate the presence of HPV DNA in archived paraffin-embedded breast cancer cases at the University Hospital of Brazzaville and to assess the association between viral HPV infections and clinicopathological features.A total of 40 formalin-fixed paraffin-embedded (FFPE) biopsies were retrospectively collected and available information was recorded. HPV detection and genotyping were performed by real-time PCR by GeneXpert technology .P>0.05). However, a statistically significant difference was observed between HPV infection and SBR grade (P=0.05). The mean age was 51.1±11.4 years . Overall, HPV DNA was detected in six (15%) breast carcinoma samples. HPV-16, the most common genotype was identified in 83.7 % of all samples. HPV porting with clinicopathological features showed no significant difference ( Our study described a high prevalence of HPV-HR in breast cancer cases in the Congolese woman. Future type case-control studies are necessary to better describe the potential role of HPV in the occurrence of breast cancer in Congo. Breast cancer is the second most common cancer worldwide and the first female cancer, with 2088849 new cases diagnosed in 2018, accounting for 12.19% of all cancers. It is the leading cause of cancer death among women in developing countries with more than 324000 deaths . TherefoThe mean age was 51.1±11.4 years (22–75 years). The median was 47 years old. The localization of tumours according to laterality indicated that the right breast was slightly more affected (52.5%) than the left breast (45%). The histological types of non-specific type carcinoma (50%) and Grade III SBR (50%) were the most represented.P>0.05).et al. (1990) were the first to postulate that HPV might be involved in breast cancer [Breast cancer is the leading cause of cancer death in women worldwide, with an incidence that is increasing every year . Severalet al. 190 were tTo our knowledge, few data are available on the prevalence of HPV in breast cancer cases in Africa. We report here the first Congolese descriptive study evaluating the presence of HPV-DNA in paraffin-embedded breast cancer biopsies using the Cepheid Xpert-HPV test.et al. (2016), Virtanen et al. (2017) and Donà et al. (2017) were the first authors to use GeneXpert technology for amplification of HPV in formalin-ﬁxed parafﬁn-embedded samples. The results obtained by these studies have shown a perfect concordance with other conventional techniques, which shows that the Xpert HPV system provides a convenient molecular method for testing FFPE samples [The Cepheid Xpert-HPV assay is a new method for HPV testing using the liquid phase cytology samples. Several studies have also evaluated Xpert-HPV assay as a powerful method for amplifying oncogenic HPV in literature using cervical smear samples . However samples .et al. (1990) was the first author to postulate that HPV could be involved in breast cancer and Di Lonardo et al. (1992) showed by molecular amplification the presence of HPV-16 in 29.4 % of breast cancer samples, corroborating a potential relationship between HPV and breast cancer [In our study, the overall prevalence of high-risk HPV infection was 15%. Our results corroborate the majority of studies that investigate the presence of viral HPV-DNA in cases of breast cancer worldwide . Howevert cancer . The HPVt cancer . It is kt cancer . In our In fact, it should be noted that the Xpert HPV technique is ideal for analysing and identifying papillomaviruses in paraffinic biopsies. This system also has the advantage of partial typing of high-risk HPV 16, 18/45 genotypes responsible for more than 90% of cervical cancer cases worldwide.Moreover, in the case of breast cancer, although our study has been able to show the presence of HPV DNA in our series, many studies have not identified HPV in cases of breast cancer The main limitation in this work is the small sample size. However, our preliminary study had the privilege of showing the presence of HPV-HR DNA in certain biopsies of breast cancer in the Congolese population. This opens the way to more important investigations for the verification of this research component.Human papillomavirus was found in 15% of breast cancer cases in our study. It is an informative study because it shows the molecular signature of high-risk HPV in breast cancer cases in our country. It also shows the predominance of HPV16 in all HPV-positive cases although no significant association with histological types was observed. A larger study is needed to confirm this hypothesis and to consider infectious causes in the management of breast cancer in Congo.A descriptive and cross-sectional study was conducted on biopsies of breast cancer included in paraffin blocks between 2014 and 2017. The data collection was retrospective by analysis of the registers of the results relating to cases of breast cancer.In fact, 62 files meeting the major selection criteria ‘breast cancer’ were identified in the service’s registers. Twelve blocks of paraffin could not be found in the archives and 10 blocks were excluded for poor conservation of the material which had led to their unexploitation . The stuHPV detection and genotyping were performed using the Cepheid GeneXpert Instrument Systems  which is a real-time PCR in the Molecular Biology Laboratory of the Marie Madeleine Foundation in Pointe-Noire.−1It was performed in two steps, dewaxed with xylene and enzymatic digestion with proteinase K. Sections of 5 µm were obtained using a microtome and the dewaxing was performed according to the xylene-ethanol method for all paraffin-embedded biopsies. The dewaxed tissue was washed with PBS solution. Then we performed enzymatic digestion with proteinase k concentrate at 2 mg mlThe ‘RNA / DNA purification kit’ (NORGEN) was used for the extraction of DNA. Once the tissue was digested, we added 800 µl of ethanol at 90 °C (Cooper) to each tube containing the samples. The mixture was homogenized and extracted according to the manufacturer’s instructions. The obtained DNA extracts were measured with a Qubit 3.0 fluorometer to evaluate the concentration of the DNA.The Xpert HPV assay is an automated test for qualitative detection and differentiation of HPV DNA. The assay is performed on Cepheid GeneXpert Instrument Systems. GeneXpert Instrument Systems automate and integrate sample processing, cell lysis, purification, nucleic acid amplification, and detection of the target sequences in clinical samples by using real-time PCR. The systems consist of an instrument, personal computer, and preloaded software for running tests and viewing the results. The systems require the use of single-use disposable GeneXpert cartridges that hold the PCR reagents, house the sample, and carry out the PCR processing. Because the cartridges are self-contained, cross contamination between samples is minimized. For a full description of the systems, refer to the appropriate GeneXpert Dx System Operator Manual or the GeneXpert Infinity System Operator Manual. The Xpert HPV Assay includes reagents for the detection of high risk HPV.in vitro test for the detection of the E6/E7 region (targets a sequence of 80–150 bp) of the viral DNA genome from high-risk human papillomavirus (HPV) in patient samples. The assay performs multiplex amplification of the target DNA rt-PCR of 14 high-risk HPV genotypes in a single run. Xpert HPV specifically identifies HPV 16 and HPV 18/45 genotypes in two separate detection channels and reports 11 other high-risk genotypes  in a pooled result.The Xpert HPV is a qualitative Following the manufacturer’s instructions for collecting cervical specimens, a Sample Adequacy Control (SAC) and a Probe Check Control (PCC) are also included in the cartridge. SAC reagents detect the presence of a single copy human gene and monitor whether the specimen contains adequate numbers of human cells to carry out a qualitative assessment of HPV status. The PCC verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. The six colour channels contain primers and probes for the detection of specific genotypes or pooled results as follows: ‘SAC; Primary’ for the Sample Adequacy Control, ‘HPV 16; Primary’ for HPV 16, ‘HPV 18_45; Primary’ for the HPV 18/45 pooled result, ‘P3; Primary’ for the pooled result of any of HPV types 31, 33, 35 52, or 58, ‘P4: Primary’ for the pooled result of either of HPV types 51 or 59, and ‘P5; Primary’ for the pooled result of any of HPV types 39, 56, 66 or 68.p-values (P≤0.05) were considered statistically significant.All statistical analyses were performed using the Epi-info V7.2 statistical package. The Chi-square test was used for the comparison of the variables with HPV infection. The


Ictalurus furcatus are an invasive, yet economically important species in the Chesapeake Bay. However, their impact on the trophic ecology of this system is not well understood. In order to provide in‐depth analysis of predation by Blue Catfish, we identified prey items using high‐throughput DNA sequencing (HTS) of entire gastrointestinal tracts from 134 samples using two genetic markers, mitochondrial cytochrome c oxidase I (COI) and the nuclear 18S ribosomal RNA gene. We compared our HTS results to a more traditional “hybrid” approach that coupled morphological identification with DNA barcoding. The hybrid study was conducted on additional Blue Catfish samples (n = 617 stomachs) collected from the same location and season in the previous year. Taxonomic representation with HTS vastly surpassed that achieved with the hybrid methodology in Blue Catfish. Significantly, our HTS study identified several instances of at‐risk and invasive species consumption not identified using the hybrid method, supporting the hypothesis that previous studies using morphological methods may greatly underestimate consumption of critical species. Finally, we report the novel finding that Blue Catfish diet diversity inversely correlates to daily flow rates, perhaps due to higher mobility and prey‐seeking behaviors exhibited during lower flow.Blue Catfish  We compared high‐throughput sequencing (HTS) to traditional morphological diet analysis methods in Blue Catfish and found that HTS vastly outperforms visual methods, even when coupled with barcoding of unidentified tissues. We identified levels of at‐risk species consumption, including Atlantic Sturgeon and alosines, by Blue Catfish higher than those previously reported. Additionally, our study details the novel finding that Blue Catfish diet diversity is inversely related to flow rates. All sampling occurred in tidal‐fresh habitats with minimal salinity influence (0–0.1 ppm) at 65 rkm (measured from the York River mouth at the Chesapeake Bay). Each collection transect was approximately 2 km each in length. We used a boat‐mounted Smith‐Root electrofishing 7.5 GPP unit, 5,000‐watt generator at 15 pulses per second. All fish were measured  and placed into containers with ice to minimize tissue degradation. On the same date, fish were taken to a controlled laboratory setting for GI tract extraction and preservation. Entire GI tracts (esophagus to anus) were carefully removed with sterile procedures, placed in 100% ethanol, and stored in a temperature‐controlled facility until further processing.2.2We briefly examined contents from the GI tract and removed them using sterile techniques. Two Blue Catfish contained empty stomachs and digestive tracts and were not included in further analysis. DNA was extracted using the Macherey Nagel Stool Sample Extraction kit. Amplification of barcoding regions was conducted for COI . The hybrid study analyzed contents found in stomachs only, which would have been consumed within the past 24 hr. Therefore, we correlated the hybrid dataset to average flow within one day of collections.  was detected per sample. We identified 8,701 COI features with a mean frequency per sample of 30,269. An average of 141.2 features (45.3 OTUs) was identified per sample. The COI gene thus detected, on average, two times more OTUs than the 18S gene in Blue Catfish.3.2F = 2.64, q = 0.008) as well as for life stage , with juvenile Blue Catfish consuming noticeably more Asian Clam than their adult counterparts  had relatively low flow rates of 9.1 and 27.0 mD Figure . ANCOM a3.3Morone saxatilis, Bay Anchovy Anchoa mitchilli, Blue Crab Callinectes sapidus, and native submersed aquatic vegetation  and American Shad Alosa sapidissima, species of concern under a moratorium in this geographical region due to low population levels .We compared our taxonomic results to those achieved using a hybrid approach by Schmitt et al. . The hybLastly, we examined hybrid diet content in relation to flow rates as performed for HTS results. As seen with HTS data, samples collected on low flow rate days exhibited increased diet diversity when compared to samples collected on higher flow rate days Figure . Thus, t4Anguilla rostrata. However, HTS methodology detected considerable consumption of these species. Our study thus supports the hypothesis that traditional morphological and even hybrid approaches may miss important prey, especially small, soft‐bodied organisms, eggs, and larvae that digest rapidly  spatial trends. Schmitt et al.  on leaf and stem surfaces that contain a diverse assemblage of algae, cyanobacteria, heterotrophic microbes, and detritus ; Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Software ; Supervision ; Writing‐original draft ; Writing‐review & editing (lead). Aaron Bunch: Conceptualization (lead); Formal analysis ; Funding acquisition (lead); Project administration ; Supervision ; Writing‐original draft ; Writing‐review & editing (supporting). Joseph Schmitt: Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Frederick Hoogakker: Data curation (supporting); Investigation (supporting); Writing‐review & editing (supporting). Kara Carlson: Data curation ; Investigation ; Methodology (supporting); Writing‐review & editing (supporting).


The treatment landscape of locally advanced HPV-oropharyngeal squamous cell carcinoma (OPSCC) is undergoing transformation. This is because the high cures rates observed in OPSCC are paired with severe treatment-related, long-term toxicities. These significant adverse effects have led some to conclude that the current standard of care is over-treating patients, and that de-intensifying the regimens may achieve comparable survival outcomes with lower toxicities. Consequently, several de-escalation approaches involving locally advanced OPSCC are underway. These include the reduction of dosage and volume of intensive cytotoxic regimens, as well as elimination of invasive surgical procedures. Such de-intensifying treatments have the potential to achieve efficacy and concurrently alleviate morbidity. Targeted therapies, given their overall safer toxicity profiles, also make excellent candidates for de-escalation, either alone or alongside standard treatments. However, their role in these endeavors is currently limited, because few targeted therapies are currently in clinical use for head and neck cancers. Unfortunately, cetuximab, the only FDA-approved targeted therapy, has shown inferior outcomes when paired with radiation as compared to cisplatin, the standard radio-sensitizer, in recent de-escalation trials. These findings indicate the need for a better understanding of OPSCC biology in the design of rational therapeutic strategies and the development of novel, OPSCC-targeted therapies that are safe and can improve the therapeutic index of standard therapies. In this review, we summarize ongoing research on mechanism-based inhibitors in OPSCC, beginning with the salient molecular features that modulate tumorigenic processes and response, then exploring pharmacological inhibition and pre-clinical validation studies of candidate targeted agents, and finally, summarizing the progression of those candidates in the clinic. The other subtype (HPV–HNSCC) is caused primarily by alcohol and tobacco usage, and is more anatomically distributed. Epidemiologically, HPV+-OPSCC has been rising substantially in the past 4 decades. In parallel, HPV–HNSCC has been declining and shifting in the opposite direction  OPSCC versus p16- negative (HPV negative status) patients (th) of The American Joint Committee on Cancer (AJCC) staging system, which downgrades the Tumor, Nodes, Metastasis (TNM) classification of HPV+-OPSCC relative to HPV-HNSCC  arise from anatomical regions of the head and neck that include the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx . Althougirection , 2. The PV–HNSCC –5. More outcomes , 6. One outcomes . They repatients . This prPV-HNSCC . EssentiPV-HNSCC . UnfortuPV-HNSCC . Many no+ patient cohort. To this end, a number of de-escalation strategies are currently being pursued. These strategies generally include reduction of target volume, dosages of RT and chemotherapy, reduction of the number of modalities in combinatorial regimens, radiation treatment guided by response to induction chemotherapy, and use of minimally invasive surgery as shown in +-HNSCC. We then highlight pre-clinical research done to validate the functional importance of these unique alterations. Finally, we briefly review the translational relevance of targetable alterations by looking at impact and progress of targeted agents currently in clinical studies and their potential utility as agents for de-escalation in the future.For these reasons, a separate therapeutic strategy is needed for the HPV- and HPV+ cancers are genetically distinct and heterogeneous diseases. The most dominant mutations in HPV–HNSCC affect cell cycle control and involve inactivation of the p53 and Rb-p16INK4A–cyclin D1 pathways  tools, setting the stage for mechanism-driven research , 13–16. pathways , 15. In nd c-Met , 18. Oth altered –16. For  altered –16. As i+-HNSCC (and HPV-unrelated HNSCC) are comparatively fewer than in other cancers. In principle, this limits the number of druggable targets and consequently, the respective translational efforts aimed at improving patient outcomes in HNSCC. That said, the integration of the HPV genome in transformed keratinocytes and the continuous expression of associated viral proteins in HPV+ tumors may provide additional molecular and biological alterations of therapeutic relevance. Not only do the HPV proteins influence the global methylation profile of HPV+-tumor cells and in turn their own expression, studies show that they modulate the expression of other cellular oncogenes including members of the ErBb/Her family . This phosphorylated lipid will then transduce the signal to major downstream effectors of the PI3K pathway, Akt and mTOR, to modulate processes that are key to the growth and metastasis of many cancers as shown in +- and 34% in HPV–HNSCC tumors . Studies using transgenic mice and cancer cells have shown that the two types of mutants activate their effectors through different mechanisms and that the H1047R substitution may be the better activator of Akt  and double strand breaks (DSBs) when applied to cells . Under n+-HNSCC has revealed that this cancer subtype harbors DSB repair defects , can also be considered a potential target , 74. Stu cancers , 81. Theactivity . Taken t+- and HPV–HNSCC tumors exhibit some functional equivalency in how they inactivate cell cycle regulators. HPV+ cancers express oncoproteins that accelerate the degradation of p53 and Rb through the ubiquitin proteasome system. In HPV- cancers, mutations and promoter methylations affecting p53 and CDK2A (which encodes p16INK4a and p14) are common , is another approach that has potential as a candidate for de-intensification. Although immunotherapies do not directly target driver oncogenic events in tumor cells, their effect on non-cancerous, immune cells in the tumor nest can hugely impact on tumor control and response to therapy. Various ICIs including anti-PD1, anti-PDL1 and CTLA-4 antibodies have been tested for the treatment of HNSCC and two ICIs, nivolumab and pembrolizumab, both anti-PD-1 antibodies, were granted FDA approval for platinum refractory patients in 2016. More recently, pembrolizumab has been approved as a first-line therapy alone or in combination with platinum/fluorouracil based on PD-L1 expression for patients with R/M disease. This progress in the last 5 years is significant and remarkable given that immune function is depleted in R/M tumors compared to earlier stages such as LA disease. Additionally, these immunotherapies have also shown to be more tolerable than chemotherapy, improving both OS and QoL . These f+- and HPV–HNSCC confirms that these two entities are immunologically distinct diseases. Part of this distinction comes from their differences in mutational profiles. HPV-HNSCC has a relatively high tumor mutation burden (TMB) and a wider spectrum of mutations  subgroup (+-HNSCC is also characterized by the presence of CD4+ CD25+ Tregs and high expression of PD-1, CTLA-4 and TIM-3 on the T cells (+-HNSCC tumor cells themselves also express higher levels of the PD-1 ligand, PD-L1 (–HNSCC have significantly lower levels of immune infiltrates and relatively lower numbers of CD8+ T cell and exhaustion markers, despite their higher mutational burden. In fact, among infiltrated tumors, a pan-cancer analysis showed that HPV+-HNSCC had the highest median of immune infiltrates (+-HNSCC corresponded well with clinical outcomes (–HNSCC, an immune regulatory character was detrimental to patient prognosis. Only high levels of CD8 T cells and high TMB correlated positively with prognosis for HPV- patients (A close look at HPVutations . A high utations . The tume robust , 110. Al cancers . Indeed,category . This ca, HLA-DR . Again, subgroup . Interes T cells . Moreoveiltrates . Interesoutcomes , 113. In+ and HPV–HNSCC patients, respectively (+ and 10.8% in HPV- patients when separated by HPV status (+ than in HPV- patients (+-HNSCC patients. As the research on this continues, studies exploring an exciting alternative strategy involving the combination of ICIs with other traditional treatments such as radiation, with or without de-escalation intent are already underway (+ cohort if it comes with less toxicity compared to platinum-based CRT. As of now, it is still too early to tell what conclusions these trials will ultimately support. So far, the results have been negative, as evidenced by the JAVELIN head-and-neck 100 Phase III trial that failed to improve OS with avelumab as compared to cisplatin/radiation therapy (+ OPSCC with and without reductions in the dosage of radiation (+ patients.Along those lines, evidence suggests that it is too early to safely conclude whether HPV status is associated with superior outcomes following treatment with ICIs. Indeed, a few studies have reported that there is no discrimination between patients by HPV status , 114. Onectively , 116. InV status . A more patients –117. Addunderway . There iunderway . Chemothunderway . Harnessunderway –123. Alt therapy . The sam therapy . However therapy –123. Mor+-HNSCC. Future paradigms may incorporate a switch from escalated therapies to less toxic, de-intensified CRT regimens for locally advanced disease, given the amplification of toxicities by chemotherapy-based radio-sensitizers (Treatment guidelines for HNSCC are currently evolving, particularly for HPVsitizers , 126. InLC wrote the manuscript. PD-H edited and supervised the manuscript. All authors contributed to the article and approved the submitted version.Funds supporting the publication of this work were provided by Loma Linda University and the Fletcher Jones Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


NCF1, which indicated autosomal recessive chronic granulomatous disease (CGD). Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Coamplification of NCF1 and its pseudogenes identified a GT-deletion (ΔGT) at the start of exon 2 in NCF1 (NM_000265.7: c.75_76delGT: p.Tyr26Hisfs*26). Estimation of NCF1-to-NCF1 pseudogenes ratio using ΔGT and 20-bp gene scans affirmed nil copies of NCF1 in the patient. While the father had a normal ratio of 2:4, the mother had a ratio of 1:5, implicating the carrier of ΔGT-containing NCF1. Discovery of a 7q11.23 deletion involving one NCF1 allele and a ΔGT in the second NCF1 allele explained the coexistence of WBS and CGD in our patient. This study highlights the capability of WES to establish a molecular diagnosis for a case with blended phenotypes, enabling the provision of appropriate prophylactic treatment.Blended phenotypes exhibited by a patient may present a challenge to the establishment of diagnosis. In this study, we report a seven-year-old Murut girl with unusual features of Williams-Beuren syndrome (WBS), including recurrent infections and skin abscesses. Considering the possibility of a second genetic disorder, a mutation screening for genes associated with inborn errors of immunity (IEI) was conducted using whole exome sequencing (WES). Analysis of copy number variations (CNVs) from the exome data revealed a 1.53Mb heterozygous deletion on chromosome 7q11.23, corresponding to the known WBS. We also identified a biallelic loss of  Williams-Beuren syndrome (WBS) OMIM ID: 194050) is a contiguous gene deletion syndrome inherited in autosomal dominant pattern. Most cases occurred sporadically with an estimated prevalence of 1/7,500 to 1/20,000 live births 94050 is . Other fOur study involved a seven-year-old patient with blended phenotypes. The patient manifested typical WBS features such as aortic stenosis, inguinal hernia, subclinical hypothyroidism and learning difficulty with developmental delay. Nevertheless, she also had recurrent infections, diarrhea and skin abscesses since the age of one year. As WBS patients are not commonly associated with immune disorders, we suspected the possibility of a primary immunodeficiency. Primary immunodeficiencies, recently termed as inborn errors of immunity (IEI) are characterized by increased susceptibility to infections, autoimmune diseases, autoinflammatory disorders, allergies and malignancies . These dPreviously, we utilized whole exome sequencing (WES) to diagnose patients suspected with IEI and obtained molecular diagnoses in 46.7% of the cohort . Among tA seven-year-old Murut girl with WBS caused by a heterozygous deletion  was recruited by the Institute for Medical Research. The patient was presented with unexpected phenotypes of WBS, i.e., multiple severe infections and abscess formation, raising clinical suspicion of a cooccurring genetic disorder. Written informed consent was collected from the patient and her parents upon blood sample collection. The study received approval from the Medical Research and Ethics Committee, Ministry of Health Malaysia (KKM/NIHSEC/P16-837) and adhered to the Declaration of Helsinki.Genomic DNA was isolated from peripheral blood mononuclear cells using QIAamp DNA Blood Mini Kit  according to the manufacturer’s protocol. Exome capture was performed using Agilent SureSelect Human All Exon V5  with target size of 50Mb. Paired-end reads of 101 base pairs (bp) were generated from a HiSeq 4000 sequencer  at mean coverage of 100x. Burrows-Wheeler Aligner-maximal exact matches (BWA-MEM) was used to align the sequencing reads to the human reference genome GRCh38 . Mapped in silico pathogenicity prediction tools, namely Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2) and MutationTaster  according to the manufacturer’s instructions. The heparinized whole blood sample was incubated with a stimulant, protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at 37°C. A negative control was prepared by substituting the stimulant with wash solution. After 10 minutes of incubation, nonfluorescent dihydrorhodamine 123 substrate solution was added to every sample. Oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123 upon reaction with reactive oxygen species (ROS) was measured by a BD FACSCanto™ II flow cytometer  using FlowJo™ v10. Neutrophils were gated to analyze the percentage of cells producing ROS during unstimulated and PMA-stimulated conditions.NCF1 (Polymerase chain reaction using published primers 2LB2 and 2RB2 was performed to screen for the GT-deletion (ΔGT) at the beginning of exon 2 in NCF1 . The ampNCF1 and its two pseudogenes  and splice sites (n=115) Figure 1NSUN5 to GTF2IRD2, including NCF1. The second allele of NCF1 was found to be deleted, resulting in biallelic loss of NCF1.ExomeDepth identified a total of 195 CNV calls in the patient, of which 19 were shared with the control and two were localized on sex chromosomes. The CNVs retained for further analysis encompassed 94 deletions and 80 duplications. The proportion of predicted CNVs with a size larger than 1 kilobases (kb) (63.8%) was higher compared to those shorter than 1kb (36.2%). A high Bayes factor of 1,100 was computed for the heterozygous deletion on chromosome 7q11.23 ranging from 73,303,201 to 74,836,526, corresponding to the WBS region . ΔGT gene scan detected fragments of two sizes, i.e., 196-bp fragment reflected NCF1 pseudogenes with ΔGT and 198-bp fragment reflected NCF1 with GTGT. The proportion of GTGT and ΔGT was found to be 2:4 in the control and father . Repeated infections and formation of skin abscesses in our patient raised the suspicion of an IEI. We performed initial screening for germline short variants associated with IEI using WES, but detected no causative variants matching the clinical presentation. Further analysis involving CNV detection from exome data uncovered a biallelic loss of sitivity . Howeversitivity , 24. ThuCYBB mutations  awarded to AMR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cervical Cancer is the 6th most common and 3rd most deadly cancer among women. Despite the fact that the majority of the countries in Asia and Africa have a similar economy and low life expectancy, the mean age-standardized incidence rate (ASIR) of cervical cancer is substantially higher in Africa than in Asia. This study identified the correlates of the higher ASIR rates in Africa relative to Asia against two timelines; 2004-2009 and 2010-2017. Peer-reviewed articles published between 2004 and 2017 were selected using the PRISMA standard. Sources of articles included Google Scholar, Scopus, PubMed Central, and EMBASE. Search keywords included: HPV genotypes, cervical cancer, HPV vaccine, and multiple infections in Africa and Asia. Twenty-nine and seventeen full-length articles were selected from Africa and Asia, respectively. The pooled prevalence of HPV infection up to 2017 was higher in Africa  than in Asia  at p< 0.001. Between 2004-2009 and 2010-2017 timelines, the pooled prevalence of HPV infection decreased from 49.1% to 36.7%  in Africa and increased from 16.9% to 20.5%  in Asia. However, the pooled prevalence of multiple HPV infections and non-vaccine high-risk HPV infections were higher among African women diagnosed with cancer (30.9% and 5.2%) than their Asian counterparts  at p< 0.001. Additionally, the pooled prevalence of the five most prevalent high-risk HPV types in Africa were HPV16 (35.3%), HPV52 (14.2%), HPV35 (12.4%), HPV18 (10.4%), and HPV58 (10.0%), while that of Asia were HPV16 (37.3%), HPV52 (16.2%), HPV58 (14.7%), HPV33 (7.4%) and HPV18 (7.2%). This study suggests that the higher prevalence of HPV, multiple HPV and non-vaccine HPV infections could be responsible for the higher ASIR in Africa than in Asia. Studies show a minor reduction in age standardized incidence rate  from 14.5 to 13.1 between 2017 and 2018, and a minor increase in the age standardized mortality rate  from 6.1 to 6.9 within the same period . Despite the fact that majority of the countries in Asia and Africa have similar economy (less developed), low life expectancy and high mortality-to-incidence rate , the mean ASIR/ASMR of cervical cancer was higher in Africa (29.4/19.8) than in Asia (11.3/6.2) as of 2018 . The findings of Canfell et al., (2017) suggest that the high mortality rate among cervical cancer patients in Africa is associated with low access rate to treatment. However the reason for the high incidence rate of the disease in Africa remains unknown. As of 2018, the ASIR/ASMR varies: 43.1/20.0, 40.1/30.0, 29.6/23.0/, 26.8/21.1, and 7.2/5.1 in Southern, Eastern, Western, central, and Northern Africa, respectively . This shows that Northern Africa had the least ASIR/ASMR in Africa, yet these values are higher than that of Western Asia, the least affected Asian sub-region (4.1/2.5) . Of note also, the ratio of cervical cancer attributable to HPV in Sub-Saharan Africa and Northern Africa/Western Asia is 9.3:1 . Abryn et al. and Martel et al. attributed the low ASIR in Northern Africa and Western Asia to low prevalence of HPV. They did not offer any reasons for the variation in ASIR of cervical cancer and prevalence. We however hypothesize that this could be related to the peculiar HPV subtypes found in Africa and the type of vaccines adopted in the African sub-regions.Cervical cancer is the 9Commonly used HPV vaccines include bivalent (HPV16/18), quadrivalent (HPV6/11/16/18) and nonavalent vaccines  . The first two vaccines are widely used in Africa but the nonavalent vaccine which has a global estimated efficacy of 87-89.5% in prevention of cervical cancer worldwide, is yet to be widely adopted in Africa . Barriers to implementation of national vaccination programs in African countries include inadequate infrastructure and ﬁnances, limited health worker training, vaccine cost, and cold chain capacity constraints . Even if nonavalent vaccine is widely distributed in Africa, majority of the countries in sub-Saharan Africa would not reach HPV elimination by vaccination alone. This is because, if HPV vaccination were to eradicate HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, the incidence of cervical cancer will still be > 4/100,000 due to other non-vaccine high risk HPV (hrHPV) types that are not currently covered . Furthermore, the effectiveness of this vaccine will depend on the rate of HIV infection in the target population which in turn drives multiple HPV infection. In Asia, the prevalence of multiple HPV infection in normal and abnormal cervix is 5.1-25.2% and 10.6-33.3%, respectively  whereas in Africa, this is 3.9%-15.8% and 22.9-35.7%, respectively . This underscores the fact that multiple HPV infection limits the efficacy of available vaccine to prevent the development of cervical cancer. This review aimed at identifying the prevalent HPV types in Asia and Africa both in the general population and among women with cervical abnormalities. It also assesses the paradigm shift in the prevalence of HPV infection in Africa and Asia between two timelines; 2004-2009 and 2010-2017. It determined the prevalence of HPV infection preventable by available vaccines.Peer-reviewed articles published in Africa (n= 29) and Asia (n= 17) between 2004 and 2017 were selected and screened using the PRISMA standard   AND  AND , AND (‘Africa’), AND (‘Asia’). We also searched for unpublished studies (grey literature) by evaluating ClinicalTrials.gov (NIH), and International Clinical Trial Registry Platform (WHO). Inclusion criteria included: Studies with frequency of HPV infection, must be full-length articles and involve cervical cancer, and the articles must involve Africa and Asia. Exclusion criteria: Articles not written in English, abstracts, non-full-length article, and articles without specific frequency of HPV types, articles not involving Africa and Asia as well as articles not involving cervical cancer. Data collection and extractionThe vital information extracted for analysis included: participant characteristics such as sample size, cases of among African and Asian women, prevalence of any HPV infection and multiple HPV infection, HPV types , mean age, recruitment method, period of data collection, and study location and region (according to WHO classification). We investigated the frequency of HPV infection in between the two continents. When calculating the prevalence of any HPV infection (women who tested positive for any HPV type), an individual may have acquired both HPV35 and HPV45 or more but it would only count as one event. The range of high-risk HPVs investigated in the selected studies varied, thus when calculating the prevalence of an HPV type, only studies or cases that investigated that particular HPV type were considered. To assess the impact of timelines on the prevalence of HPV infection, data points were categorized into pre-and up to 2009, and from 2010 and up to 2017.Data analysesX2) and Odds ratio analyses were used in calculating the difference in HPV types between Africa and Asia against different timelines . Significance was set at p< 0.05Pooled prevalence of HPV prevalence were presented in forest plots. Chi-square , followed by East Africa , Central Africa , North Africa , South Africa . One mixed study involving Tanzania and South Africa (with 194 participants) was also included. Prevalence of HPV types in multiple infection Considering the general population, significant differences in the prevalence of HPV types were observed between Africa and Asia, except for HPV6 (p< 0.001). High differences were observed between the two continents with regards to HPV16, HPV35, HPV18, HPV31 and HPV53/66 prevalence, in descending order of rank . There wPrevalence of HPV types among women with cervical abnormalitiesThe prevalent HPV types in Africa were HPV16, HPV52, HPV35, HPV18 and HPV58 while the prevalent HPV types in Asia were HPV16, HPV52, HPV58, HPV33, and HPV53, in descending order of rank. High significant differences were observed between Africa and Asia with regards to HPV35, HPV6, HPV45, HPV31, HPV58, HPV66 and HPV68, in descending order of rank . Result Difference in HPV prevalence between 2004-2009 and 2010-2017Overall, the prevalence of HPV infection in Africa decreased between 2004-2009 and 2010-2017 whereas it increased in Asia between the same periods. Up to 2009, the pooled prevalence of HPV and multiple HPV infections were significantly higher in Africa (49.1% and 32.4%) than in Asia  at p< 0.001. Up to 2009, there were significant differences between Africa and Asia with regards to the prevalence of HPV types in (p≤ 0.01), except for HPV-82 (p> 0.05). Within the same period, the prevalence of vaccine and non-vaccine HPV infection were higher in Africa (5.9% and 3.3%) than in Asia . As of 2010 and later, the pooled prevalence of HPV infection and multiple HPV infection were significantly higher in Africa (36.7% and 14.3%) than in Asia  at p< 0.001. This reveal that the multiple HPV infection decreased by 18.1% in Africa and increased by 0.3% in Africa between the timelines of 2004-2009 to 2010-2017 . No significant difference was observed between the two continents in terms of the prevalence of HPV6 and HPV11 (p> 0.05 and p< 0.05), respectively (Table 4). As of 2010 and later, the prevalence of vaccine and non-vaccine HPV were higher in Africa (5% and 2.5%) than in Asia  at p< 0.001 . Both inPrevalence of HPV types in the African sub-regionsThe highest and lowest prevalence of HPV infection as well as highest and lowest multiple infections were found in South Africa and Central Africa respectively. South Africa had the highest prevalence of HPV18, HPV45, HPV58, HPV35, HPV39, HPV51, and HPV59 while West Africa had the highest prevalence of HPV52, HPV53, HPV56, HPV66, and HPV82. East Africa had the highest prevalence of HPV31, HPV33, and HPV11 while North Africa had the highest prevalence of HPV16 and HPV6. Central Africa had the highest prevalence of HPV68. North Africa had the lowest prevalence of HPV45, HPV52, HPV58, HPV35, HPV66, HPV68, and HPV82 . The pool prevalence of HPV in North, East, Central, West and South Africa were 32.0%, 30.6%, 30.1%, 44.9% and 58.2%, respectively while the prevalence of multiple HPV infections were 9.1%, 24.4%, 3.5%, 20.0% and 26.1%, respectively (see supplementary data). Result showed that the prevalence of non-vaccine hrHPV types were lower in the North and East Africa  while thThe incidence of cervical cancer in a population majorly depends on the prevalence of HPV infection and cervical cancer awareness, extent of high-risk sexual behaviour, prevalence of HPV infection and rate of HPV vaccination . In Asia, although the rate of vaccination status is low (15.0%), knowledge of risk factors for cervical cancer and willingness to get vaccinated among females aged 19-23 years are high; 98.8% and 92.5%, respectively . However, in Africa, HPV vaccine coverage in Africa (1.2-4.1%), the knowledge of risk factors for cervical cancer (7.8-11.8%) and willingness to get vaccinated (26.0%-58.8%) among females aged 16-20 years are all low . The low HPV vaccine coverage in Africa has been associated with high cost of vaccine and skepticism of its safety . Considering the lack of implementation of World Health Organization (WHO) and Center for Disease control and prevention (CDCP)’s recommendations , a high ASIR of cervical cancer is expected in Africa . However, there is a need to indirectly assess the past and ongoing cervical cancer related interventions. There is also the need to identify major contributors to the high ASIR and ASMR in Africa for appropriate channeling of future interventions following recommendations. In the general population, this study revealed that HPV16, HPV52 and HPV58 were the most prevalent hrHPV types in Asia whereas HPV16, HPV18 and HPV52 were the most prevalent hrHPV infections in Africa. Considering the prevalence of hrHPV in abnormal cervix, this study revealed striking differences between Africa and Asia with regards to the prevalence of HPV35, HPV45, HPV31, HPV58, and HPV66. According to Hashim et al. (2020), the risk of developing CIN2+/CIN3+ among HPV16 and HPV18 positive women following a regular follow-up of 21 months are 24.4/19.9 and 16.2/10.8, respectively while the risk for women who are positive for other hrHPVs is 9.6/5.5. Based on the latter, the substantial higher prevalence of HPV16 and HPV18 in Africa relative to Asia might also be an explanation for the difference in cervical cancer ASIR between the two continents as revealed by Fitzmaurice et al. (2019) and Arbyn et al. (2020). Considering the fact that HPV16 infection is associated with high mortality among HPV positive women , it could be argued that the high prevalence of HPV is linked to the high ASMR or poor prognosis among African women . Namujju et al. (2011) stated that HPV16- and HPV18- positive women have 11-22-fold and 45-58 increased risk of acquiring other hrHPVs, respectively. Thus, the higher prevalence of multiple HPV infection observed in Africa relative to Asia could be due to the higher prevalence of HPV16 and HPV18 in African women. The multiple HPV infection could be responsible for the higher ASIR in Africa than in Asia since higher prevalence of multiple infection has been reported in abnormal cervix than in normal cervix . In addition, multiple HPV infection has been linked to increasing prevalence of human immunodeficiency virus (HIV) which in turn contributes significantly to cervical carcinogenesis . Thus, it could be suggested that the high ASIR of cervical cancer in Africa when compared with Asia stems from a cascade of event beginning from non-vaccine HPV infection and high multiple infection. In this study, South Africa had the highest prevalence of any HPV and multiple HPV infection while Central Africa had the least of both parameters. The difference between the African sub-regions in terms of ASIR of cervical cancer as revealed by Martel et al., (2017) could be due to high multiple HPV infections and HIV infection rate . According to Huh et al. (2017), nonavalent vaccine prevents 90% of cervical cancer worldwide. Their estimate is substantially higher than the estimate of this study (69%). This could be attributed to their study not including any participants from African countries. Cervical cancers could be attributed to a single hrHPV type or multiple types such as HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 which are preventable by available vaccines . However, the vaccine hrHPV and non-vaccine hrHPV may not be effective in preventing multiple infection mediated cervical cancer involving HPV16/35, HPV16/53, HPV16/66, except in the event of cross-protection. Pirek et al. (2015) stated that there is an increased rate of malignant transformation in women with normal cervix and multiple HPV infection (< 365days) than in women with normal cervix and single HPV infection, hence the need for continual screening, even among vaccinated women and immunocompromised women. Given the high rate of multiple infections in Africa, especially West and South Africa, nonavalent vaccine may offer better protection against cervical cancer than other vaccines. Additionally, the hrHPV type with the highest difference between African and Asian women diagnosed with cervical abnormalities was HPV35. Consequently, the high prevalence of HPV35 and its involvement in multiple infection may account for the higher ASIR in Africa than in Asia . Sadly, none of the available vaccines offer protection against HPV35, the second most prevalent HPV types, which accounts for 12.4% of cervical abnormalities in Africa. Since HPV35 appears to be the major correlate of cervical carcinogenesis in Africa, continual Pap smear screening is recommended.In this study, between 2004 and 2017, the prevalence of HPV infection and multiple infection decrease by 12.4% and 18.1% in Africa while they increased by 3.5% and 0.2% in Asia, respectively. The reason for the observed decrease in Africa could majorly be due to increasing awareness. On the other hand, considering the fact that the widely distributed vaccine in Africa are bivalent and quadrivalent vaccines , the decrease could be attributed to HPV vaccination since the prevalence of HPV16 substantially decreased (5%). Despite the fact that the overall prevalence of HPV infection decreased between 2004 and 2017 in Africa, the prevalence of HPV 33, 18, 6, 11, 82, 68, 31, 53, and 66 increased by 0.2%, 0.3%, 0.4%, 0.5%, 0.5%, 0.8%, 0.9%, 1.1%, and 1.3%, respectively within the same period, majority of the HPV types  are not covered by available vaccines. This may severely impact on the prevalence of cervical cancer in Africa. The reason however for the increased prevalence of HPV in Asia from 2004-2017 is still unknown. Interestingly, in Asia and Africa, the prevalence of HPV16 decreased by 16% and 5%, respectively while the following HPV types increased by certain percentage in 2017: HPV31 (0.2% vs 0.9%), HPV53 (0.2% vs 1.1%), HPV33 (0.6% vs 0.2%), HPV68 (0.8% vs 0.7%), HPV11 (1.3% vs 0.5%), and HPV6 . Differentially, the prevalence of HPV 52 and 58 increased in Asia by 0.5%, and 1.5%, respectively while in Africa the prevalence of HPV 18, 82 and 66 increased by 0.3%, 0.5% and 1.3%, respectively (Table 4). Though the prevalence of HPV 6, 11, 31, 33, 53, and 68 increased in both continents, the findings of this study suggest that if both continents adopt only nonavalent vaccines, it would still take a longer time to eradicate or significantly reduce cervical cancer in Africa than it would in Asia. This reiterates the fact that cervical cancer screening using Pap smear should still be an integral part of preventive measure in order to eliminate the disease in Africa by 2090 .The prevalence of vaccine and non-vaccine hrHPV were higher in South Africa than in other African sub-region while the prevalence of vaccine and non-vaccine hrHPV were lowest in Central Africa and North Africa. This study suggests that majority, over 72%, of cervical cancer attributable to HPV in North and East Africa could be prevented by vaccine, especially by using quadrivalent and nonavalent vaccines. Since 7 out of 12 countries are currently providing HPV vaccination at no cost for girls in East Africa, there is a higher chance of eliminating cervical cancer in the sub-continent by 2090 . The findings of this study show that though nonavalent vaccine could prevent approximately 52% of cervical cancer attributable to HPV in Central, Western and Southern Africa, a tangible number of cervical cancer will still be observed in the sub-regions due to high prevalence of non-vaccine hrHPV.LimitationNo grey literature was identified following our searches. This may constitute a limitation in this study.In conclusion, This study revealed that the disparity between African and Asian women with regards to ASIR could be linked to high-risk HPV infection and multiple HPV types. It suggests that nonavalent vaccination could prevent over 90% of the cervical abnormalities in Africa. This review was carried out and approved in collaboration between all the authors. JOO and AAN conceptualized and developed the study protocol. JOO, AAN, CFC, INO, SIO and ISO identified records for full-text review and data extraction. JOO and AAN drafted the manuscript.This study is not part of an approved student thesis. It is exempt from ethical review and approval, since the data that were reviewed and used for meta-analysis were retrieved and synthesized from studies published in indexed journals. Informed consents and ethical clearance were individually sought and obtained by authors of the included articles. Hence, ethical approval to conduct the study was not sought from any scientific bodies.Available at: Racial disparities in the prevalence of vaccine and non-vaccine HPV types and multiple HPV infection between Asia and Africa: A systematic review. doi: https://doi.org/10.1101/2020.11.02.20224857The authors declare that they have no financial or personal relationships which may have inappropriatelyThe authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.