Emerging evidence has demonstrated a close association between perturbations in vaginal microbiota composition in women and human papillomavirus (HPV) infection, cervical lesions, and cervical cancer (Ca); however, these findings are highly heterogeneous and inconclusive.To perform a comprehensive systematic review of the global disturbance in the vaginal microbiota, specifically in women with HPV-associated cervical diseases, and to further conduct within- and across-disease comparisons.Twenty-two records were identified in a systematic literature search of PubMed, Web of Science, and Embase up to February 28, 2022. We extracted microbial changes at the community  and taxonomic (relative abundance) levels. Within- and across-disease findings on the relative abundance of taxonomic assignments were qualitatively synthesized.Gardnerella, Megasphaera, Prevotella, Peptostreptococcus, and Streptococcus showed the greatest abundances with HPV infection; Sneathia and Atopobium showed inconsistent abundance with HPV infection, and Staphylococcus was observed in Ca. Across diseases, we find increased levels of Streptococcus and varying levels of Gardnerella were shared across HPV infections, high-grade squamous intraepithelial lesions, and Ca, whereas Lactobacillus iners varied depending on the HPV-related disease subtype.Generally, significantly higher alpha diversity was observed for HPV infection, cervical lesions, and/or cancer patients than in controls, and significant differences within beta diversity were observed for the overall microbial composition across samples. In within-disease comparisons, the genera Lactobacillus, enrichment of anaerobes, and increased abundance of aerobic bacteria in HPV infection and related cervical diseases. Moreover, L. iners may exert either protective or pathogenic effects on different HPV-related diseases.This systematic review reports that vaginal microbiome disturbances are correlated to the depletion of  Cervical cancer (Ca) remains the fourth most prevalent cancer in women worldwide, with 6,04,127 new cases just in 2020, and more than 3,41,831 deaths, accounting for nearly 8% of all female cancer-related deaths every year , endometriosis, and HPV-related cervical diseases. Bacterial vaginosis-related (BV) microbes have been detected in PCOS and endometriosis in previous studies , 4. EpidG. vaginals, Megasphaera, Prevotella, and Sneathia)  have also been reported to be associated with cervical lesions and Ca  , whereasabundant , 16. Oths and Ca –19. Addius iners , 20 and Therefore, we conducted an updated systematic review of vaginal microbiome studies to characterize the microbial disturbances in women with HPV infection, HPV persistence, cervical lesions, and cervical cancer. This review aims to synthesize the findings of previous studies and conduct comparisons between- and across-diseases.The present review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.A systematic literature search of PubMed, Web of Science, and Embase was carried out for articles published between January 01, 2000, and February 28, 2022. The keywords included HPV [all fields], HPV infection [all fields], human papillomavirus [all fields], cervical intraepithelial neoplasia (CIN) [all fields], cervical lesion [all fields], cervical dysplasia [all fields], cervical neoplasm [all fields], cervical cancer [all fields], cervical carcinoma [all fields], microbiome [all fields], microbiota [all fields], and flora [all fields].The inclusion criteria for the study, were as follows (1): all observational studies  (2); women with HPV infection (HPV+), HPV persistence, low-grade squamous intraepithelial lesions (LSIL), HSIL, and Ca – categorized according to the results of HPV testing and histology of cervical biopsy, compared to healthy controls (HPV-negative and cytology-negative women) or HPV-negative women (HPV-), cervical cytology was used for classification if histology was not reported (3); studies reporting microbiota analyses of vaginal samples based on high-throughput sequencing ; studiesRecords were excluded if (1) they did not use high-throughput sequencing approaches (2), sequenced the microbiota from cervical or cervicovaginal swabs [because of anatomy-associated discrepancies in the microbiome ] (3), anProspective or interventional studies reporting altered microbial composition without relevant baseline measurements were also excluded.Study characteristics and microbiome quantification methods were extracted from the included references. Study characteristics include the following details: publication information, subject demographics, and clinical features, whereas the latter consists of a sequencing platform, sequencing target, DNA extraction, data analysis pipelines, and reference databases. As major outcomes of the analysis, we extracted microbial alterations at the community level  as well as taxonomic levels . Within- and across-disease findings for the relative abundance of taxonomic assignments were qualitatively synthesized and categorized as increased, decreased, or inconsistent abundancies. No findings were consistent, with less than 75% agreement between studies reporting this taxon .We utilized the National Institutes of Health (NIH) National Health, Lung, and Blood Institute Study Quality Assessment Tool for Observational and Cross-sectional Studies to evaluate the internal validity and potential bias of the included studies. Study quality was rated as “good”, “fair, or poor” by two authors (MW and HL), with differences addressed through discussion.A total of 3322 relevant records were obtained in the initial electronic search, including 1042 from PubMed, 912 from Web of Science, and 1368 from Embase. Six hundred ninety-nine studies were imported after removing duplicates and screening the titles and/or abstracts. Of those, studies were excluded due to non-vaginal samples (n=15), non-high-throughput sequencing approaches (n=2), no information on any change in taxa compared with HPV- (n=1), and longitudinal studies in the absence of controls (n=2) after assessing the full text (n=42). Ultimately, 22 studies were selected for the final analysis. A flowchart of this process is shown in The 22 studies consisted of 17 case-control, three cross-sectional, and two longitudinal studies. Most studies  were conducted in Asia , five (22.7%) were performed in Western countries , and one (4.5%) was performed in Africa (Nigeria). In the 17 case-control comparisons, six studies compared HPV+ or HPV persistence to the controls, ten studies compared women with cervical lesions and/or Ca to the controls, and 3 included women with HPV infection. Only one study compared ≥ HSIL with LSIL. Of these, one study grouped samples into LSIL, HSIL, and normal cytology groups based on observed cytology. In the two prospective studies, comparisons were performed between cases before treatment  and untreated controls. The detailed information is provided in Control samples were defined as healthy women in most studies, whereas in two studies the controls were defined as having normal cytology. The controls were HPV positive in one study.The different microbiome analysis methods are shown in A total of 57 alpha-diversity assessments were performed across 21 studies Figure 2When comparing HPV+ vs. controls or HPV−, five studies examined community richness using different metrics. Two studies indicated that there were no significant differences, two found significantly higher richness indices, whereas one found a non-significant decrease in richness indices. Regarding diversity, combining richness and evenness, significantly higher diversity was observed in three studies, significantly lower diversity was observed in one study, and no significant differences were observed in three studies. One study found a significant increase in Faith’s phylogenetic diversity.When comparing HPV persistence vs. controls, lower richness indices were reported in two studies, and lower alpha diversity based on a combination of richness and evenness data was obtained in one study.In comparisons between CIN and controls, two studies investigated higher richness and diversity by incorporating richness evenness indices in LSIL, four studies reported increases in both metrics in HSIL, and four studies reported a similar pattern in CIN. Among women with Ca, 4 studies reported differential richness data. Higher richness was reported in 2 studies, and lower richness was reported in the remaining studies. Differential diversity indices combining richness and evenness were reported in five studies, four of which reported higher diversity and two of which reported lower diversity. Four studies examined diversity based on a comparison between HSIL and LSIL, two of which reported no significant observations in terms of richness and/or evenness data, with the remaining studies providing higher indices. In addition, a significantly lower Good’s coverage was reported in one study comparing HSIL and controls.Beta diversity comparisons were evaluated in 15 studies, with 13 studies comparing subgroup vs. controls, one study comparing combined subgroups vs. controls, and one study comparing within-subgroup results. Six of 13 studies identified significant differences between different samples, with 4 of 13 showing visually separated components with no significance, 2 finding no clear separation between groups, and 1 study without a report. In the across-group (controls excluded) comparisons, two studies reported no significant differences between subgroups . One subgroup (HPV+ + LSIL + Ca) was significantly different from the controls. This study also showed a significant difference between the combined subgroup (LSIL + HSIL) and HPV infections. The beta diversity assessments are presented in Twenty-one studies have reported the relative abundance of vaginal microorganisms at the phylum, family, genus, and species levels. Between-subgroup comparisons of CIN were conducted relative to controls or in the order of abundance in subgroups reported by previous studies.Actinobacteria, Bacteroidetes, and Fusobacteria; the genera Gardnerella, Megasphaera, Peptostreptococcus, and Streptococcus; the species L. jensenii and Veillonella montpellierensis); the four taxa with a decrease in abundance included the phylum Firmicutes, the genus Lactobacillus, and the species L. crispatus and L. iners). Prevotella, Sneathia, Atopobium, Anaerococcus, and Gardnerella vaginalis were inconsistently identified ((1)In HPV+, the abundance of thirteen taxa increased or decreased: the nine with increased abundancies included the phyla entified Figure 3Lactobacillus numbers were decreased ((2)In HPV persistence, ecreased Figure 3Sneathia) and two decrease in abundance in taxa (the phylum Firmicutes and the genus Lactobacillus) were observed in LSIL; five increased taxa , two decreased taxa (phylum Firmicutes and genus Lactobacillus), and three taxa with inconsistent numbers  were reported in HSIL ((3)In CIN, one increase in abundance in taxon In Ca, the numbers of ent data Figure 3Bacteroidetes was common to both HPV+ and HSIL; lower Firmicutes was common to HPV+, LSIL, and HSIL; higher Streptococcus was common to HPV+, HSIL, and Ca; lower Lactobacillus was common to all subgroups. The genera Gardnerella and Prevotella were inconsistently present in HPV+, HSIL, and Ca; Sneathia was inconsistent in HPV+ and LSIL; L. iners was inconsistent in HPV+ and HSIL . Only four studies were rated as ‘Good’ , which not only included study features, microbiome methods, and rich reporting of major outcomes but also considered several potential confounders in the reports. Four studies were rated as ‘poor’ quality . The major sources of potential bias were the lack of reporting of tools or methods for measuring outcomes, lack of detailed information on demographic or clinical characteristics, and lack of detailed definition of cases. The internal validity of the included studies is shown in Our systematic review evaluated vaginal microbiome disturbances across a spectrum of HPV-associated diseases to analyze compositional alterations and perform within- and across-disease comparisons. A total of 22 studies were included in the meta-analysis. First, we outline the variances in both the study features and methodology. Next, a qualitative synthesis indicated that the alpha diversity was significantly higher than that of the controls, whereas the beta diversity showed a significant difference in the overall microbial composition across samples. The patterns of observed taxonomic changes revealed a large heterogeneity at the within- and across-disease levels. When considering alterations in abundance in at least two studies, we analyzed the alterations in consistent and inconsistent directions, within the disease. In addition, our results revealed an overlap across diseases in consistently and inconsistently altered taxa. The underlying mechanisms explaining the observed disturbances are discussed for both comparisons. The confounding effects of study characteristics and microbiome methodologies were also assessed.Overall, our synthesis indicated a significant increase in richness and diversity in women with cervical lesions and cancer, but no difference was observed between HSIL and LSIL. Medium and large studies indicated a non-significant relationship between richness and/or evenness indices in women with HPV infection, suggesting that alpha diversity indices are of limited utility as a measurement of vaginal health, or for distinguishing between HPV infection and controls. A large number of intrinsic and extrinsic factors account for variations in diversity at the baseline, including race, age, menstrual cycles, sexual activities, contraception use, smoking, and diet, thereby complicating the measurement , 41–46.However, reports with inconsistent data have been published. A few studies have reported a reduction in the richness and diversity of HPV infections. A similar pattern was observed in women with HPV persistence and Ca. These residual heterogeneities for certain diseases may be attributed to methodological effects and the study design. Alpha diversity is particularly affected by measurement errors in microbiome research, owing to the heavy bias of commonly used estimators . AlthougRegarding beta diversity, most studies have observed a significant cluster between the samples. In summary, two of the studies reported no significant differences between HSIL and LSIL, which is consistent with the pattern observed for alpha diversity.Staphylococcus was the only taxon enriched in Ca whereas, the genera Gardnerella, Megasphaera, Prevotella, Peptostreptococcus, and Streptococcus showed the most common increase in HPV infections, most of which were BV-related anaerobes, although they were also reported in HSIL and Ca. These associations are likely mediated by biological amine-induced oxidative stress since BV-related bacteria are linked to their production  IV-dominated communities . At the same time, divergence was observed in the categorization of subgroups and controls, which is problematic in disentangling the panel of vaginal microbial disturbances observed in HPV infections and cervical neoplasms. For example, Laniewski et al.  clustering tool  and data preprocessing methods . Our analyses may also suffer from the use of different sequencing platforms as well as differences in hypervariable regions since heterogeneity in evaluating microbial diversity has been reported . For exaing tool  is a novGardnerella, Prevotella, and Atopobium , the General Project of the National Natural Science Foundation of China (Grant No. 82071674), the National Natural Science Foundation of China (Grant No. 82101705), the Natural Science Foundation of Tianjin Municipal Science and Technology (Grant No. 20JCYBJC00440), the Tianjin Health Science and Technology Project , the Scientific Research Project of Tianjin Education Commission (Grant No. 2020KJ158), and Tianjin Key Medical Discipline  Construction Project.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.


A correlation between OSCC and oral HPV among analyzed samples could not be demonstrated in our small cohort.Highly oncogenic human papillomavirus (HPV) is well known to be associated with and a risk factor for various types of oral carcinomas such as oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate and describe the HPV-induced OSCC prevalence and genotyping in the city of Belém, northern Brazil. This cross-sectional study features 101 participants who attended an oral pathology referral center in a dental college looking for diagnoses of oral lesions (OL). After signing the consent term and meeting the inclusion criteria, all participants went through a sociodemographic and epidemiological questionnaire. Then, OL were collected by excisional or incisional biopsy depending on OL size; after that, OL tissues were preserved in paraffin blocks to histopathological diagnoses. Afterwards, paraffin blocks were divided into benign and malignant/premalignant lesions based on the classification of potentially malignant disorders of the oral and oropharyngeal mucosa. Then, the paraffin blocks had DNA extraction performed by the ReliaPrep FFPE gDNA Miniprep method in order to identify HPV DNA of high oncogenic risk and low oncogenic risk. Then, the viral DNA was amplified and typed using the Inno-Lipa genotyping Extra II method, and the collected data were analyzed by Chi-square and G-tests. In total, 59/101 (58.4%) OL were malignant/premalignant lesions, of which OSCC was the most prevalent with 40/59 (67.7%) and 42/101 (41.6%) benign lesions. The most common area of OL incidence was upper gingiva 46/101 (45.5%). Regarding HPV DNA detection, approximately 27/101 (26.7%) had positive results; of these, 17/59 (28.8%) were malignant/premalignant lesions, and the most prevalent genotypes detected were 16, 18, 52 and 58, while among benign lesions, 10/42 (66.6%) had HPV-positive results, and the most prevalent genotypes detected were 6, 11 and 42. Age range was the only risk factor with a significant association between HPV and OSCC presence ( The human papillomavirus (HPV) is a member of the Papillomaviridae family. According to Monteiro et al. , there aHPV can infect the epithelial surface through microlesions at the basal layer, during unprotected sexual behavior, which then conduct to a proliferative benign or malignant lesion both in skin, oropharyngeal mucosa and the vaginal or anal tract due to morphological similarities . CurrentHPV oncogenic potential is based on the virus capacity to lodge two specific encode viral oncoproteins, E6 and E7 genes, into hosts, infecting the cells’ genome ,7,8. ThiWorldwide, HPV infection is a major public health issue, mainly because it is one of the most common sexually transmitted infections (STIs). Globally, HPV infection prevalence is approximately 12% with continental discrepancies due to the socioeconomic development and vaccination program of each country, and HPV-induced cancer has a prevalence of 5.1%, which ranges through genders and innumerous anatomic sites . In BrazSCC is one of the main malignant lesions of invasive skin cancer and is easily identified by an atypical, accelerated increase in squamous cells. Following the morphological similarities with oropharyngeal mucosa tissue, the presence of an oral SCC (OSCC) is possible; thus, OSCC can arise from any location of oropharyngeal mucosa ,17. AccoIn general, for OSCC, well-established risk factors are smoking, alcohol overconsumption and tobacco chewing, although since 1983, it was hypothesized that OSCC can emerge from HR HPV subtypes; in particular, HPV subtype 16 (HPV-16) is associated with unprotected sexual behavior. However, the specific role of HR HPV regarding OSCC risk factors is not fully understood. Therefore, furthermore information is needed to clarify the relationship between HR HPV infection and OSCC in northern Brazil. So, in this context, this study aimed to evaluate and describe the HPV-induced OSCC prevalence and genotyping in the city of Belém, northern Brazil.This descriptive, cross-sectional single-center study was population-based on clinical symptoms, sociodemographic and epidemiological data from individuals who attended an oral pathology and malignant lesions referral center at a dental college (CESUPA) located in the city of Belém, Pará, northern Brazil . All indAll interventions were performed in accordance with the guidelines and regulatory standards for research involving human subjects of the National Health Council and Papilloma Virus laboratory of Instituto Evandro Chagas (IEC). This study was approved by the Ethics Committee on Human Research of the University Center of the State of Pará—CESUPA under protocol number 4.197.815. Written informed consent was obtained from all 101 patients for the publication of any potentially identifiable images or data included in this paper.The benign and malignant lesions were established according to the classification of potentially malignant disorders of the oral and oropharyngeal mucosa . OL wereThe sample consisted of patients registered and treated at CESUPA. In total, 101 individuals were informed about the purpose of the study and invited to participate. Then, they all agreed to and signed a written consent form before data collection and oral evaluation. The study eligibility criteria were: (i) ≥18 years old; (ii) have a conclusive diagnosis of oral benign lesion or OSCC; (iii) resident of Pará State; (iv) medical records filled in; and (v) signed the free and informed consent form. The exclusion criteria were: (i) individuals who transferred to other cities, affecting follow up; (ii) individuals with neurological and/or cognitive impairment; (iii) medical records not filled out correctly; and (iv) refusal to sign the consent form. Individuals who met the inclusion criteria were invited to participate in this study and signed the consent form.Each participant was orally evaluated in a private location in the oral pathology department. Clinical data were collected by a single researcher, a specialist in oral pathology who had previous experience in clinical studies. The intraoral clinical examination was performed in a dental office, in a dental chair, under indirect and artificial light, using a dental mirror and clinical tweezers, which were all sterile, consisting of disposable materials; the OL evaluations were performed daily.Demographic and epidemiological data were obtained through a pre-tested standardized semi-structured questionnaire and medical records. Regarding oral biopsies, all lesions included had been submitted either to excisional biopsy, when OL were sized approximately to ≤1 cm, or incisional biopsy, when they were sized approximately to size ≥ 1 cm with a scalpel. All biopsies were performed by only one researcher who had previously experience in clinical biopsy. Examinations occurred under local anesthesia, and for excisional biopsies, a 3 mm margin of normal tissue was included. Then, the biopsy material was preserved in 10% formaldehyde then transported to histopathologic exam.All pieces were processed in paraffin blocks for histological analysis. At first, it was necessary to replace tissue liquid with paraffin; then, a sequence of ethyl alcohol baths at increasing concentrations (70–99%) for approximately 6 h for tissue dehydration was made. Subsequently, the pieces went through the process of diaphanization in xylol for 3 h and impregnation in molten paraffin at 60 °C for 2 h. Finally, the pieces were transferred to steel molds, which were bathed in liquid paraffin at 65 °C and taken to rapid cooling at 0 °C . After pEach block of paraffin samples was cut into 10 “slices” with 5 µm thickness each, and viral DNA extraction was performed using the “ReliaPrep FFPE gDNA Miniprep Syste . This system is based on whether in the use of cellulose membranes in columns, where the lysed biological material is subjected to centrifugation, the DNA is bound to membranes charged with (+) charge so that there is binding to the (−) DNA. Subsequently, washes were carried out with alcoholic solutions, and the DNA elution was carried out in a saline medium. All conditions described were specified in the manufacturer’s protocol.Each “sample” was heated in a thermoblock at 80 °C for approximately 2 min, and 500 μL of mineral oil was added to dissolve the paraffin—a process which was repeated for all “slices”. Subsequently, 300 μL of PBS buffer and 20 μL of Proteinase K were added for sample digestion. Soon, the samples were incubated at 65 °C for 1 h. After the complete digestion process, the DNA samples were considered homogeneous; then, they were transferred to another Eppendorf tube and were incubated at 95 °C for 15 min. After this process, the samples were kept at room temperature, and later, 220 μL of lysis buffer and 240 μL of absolute ethanol were added to assist in the aggregation of precipitated DNA. All samples were transferred to kit columns and then centrifuged at 10,000 RCF for 3 min. The fluid remaining in the tube was discarded, and 500 μL of washing solution was added, centrifuging at 10,000 RCF for 30 s with the cap closed and 16,000 RCF for 3 min with the tube cap open. Subsequently, the column was transferred to another Eppendorf tube, discarding the collection tube. Finally, 50 μL of elution buffer was added directly to the column, and it was centrifuged again at 16,000 RCF for 1 min. The column was discarded and the Eppendorf tube with the filtered sample was stored at −70 °C.The extracted viral DNA was identified and typed in the analyzed samples using the “Inno-Lipa Genotyping Extra II System , which is able to amplify a portion of the HPV genome in the viral L1 gene region. Through the reverse hybridization system, this generated fragment is able to identify infection by up to 32 viral types. Therefore, the presence of up to 28 genotypes of HPV  according to the manufacturer’s instructions.This system is based on the hybridization of the PCR-amplified fragment through the base homology of the amplified fragment with the probe contained in a “nylon” strip that corresponds to the specific sequence for each of the mentioned types, which defines, in addition to positivity, the infecting viral type. So, the viruses were cataloged on a decreasing scale as to their oncogenic risk.p value (p < 0.005) by the Chi-square, Fisher Exact Test and G Test, in the selected groups.The collected data were analyzed by the BioEstat program and evaluated with respect to mean, standard deviation and absolute and relative frequency, as well as In total, 101 individuals were recruited for study assessment, and the sociodemographic and behavioral data are presented in p value of 0.0004 (p < 0.005); the 60–69 age range was the most prevalent among individuals with 25/101 (24.7%). Of these, 19/59 (32.2%) were malignant lesions and 6/42 (14.2%) were benign lesions. Thus, we may infer, in our sample, that age can influence OSCC prevalence. Otherwise, most of the samples were: female 55/101 (54.4%)—of these, 29/59 (49.2%) were malignant lesions and 26/42 (62%) were benign lesions; mixed ethnicity 46/101 (45.5%)—of these, 26/59 (44%) were malignant lesions and 20/42 (47.6%) were benign lesions; smoking habits 71/101 (70.3%)—of these, 37/59 (62.7%) were malignant lesions and 34/42 (81%) were benign lesions; and alcohol overconsumption 71/101 (70.3%)—of these, 44/59 (74.5%) were malignant lesions and 27/42 (64.3%) were benign lesions. In addition to age range, no other significant association between risk factors, sociodemographic factors and type of lesion was found.The average age was 39.9 years and had a significant p < 0.4713), although upper gingiva was the most common area of incidence  of both malignant lesions  and benign lesions .In p value of 0.0001 (p < 0.005) was established.In n = 101), 27 (26.7%) had positive results for HPV DNA and 74 (73.3%) had negative results for HPV DNA (n = 17), several cases of LO were detected. OSCC  predominated, but cases of leukoplakia  and erythroplakia  were also recorded. HPV genotypes 16, 18, 52 and 58 were detected, and all were classified as high oncogenic risk. In the ten samples with HPV-16, OSCC , leukoplakia  and erythroplakia  were detected. OSCC was also detected in five (29.4%) samples of HPV-18, and leukoplakia was recorded in each of the HPV-52 (5.9%) and HPV-58 (5.9%) positive samples.In total , papilloma (33.3%) and condyloma acuminatum (16.7%) were detected in samples with HPV-16. Condyloma acuminatum was detected in all HPV-11 samples, and papilloma was detected in the HPV-42 sample.Since 1983, the increasing number of papers indicating a correlation between HPV infection with oropharyngeal tumors or lesions has increased ,28,29,30Through the years, OSCC-related HR HPV has been well documented, although different important factors could affect the outcomes of HPV-induced OSCC. The most common associations are smoking and alcohol overconsumption, which according to the literature influences various populations worldwide. Madathil et al.  evaluateSmith et al.  evaluateIn this study, risk factors such as smoking (70.3%) and alcohol overconsumption (70.3%) proved to be an important etiology factor to OSCC, confirming the results of all the studies mentioned above, although among our samples, the smoking and alcohol overconsumption associated with HR HPV prevalence could not be inferred, which was similar to the other studies prior. Perhaps the influence and the prevalence of HR HPV over OL might be impacted by other risk factors besides smoking and alcohol overconsumption, such as unsafe sex, multiple sexual partners, drug dependence, lack of access to public health services, co-infections such as HIV-HPV, and HPV non-vaccination, which would facilitate HPV throughout the state ,25.p < 0.05). According to our results, age range (60–69) could be considered as a major influencing factor for high OSCC prevalence; however, a cause–effect relationship could not be established.Bezerra et al.  demonstrIn the present study, the OSCC prevalence was 58.5% (59/101) and the HPV prevalence was 27/101 (26.7%). Among HPV positive samples, the most prevalent HR HPV subtype was HPV-16 (10/17—58.8%), which was exactly the same subtype as previous studies. When comparing the HPV prevalence ratings to our studies, ours is the smallest prevalence, which is mainly because our cohort had a smaller sample size. Other influencing risk factors might be demographic region, socioeconomic status, anatomical sites and access to quality public health services. Bean et al.  demonstrDespite the fact that OSCC prevalence was the main objective of our study, during the results analysis, the decreased prevalence of HPV was interesting, because it is different from the literature. The higher prevalence of HR HPV subtypes among HPV prevalence highlighted the urgency to improve the distribution of specialized oral pathology services, HPV vaccination and implementation of oral cancer prevention and treatment programs in northern Brazil cities. Although there are some interesting results, this study has some limitations. The small sample size, the short study interval and the paraffinization process may degenerate HPV DNA.This study managed to determine the prevalence of OSCC associated with HPV infection, despite the fact that HPV prevalence had a low presence. That is a good result when we consider that HPV is an important etiological factor to oral carcinoma. Our major concern was the high prevalence of 58.5%, which presents concerning data to our region, demonstrating that the lower prevalence presented in previous studies might have some bias that directly influenced the results. So, we identified the need to improve diagnosis and therapy services for various lesions of the oral cavity in the Pará state in order to provide greater assistance to the population of Pará and prevent oral cancer.


A subset of human papillomaviruses (HPVs), so-called high-risk (HR)-HPVs are the causal agents of cervical, anogenital and a group of head and neck carcinomas. Additionally, HR-HPVs have been detected in extragenital tumors including in lung cancer, though their role in this heterogeneous group of malignancies remains controversial. In this review, we address the epidemiological and experimental studies regarding the role of HR-HPV in lung cancer, worldwide, and we propose potential mechanisms. The evidence suggests that HR-HPVs are involved in the development of a variable subset of lung carcinomas in both smoker and non-smoker subjects. Lung cancer is a very prevalent and heterogeneous group of malignancies, and most of them are etiologically associated with tobacco smoking. However, viral infections have been detected in lung carcinomas, with high-risk human papillomaviruses (HR-HPVs) being among them. The role of HR-HPVs in lung cancer has been considered to be controversial. This issue is due to the highly variable presence of this virus in lung carcinomas worldwide, and the low viral load frequently that is detected. In this review, we address the epidemiological and mechanistic findings regarding the role of HR-HPVs in lung cancer. Some mechanisms of HR-HPV-mediated lung carcinogenesis have been proposed, including (i) HPV works as an independent carcinogen in non-smoker subjects; (ii) HPV cooperates with carcinogenic compounds present in tobacco smoke; (iii) HPV promotes initial alterations being after cleared by the immune system through a “hit and run” mechanism. Additional research is warranted to clarify the role of HPV in lung cancer.  Lung cancer is the main cause of cancer deaths around the world, with there having been 1.8 million deaths in 2020 and an estimated nearly 2 million cases by 2022 . Due to A plethora of persistent viral infections have been detected in lung carcinomas worldwide, including high-risk human papillomaviruses (HR-HPVs) , Merkel Papillomaviridae family which includes five genera: alpha-HPV (α-HPV), beta-HPV (β-HPV), gamma-HPV (γ-HPV), mu-HPV and nu-HPV P)-induced DNA adducts, in turn, contributing to lung tumorigenesis. B[a]P is a polycyclic aromatic hydrocarbon (PAH) that is a recognized as a class I carcinogen that is present in tobacco, industrial exhaust, and environmental contamination. Additionally, arsenic , radon  and otheImportantly, the viral load in lung tumors can shed light in respect to its role in this malignancy. Indeed, the very low viral load in lung carcinomas (less than 1 copy/cell) suggest the possibility that HPV is a mere bystander in the clinical samples. However, we cannot deny the possibility of a “hit and run” mechanism, by which the viral infection is involved in the initial events in carcinogenesis, and then the virus is cleared as it is no longer required for the cancer progression. Indeed, previous evidence shows that HPV can work by this “hit and run” mechanism ,164,165.An etiological role of HR-HPV in a subset of lung cancers including both AdCs and SQCs is plausible. However, there is a high variability in HPV detection rates among different countries, and moreover, in the same countries (from 0 to 100%). Probably, the factors related to the differential sensitivity among the detection methods can account for these differences because we lack a gold standard method for HPV detection in human tissues. Additionally, ethnic, lifestyle and sociodemographic factors can be involved in explaining such differences. The molecular mechanisms by which HPV promotes lung cancer are not fully known. As in cervical cancer, HR-HPV E6/E7 overexpression by HPV genome integration is an important event, though the completion of additional studies is necessary to address the consequences of such events for lung epithelial cell transformation. Thus, the analysis of some viral parameters such as the HPV copy number and the viral physical status in the lung clinical specimens is warranted in future studies. Because a “hit and run” mechanism cannot be denied, studies including the preneoplastic lesions of lung cancer will be valuable for the further characterization of this virus in this malignancy. As an HR-HPV infection is not a sufficient condition for carcinogenesis, the role of additional co-factors such as xenobiotics, including environmental factors  which can potentially cooperate with HPV for lung tumorigenesis remains an interesting point for future research.


Background: As the human papillomavirus (HPV) infections are detected in healthy oral mucosa as well as in oral lesions, dental practitioners have an important role in detecting any possible lesions that might be caused by this virus. Therefore, the aim of this study was to investigate the outcomes of orthodontic treatments and HPV infections and to report a rare case of ongoing orthodontic treatment superposed on an HPV infection. Methods: An electronic English literature research of the articles published between the years 2011–2021 was conducted between December 2021–February 2022, accessing PubMed, Web of Science, Embase, Scopus, and Google Scholar. The terms “HPV”, “orthodontics”, “orthodontic treatment”, “tooth movement”, “tooth mobility”, and “malocclusion” were searched. The following inclusion criteria were pursued: articles published in English language; studies reporting HPV infection in subjects with past or ongoing orthodontic treatment; and case reports of subjects with HPV and orthodontic treatment. Exclusion criteria were: articles in languages other than English, studies related to malignancies other than HPV and orthodontic treatment; and studies reporting patients with HPV and no orthodontic treatment. Results: Following the systematic review, which includes six papers, a case of orthodontic treatment superposed on a HPV infection is presented. Conclusion: Incumbent, postponed HPV infection on an ongoing orthodontic treatment might affect treatment outcome and patient compliance. Human papillomavirus (HPV) infections have become more remarkable during the last years, with oropharyngeal manifestations that have to be considered when planning a complex dental treatment plan, especially when the infection occurs during the treatment period. There are more than 200 different HPV genotypes with high or low risk of malignancy ,2. Oral However, new perspectives have been developed for the prevention of these infections by the application of HPV testing technologies and vaccines . There aHPV infections are detected in healthy oral mucosa as well as in oral lesions , and theHuman papillomavirus is rare in children and patients with orthodontic treatment need. Usually, orthodontic treatment is not initiated in cases with positive HPV infection, but when the infection is discovered, by the presence of oral condyloma, warts, or papilloma, orthodontic treatment has already been initiated. There are a few publications worldwide focused on the of HPV infection in subjects with ongoing orthodontic treatment since orthodontics is contraindicated in subjects with malignancies. To the best of our knowledge, there has not yet been published a paper related to the possible impact of the genital HPV-58 infection on the outcomes of orthodontic treatments. Therefore, the aim of this study was to review the English literature related to human papillomavirus infections in subjects with ongoing orthodontic treatment and to report a rare case of HPV infection. This systematic review was performed in accordance with the recommendations of the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA) statement” .A structured search was conducted (between December 2021–February 2022) on articles published between the years 2011–2021, accessing PubMed, Web of Science, Embase, Scopus, and Google Scholar databases. In addition, a handsearching of the reference lists of included studies or relevant reviews was performed.® .The terms “HPV”, “orthodontics”, “orthodontic treatment”, “tooth movement”, “tooth mobility”, and “malocclusion” were searched in combination with the Boolean operators “AND” and “OR” All references were imported and organized in the bibliographic software MendeleyThe following inclusion criteria were pursued: (1) articles published in English language; (2) studies reporting HPV infection in subjects with past or ongoing orthodontic treatment; and (3) case reports of subjects with HPV and orthodontic treatment. Exclusion criteria were: (1) articles in languages other than English; (2) studies related to malignancies other than HPV and orthodontic treatment; and (3) studies reporting patients with HPV and no orthodontic treatment . ®, MS, Redmond, WA, USA) spreadsheets were compiled. This way, data were extracted using a standardized form, which included the following information: (1) authors’ names and publication year; (2) study design; (3) aim of the study; (4) methodology; (5) key findings; and (6) conclusions. Afterwards, both authors compared their assessments and confirmed the data on the basis of the compiled spreadsheets. Both researchers compared their assessments and confirmed the data. When in doubt regarding the study data, the two researchers resolved disagreements by discussion, or a third researcher solved discrepancies.All the eligible citations imported into the bibliography were checked, and all the duplicates were removed. Two reviewers carried out the evaluations independently. For the assessment of each publication, Excel   publisheNoonan et al. (2017) presented a series of seven cases of gingival papillary keratosis with unknown etiology. The lesions were bilateral and symmetric, characterized by yellow-white plaques. While the authors did not exclude a HPV infection, the authors suggested that identification of additional patients diagnosed with such lesions may help in the understanding of their etiology . Henn et al. (2014) presented a case of condyloma acuminatum lesion on a HIV-positive patient (undergoing an orthodontic treatment) and HPV induced. For this case, the treatment plan included surgical removal and chemical cauterization using trichloroacetic acid (TA). The authors emphasized the importance of correct diagnosis and planning for HPV-induced lesions, as there is a high risk for recurrences . The useMagalhaes et al. (2016) presented a case of oral squamous cell carcinoma on an 8-year-old patient undergoing orthodontic treatment. Histopathological exam was p16-positive in a patchy pattern, which is suggestive of HPV. The lesions were located in the gingiva and alveolar ridge, a common location for this demographic group; the post-operative evolution was without events, and the patient was considered disease free at 16 months after surgical resection .Schott et al. (2019)  conducteFurther, we will present a case-report of an HPV infection interposed with an orthodontic treatment.A 25-year-old female, with a skeletal class II relationship and who had crowding on both arches, seeking orthodontic treatment, presented to our clinic with the main complaint of crowding in both arches , Table 4Orthodontic treatment was initiated before the onset of the SARS-CoV-2 pandemic. At that time, the patient did not have the HPV or SARS-CoV-2 infection and followed her regular orthodontic appointments at about 4–6 weeks. After the pandemic onset, which comprised also a period of two months of closed dental offices in our country (mid-March to mid-May 2020), treatment visits became rare, about 8–10 weeks, and the patient missed a few appointments. Protocols for treating patients during the pandemic have been continuously updated. Risk assessment of the pandemic situation has to be adhered , and theDuring the above mentioned period, the patient failed to follow a few appointments, which usually were scheduled monthly, due to the overlap of a genital HPV infection (May 2020). The patient achieved a genital HPV 58 infection, which was diagnosed by real-time PCR multiplex technology, a test that allows simultaneous detection of 19 HPV high-risk types  and 9 low-risk HPV types  as well as intern control. Intern control inspected PCR reaction to each sample. Every reaction was monitored by using six positive intern controls and two negative intern controls. Biopsy of the cervix revealed cervical intraepithelial neoplasia grade 1. Immunohistochemistry using Benchmark Gx Ventana-Roche platform showed intense inflammatory cervicitis and pavement epithelium with nuclear atypia, suggestive for a low-grade squamous intraepithelial lesion (LSIL). No signs or symptoms of oral HPV were noted. During the infection and treatment for HPV, the patient missed three appointments due to her medical condition; whilst she had her surgery, the orthodontic treatment time was prolonged and tooth movement difficult; in addition, the response of the periodontium to the orthodontic forces was impracticable and incompliant. An increase of tooth mobility was observed clinically. Unanticipated difficulties, such as debonded brackets, space appearance, broken wires, lower incisor proclination, lost springs, and exposed end of wire, occurred. In spite of the HPV onset, leveling and alignment of the arches were acceptable, and the occlusal plane was successfully corrected. However, no significant periodontal pathology  occurred at the end of the treatment .As previously stated, HPV infections became more frequent among young patients in an oropharyngeal manifestation, which have to be considered when planning a complex treatment plan, especially an orthodontic therapy. While this systematic review aimed to identify any correlations between ongoing orthodontic treatments along with their outcomes and HPV infection, the literature suggests a lack of such data so far. Out of the six articles included, five were case reports or case series of different oral lesions superposed with an HPV infection (confirmed or not). These studies do not concentrate whatsoever on the effects of the orthodontic therapy but rather on the diagnostic, treatment, and prognostic of the oral manifestations presented. In addition, an interesting approach was identified in the paper published by Schott et al., which investigated, through a questionnaire form, 233 women’s personal history of orthodontic care, long-term satisfaction, as well as adherence to dental and gynecological screening [Orthodontic tooth movement is consequent of an alternation of bone resorption and bone formation, which takes place yearlong , and couTreating class 2 malocclusion is challenging relating to the dental and skeletal problems as regards achieving stable results and sparing the periodontal tissues. This prolonged time interval could influence the teeth mobility as well as the support tissues: the periodontal ligament and alveolar bone. Associated pathology or general disease may also have a major role on tooth movement and the response to orthodontic forces. Prolonging the appliance activation period for better healing and optimal stability could improve prevention of alveolar bone loss or tooth ankylosis. Another aid in delivering a safe treatment strategy is to shorten the treatment time. Procedures that could shorten the treatment time have been described, such as distraction osteogenesis , corticoThe HPV infection of the patient was discovered due to the appearance of a characteristic macroscopic wart in the genital area. Cervical cytology by Pap test revealed atypical squamous cells of undetermined significance (ASC-US). After that, orthodontic treatment became subsidiary. Deferring orthodontic treatment until the cure of the HPV infection will prolong the treatment period, with possible repercussions. Ankylosis, root resorption, and marginal bone loss can be associated with prolonged orthodontic treatment time due to the superposition of the HPV infection. The increased treatment time may also lead to unsatisfactory treatment outcome, as also reported by Umeh et al. . ShortenHuman papillomavirus was first described as “human warts virus” implicated in the pathogenesis of laryngeal papillomatosis and genital warts, also being described as having a potential carcinogenetic role . In as fRoutine screening of patients needing orthodontic treatment is not performed although screening for HPV infection is extremely important. We want to emphasize this aspect and draw attention on this subject, as such rare cases can occur by chance. It is essential to know about the etiology, prevention, and treatment of HPV related tooth mobility in as much as HPV is a risk factor for increasing tooth mobility in patients with ongoing orthodontic treatment. Screening for HPV is extremely important, as HPV genotyping significantly improves detection rate of high-grade cervical intraepithelial lesions . It is sUnfortunately, there are no available guidelines regarding screening for HPV-related malignancies in other anatomic sites . In lighFuture recommendations: screening children and young adolescences for HPV is an area of great interest, which necessitates future research since HPV may relate to the treatment encroachment. Complete oral examinations as a routine in orthodontic treatment may contribute to the early diagnosis of HPV-related symptoms. A limitation of this study may be the absence of any oral manifestations of the HPV infection and the lack of communication between patient and practitioner due to the embarrassment associated with HPV infection. Based on the findings obtained through the systemic review, literature suggests that HPV infections are increasing in young patients, and the oral manifestations might be identified via intraoral examinations by dental practitioners of any specialization, including orthodontics. Incumbent, postponed ongoing orthodontic treatment because of HPV infection might affect treatment outcome and patient compliance. Treatment time lengthens, and unwanted impediments can come into existence. Orthodontists should treat patients while taking into account superposed disease that might influence treatment outcome by prolonging treatment time and influencing the responding support tissues.


However, its reliability has been extensively discussed, and its applicability in every context, especially the enrollment of patients in de-intensification protocols, is debatable. Here, we discuss its limits, especially in populations with a low prevalence of HPV-driven oropharyngeal squamocellular carcinoma, and suggest the possible actions to be taken to overcome such limitations.High-risk human papillomavirus (HPV) infection is a defined etiopathogenetic factor in oropharyngeal carcinogenesis with a clear prognostic value. The P16 IHC (immunohistochemistry) is a widely accepted marker for HPV-driven carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC); in the present paper, we discuss its reliability as a standalone marker in different populations. The literature suggests that rates of p16 IHC false positive results are inversely correlated with the prevalence of HPV-driven carcinogenesis in a population. We propose a formula that can calculate such a false positive rate while knowing the real prevalence of HPV-driven OPSCCs in a given population. As it has been demonstrated that p16 positive/HPV negative cases  have the same prognosis as p16 negative OPSCC, we conclude that despite the valuable prognostic value of p16 IHC, relying only on a p16 IHC positive result to recommend treatment de-intensification could be risky. For this aim, confirmation with an HPV nucleic acid detection system, especially in areas with a low prevalence of HPV-related OPSCCs, should be pursued. Evidence accumulated over the last 20 years ,4,5,6 onSince HPV-positive OPSCCs patients have markedly longer survival, the issue of long-term morbidity and quality of life (QoL) deterioration, deriving also from the aggressive multimodality treatments employed, becomes definitely more relevant ,16. The However, despite the fact that the concept of treatment de-intensification has become very popular with several studies hypothesizing treatment modulation according to HPV status in OPSCCs, the National Comprehensive Cancer Network (NCCN) justifies, at present, such an approach only in clinical trials . On one At any rate, we believe that the lack of consensus about the best diagnostic method(s) also contributes to hampering the safe introduction of HPV in head and neck clinical practice .The mere detection of HPV in a tumor sample does not imply a transcriptionally active virus, nor that the cancer is virus related. A diagnostic method that is to be utilized in clinical practice for the characterization of OPSCCs must detect a clinically relevant number of copies of transcriptionally active viral oncogenes. In fact, we need proof that HPV has impacted the carcinogenic process and currently contributes to the transformed phenotype (HPV-driven carcinogenesis), giving the tumor the typical clinical features of HPV-induced OPSCC. On the other hand, an assay detecting a small number of copies of the HPV DNA (which may come from a transient/not relevant infection or from contamination) may have a high rate of false positive results.E6 and E7 appear to be the main drivers of cancerogenesis in HPV + HNSCC ,22,23,24On the other hand, when it comes to methods that diagnose HPV-induced carcinogenesis in standard formalin-fixed paraffin-embedded (FFPE) samples, a gold standard is actually still missing . Even ifThe detection of viral DNA with PCR is a non-quantitative method that is rather sensitive but poorly specific, as it tends to amplify contaminating material coming from non-relevant fragments of HPV DNA (non-cancerogenic strains or bystander infections) ,30,31,32ISH-based assays, despite being considered highly specific and having gained popularity, especially in the USA, share with other IHC-based techniques a certain dependance on an experienced histopathologist to correctly interpret the results, along with high costs and lengthy and complex procedures. This relative liability is probably behind the lack of diagnostic and prognostic reliability (particularly concerning sensitivity) and is sometimes lower than that of p16 IHC ,26,30,34INK4A protein (p16 IHC) has been used since 2003 as a surrogate marker [Immunohistochemistry for the p16 e marker  and rapie marker . Despitee marker ,37 and ie marker .An alternative sequential strategy, including two highly sensitive methods, has already been validated by Dutch ,31,38 anAdditionally, in Denmark and Sweden, combining HPV DNA and p16 is already recommended as essential for correct prognostication ,40.By analyzing the results of the Dutch and Swedish groups, it has been demonstrated that p16-positive OPSCCs that turn out to be HPV DNA-negative actually have the same prognosis  or slighTherefore, it is obvious that the specificity, or more precisely, the minimal false positive rate, is a fundamental requirement for any assay used to recommend treatment de-intensification in supposedly HPV-driven OPSCC, raising concerns about the use of p16 IHC for this aim ,42.In the literature, the rate of false positive cases with p16 IHC is extremely variable in the different series  overexpress p16 ,42,45,47As shown in This means, for example, that about one-fourth of p16-positive OPSCCs will turn out to be HPV-negative in Sardinia compared to only 0.6% in Scandinavian countries. The total of 0.6% for a false positive rate is acceptable in the perspective of treatment de-intensification, but 25% is not because a high percentage of p16 positive cases would be undertreated with a relevant impact on prognosis. It means that p16 IHC may be deemed sufficient to recommend treatment de-intensification in Scandinavia or in the US, but not in Sardinia or in most of the world’s population, considering that in the most densely populated areas as India, Southern China, and Brazil a much smaller proportion of OPSCCs are HPV-driven. A graphical representation of what happens in a “best case scenario” (5.5% of HPV-negative tumors overexpress p16) can be seen in To conclude, if we aim to safely implement treatment de-intensification for OPSCC, the above data and considerations must be kept in mind. We believe that the scientific community has two options in front of this evidence.1. To accept p16 IHC as a standalone test only in populations with a high prevalence of HPV and defining a threshold  = 5% in the best case scenario, according to the above formula for the prevalence of HPV-driven OPSCC, above which the expected rate of false positive p16IHC is considered acceptable.2. To always confirm p16 IHC with hr-HPV nucleic acid detection before recommending treatment de-intensification in OPSCC. Notably, this is already the recommendation in Denmark and Sweden, even if these are countries with a notoriously high rate of HPV-induced OPSCCs.


Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments.Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity.We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity  and 100% specificity (95% CI: 66–100%) for detection of HPV.The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture. Penaeus monodon densovirus (PmoDNV), P. merguiensis densovirus (PmeDNV), and P. chinensis densovirus (PchDNV) [F. merguiensis) [P. japonicas) [P. monodon) [F. merguiensis), and inferred that a reduction in virus levels could lead to a 14.5% increase in production [Viral diseases are a major concern in aquaculture, especially in shrimp rearing. The hepatopancreatic parvoviruses (HPV) are common viruses of shrimp and fish comprising (PchDNV) . Recentl(PchDNV) , these vuiensis) , and canponicas)  and farmmonodon) ,6. In paoduction . HPV hasoduction . Moreoveoduction –9, and tF. merguiensis) was recently analysed, demonstrating that after three years the average pond HPV levels increased over time [Management of HPV in aquaculture is difficult, and usually performed through challenge tests based on survival and death ratios, with viral titres determined using pleopods. Viral levels are controlled by choosing broodstock from ponds with lower HPV levels, or by selecting lineages or families known to have lower levels of HPV . The effver time . ChoosinFlavobacterium columnare causing columnaris affecting numerous freshwater fish [Vibrio parahaemolyticus strains [Enterocytozoon hepatopenaei [P. stylirostris densovirus [F. merguiensis) can be performed using only homogenization pestles, a few pipettors and a heating block, to enable portable testing amenable for field situations.Aquaculture management verification studies are performed using qPCR, which enables accurate identification of appropriate broodstock. However, qPCR requires time-consuming sample preparation and purification with laboratory-based equipment that is not suitable for field implementation. Molecular methods suitable for field detection are becoming more sophisticated through the implementation of isothermal nucleic acid amplification . Among tter fish , and bot strains  and Entetopenaei , causingtopenaei , infectitopenaei , decapodtopenaei , and P. nsovirus , resultiF. merguiensis) by morphological keys [later Stabilization Solution  and stored at -80°C until used. Samples of farmed banana shrimp were collected at harvest time after 140 days of grow-out [2−103, n = 6), medium , and high  HPV copy number [All the specimens were identified as banana shrimp  were used for qPCR: HPV140 forward, 5’-CTA CTC CAA TGG AAA CTT CTG AGC-3’, and HPV140 reverse, 5’-GTG GCG TTG GAA GGC ACT TC-3’ [Genomic DNA was extracted using a DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA concentration was measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific) and normalised to a concentration of 40 ng/μL. Extracted DNA was stored at -80°C. HPV titres were quantified by qPCR using the SensiMix HRM kit (Bioline) with a Rotor-Gene 6000 thermal cycler (Corbett Research) as described previously ,23. QuanCT TC-3’ .,23. By analysing multiple sequence alignments of hepandensovirus sequences from a variety of Penaeus and Fenneropenaeus sp., highly conserved regions were chosen for primer and probe design suitable for use in an RPA reaction. Primers were designed manually via a multiple sequence alignment  using the free to use GeneDoc software program version 2.7 [GGAAACYTCTGARYCMGGRVYYACCGCCGCRCCSC-3’) and FAM-labelled probe  were HPLC purified, and unlabelled reverse primer (5’-GTGTTKBAGRTCRAYDGGYTKRTTGCGGKGGC-3’) was desalted. An RPA test uses the recombinase enzyme to insert biotin and FAM-labelled primers and probes into the double stranded template at a single temperature of 39°C for 25 min, enabling low-resource capture of amplicons using simple LFD of the biotin-FAM dual-labelled amplicon [P. chinensis hepandensovirus  was synthesized as a double-stranded DNA gBlock® from Integrated DNA technologies  and quantitated using the Qubit™ dsDNA HS Assay Kit .A rapid, low-resource RPA-LFD test for detection of HPV in shrimps was developed by targeting the structural capsid protein gene of HPV, as this region has previously been used for development of qPCR assays sion 2.7  while adsion 2.7 . Six foramplicon . A positFig 1A).Rapid, field-amenable sample preparation was achieved by collecting ~ 40 μg hepatopancreas tissue and adding 50 μl of TNA-Cifer Reagent  to each 40 μg tissue (resulting in a concentration of 800 ng/μL after processing). The TNA-Cifer Reagent and tissue mix was then homogenized with a pestle in a 1.5 mL tube and incubated for 5 minutes at room temperature. Samples were then diluted 1:5 in nuclease-free water   as described by the manufacturer with minor adaptions. Briefly, 1x rehydration buffer & 1/5 rehydrated lyophilized pellet, forward primer (420 nM), reverse primer (420 nM), probe (120 nM) and magnesium acetate (14 mM) were mixed with 1 μL diluted sample (~160 ng) or positive control DNA template, to a final reaction volume of 10 μL. The reaction mix was incubated in a heating block for 25 minutes at 39°C, with brief mixing after 10–15 minutes of incubation and at the end of the 25 minutes incubation period . After amplification, 2 μL of RPA amplicons were dropped onto the middle of the sample application area of a preactivated [3BO3, 100 mM Na2B4O7, 1% BSA, 0.05% Tween 20, pH 8.8) [Fig 1C). All experiments were performed at least three times if not specified otherwise.RPA tests were performed using a TwistAmpctivated  HybriDet pH 8.8) , incubatTM Digital Imaging System  and analysed using ImageJ software . Greyscale-converted images were used to determine band-intensity, by measuring the mean grey value (limit to threshold), using a fixed area measurement, and subtracting from the maximum mean grey value (255). For each test band, the average of two neighbouring relative white spaces was subtracted from the band intensity to normalize the results. A sample was defined as positive if the normalised band intensity was 3 standard deviations higher than the two neighbouring white space values, representing the cut-off value. Diagnostic test evaluation and comparison used the EpiTools online epidemiological calculator (https://epitools.ausvet.io/comparetwotests) and MedCalc online . Kappa value interpretation is suggested as: values ≤0 as indicating no agreement and 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreement.Lateral flow strips were imaged using the MultiDoc-It4 copies of DNA, with a fainter positive band visible at 103 copies . With digital analysis the HPV RPA-LFD test robustly detected down to <242 genome equivalent copies per reaction. Further, in 2 out of 4 separate runs, the test showed a positive result with 24 copies per reaction. We note that a faint band in the negative controls was observed when running time exceeded 5 minutes, however, the included digital analysis enabled a definitive differentiation between positive and negative results.To test the analytical sensitivity of the developed shrimp HPV RPA-LFD test, serial dilutions of a quantified synthetic template DNA were assessed. Strong bands were observed by eye down to 10Fig 3) indicated our rapid shrimp HPV test consistently detected all HPV-positive samples as HPV-positive (n = 24), regardless of high, medium, or low HPV copy number. In addition, all samples containing non-detectable amounts of HPV by qPCR were also identified by the rapid shrimp HPV test as negative (n = 9). These results (n = 33) indicated the Rapid shrimp HPV test had a high diagnostic accuracy, with 100% diagnostic sensitivity  and 100% specificity (95% CI: 66–100%). Further, a Kappa value (95% CI) of 1.00 confirmed rapid HPV test and qPCR results were highly congruent.Diagnostic sensitivity testing of stored hepatopancreas tissues  were processed using our sample preparation method, and mixed 1:1 with a HPV-positive sample  that had been similarly processed prior to the required dilution step for RPA-LFD. The test successfully detected HPV in the mixed samples, but not in the unmixed controls , demonstrating that the negative results were not due to interfering substances in the samples. We then determined the analytical sensitivity of the rapid shrimp HPV test in the background shrimp sample environment by serially diluting an HPV-positive sample  that had been processed using our sample preparation method in negative-tested wildtype banana shrimp samples processed the same way. Results demonstrated the lowest detectable HPV titre was 34.8 genome equivalents, indicating the test could consistently detect <35 genome equivalent copies per reaction  in the banana shrimp sample matrix.To confirm the rapid shrimp HPV test was not affected by interfering substances, three negative-tested wildtype banana shrimp samples , medium (104−105) and low (102−103) copy number of the virus. The test did not detect known qPCR HPV-negative shrimp samples, and this lack of detection was confirmed by spiking positive sample into negative samples to confirm a lack of interfering substances. Retrospective testing of 33 stored hepatopancreas tissues indicated our rapid shrimp HPV test had the requisite diagnostic parameters to ensure accurate estimation of negative shrimp numbers in ponds, with 100% diagnostic sensitivity  and 100% specificity (95% CI: 66–100%).Aquaculture management of HPV infections critically requires selection of negative or low-HPV infected broodstock to prevent increasing pond HPV levels over time. Methods to measure HPV levels are difficult, and while qPCR has been demonstrated useful for accruing identifying HPV levels in broodstock, the laboratory and infrastructure requirements of the test precludes implementation for broodstock testing. Here we describe a rapid shrimp HPV test amenable to low-resource implementation, with sensitivity sufficient to detect HPV in stored shrimp hepatopancreas samples with high  heating block. Our entire workflow time is much faster than the 1–2 hour timeframe of qPCR, which requires expensive thermocycling instruments for implementation, and was slightly faster than the 45 minutes incubation at 37°C reported for RPA detection of excluded . Anotherexcluded  as well excluded –37. ThesLimitations of this study include (1) lack of in-field testing for validation to show operational suitability for farmers to screen for HPV, (2) our tests demonstration with dissected hepatopancreas rather than non-invasive testing using faecal matter, (3) lack of analytical specificity testing using other shrimp viruses, and (4) appearance of a faint band in the no template water controls or HPV-negative banana shrimp hepatopancreas samples, if running time of the rapid HPV test lateral flow detection strip exceeded 5 minutes. For the latter point, these faint bands most likely appeared due to sample matrix-induced aggregation of gold nanoparticles or formation of dual-labelled primer dimers, rather than contamination ,38–40. NF. merguiensis shrimp sample matrix, and demonstrated 100% diagnostic sensitivity  and 100% specificity (95% CI: 66–100%) in retrospective testing of 33 stored F. merguiensis hepatopancreas tissues with known HPV qPCR titres. Compared to the current gold standard qPCR, the developed test has the advantage of providing a simple, robust, rapid and affordable tool suitable for field-implementation and low-resource environments, which would facilitate on-site management of shrimps in aquaculture.In summary, here we describe a rapid and low-resource amenable test for detection of shrimp HPV. The rapid shrimp HPV test described is a sensitive and specific method for rapid detection of HPV within 40 minutes, including sample preparation. This was achieved by developing, for the first time, a simple tissue sample preparation protocol that enabled 5-minute sample preparation, and required only a pestle and some pipettors for liquid handling. The test was able to detect as little as 35 copies of HPV in a S1 FigPenaeus chinensis hepandensovirus; DQ458781: Penaeus merguiensis hepandensovirus; EU073937.1: Penaeus merguiensis densovirus; EU247528.1: Penaeus monodon hepandensovirus; EU346369.1: Penaeus merguiensis densovirus; GU371276.1: Fenneropenaeus chinensis hepandensovirus; JN082231.1: Fenneropenaeus chinensis hepatopancreatic densovirus; NC007218:Penaeus monodon hepandensovirus 1; EU290601.1 Hepatopancreatic parvovirus of penaeid shrimp structural protein gene; AF456476.1: Hepatopancreatic parvovirus of penaeid shrimp Thai strain unknown gene; EU588991.1 Penaeus monodon hepandensovirus 3 non-structural protein 2 gene; EU617324.1: Penaeus monodon hepatopancreatic parvovirus structural protein (HPSP) gene; FJ410797.2 Penaeus monodon hepandensovirus 4; JN788268.1 Penaeus merguiensis densovirus capsid protein gene; DQ415282.1 Hepatopancreatic parvovirus of penaeid shrimp isolate HPV-3 SDDL nonfunctional capsid protein gene; DQ002873.1 Penaeus monodon hepandensovirus 1; AY497195.1 Hepatopancreatic parvovirus of penaeid shrimp strain HPV-4 capsid protein-like gene; AY497193.1 Hepatopancreatic parvovirus of penaeid shrimp strain HPV-2 capsid protein gene; AY497194.1: Hepatopancreatic parvovirus of penaeid shrimp strain HPV-3 capsid protein gene; AY497192.1: Hepatopancreatic parvovirus of penaeid shrimp strain HPV-1 capsid protein-like gene; AY541606.1: Hepatopancreatic parvovirus of penaeid shrimp nonfunctional capsid protein gene; AY945941.1 Hepatopancreatic parvovirus of penaeid shrimp capsid protein gene. * indicates position for internal dS spacer on the probe.AY008257.2: (TIF)Click here for additional data file.


Animal behavior is motivated by the fundamental need to feed and reproduce, and these behaviors can be inferred from spatiotemporal variations in biological signals such as vocalizations. Yet, linking foraging and reproductive effort to environmental drivers can be challenging for wide‐ranging predator species. Blue whales are acoustically active marine predators that produce two distinct vocalizations: song and D calls. We examined environmental correlates of these vocalizations using continuous recordings from five hydrophones in the South Taranaki Bight region of Aotearoa New Zealand to investigate call behavior relative to ocean conditions and infer life history patterns. D calls were strongly correlated with oceanographic drivers of upwelling in spring and summer, indicating associations with foraging effort. In contrast, song displayed a highly seasonal pattern with peak intensity in fall, which aligned with the timing of conception inferred from whaling records. Finally, during a marine heatwave, reduced foraging  was followed by lower reproductive effort (inferred from song intensity). Our work links animal behavior and oceanography to provide insights into species life history and investigate the impacts of environmental change. We examine environmental correlates of blue whale vocalization patterns in Aotearoa New Zealand. Our results demonstrate the vulnerability of marine predator populations to marine heatwaves due to the links between environmental factors that control prey availability, foraging effort, and reproductive effort. Here we focus on the pygmy blue whale (B. m. brevicauda) population that utilizes the South Taranaki Bight (STB) region between the North and South Islands of Aotearoa New Zealand . We anticipate different seasonal patterns in song and D calls, with song showing a clear seasonal cycle in intensity that is less correlated with environmental conditions and more temporally stable. We expect D calls to occur more often in the spring and summer during periods of enhanced productivity that support prey aggregations. If D calls indeed indicate foraging effort, we expect lower D call activity during marine heatwaves when upwelling is reduced. Finally, we predict a positive correlation between the number of D call detections and subsequent song intensity, under the premise that more foraging enables more reproductive effort. Therefore, we anticipate that song will be reduced following marine heatwave events when blue whales were not able to obtain adequate energetic stores from feeding. In summary, we examine acoustic signals of blue whales in the STB to describe year‐round occurrence and behavioral patterns, assess correlations with variable ocean conditions, explore relationships between foraging and reproductive effort, and ultimately shed light on potential consequences of climate change for the viability of blue whale populations.22.1Acoustic data were recorded using five marine autonomous recording units  relative to the energy in selected background frequencies , and a similar approach has been effectively used to describe behavior and phenology of blue whales from changes in this acoustic signal  at varying distances surrounding each hydrophone, and detection area was estimated given the ambient noise recorded at the hydrophone. To summarize the modeled detection distances, we calculated the mean daily detection area for each call type at each hydrophone across the entire recording period.Sound speed profiles were based on the World Ocean Atlas WOA2009), bathymetry was extracted at a 1‐arc minute resolution from the ETOPO1 data set  using the R package “rerddapXtracto”  and pygmy blue whales (B. m. brevicauda). Catches north of the following latitudes and south of the equator were assumed to be pygmy blue whales: 20–30° E (46°S), 30–70°E (52°S), 70–80°E (53°S), and 80°E–180° (52°S). All catches longer than 24.2 m were assigned to Antarctic blue whales and catches south of this pygmy region but specifically recorded as “pygmy” were assigned to pygmy blue whales. Catches in the Atlantic Ocean (west of 20°E) were classified as Antarctic, while those off the west coast of South America were excluded as they belong to the Chilean blue whale population  or the Soviet Union (1962/63–1971/72), and lengths were recorded in metric units, mostly to the nearest cm. Note that for the Soviet data, information has been reconstructed from original logbooks for nearly all expeditions and replaces the original falsified records that were submitted to the International Whaling Commission from 1949/50 , 6442 and 6445 (both Sovetskaya Ukraina), and 6490 (Sovetskaya Rossia).The pygmy data in the New Zealand region  were assessed to determine whether these blue whales followed similar seasonal patterns of fetal growth as other blue whales in the Southern Hemisphere. All catches in this region come from Soviet expeditions and were caught while the Soviet fleets were returning from the Antarctic during February–May in the years 1964–1968 and 1973. The Soviet fleets in the database are listed under the following codes: 6300  are a machine learning multivariate modeling approach that combines decision tree methods with a boosting algorithm that iteratively optimizes model performance by combining a large number of decision trees  models performed well overall, with some variability among models fit to different call types, seasons, and hydrophones Table . The modSong intensity showed the strongest relationship with month, which contributed 59%–77% of the explained deviance in the full models Table . As was Environmental correlates had stronger contribution to the full models for D calls than song Table . Month s3.4Song intensity was at its highest between April July, with the peak taking place in May–June Figure . Examinaχ2 = 2131.2, p < 2.0 × 10−16), NPP , and D calls . Furthermore, pairwise post hoc comparisons showed significant differences between 2016–2017 and 2017–2018, but not 2016–2018 , they reflect the pattern in measurements from all other pygmy blue whales taken in the Southern Hemisphere, and Antarctic blue whale catches, both of which are far more numerous ; formal analysis (lead); investigation (lead); methodology ; visualization (lead); writing – original draft (lead). Holger Klinck: Investigation (supporting); methodology ; supervision (supporting); writing – review and editing (supporting). Dimitri Ponirakis: Formal analysis (supporting); investigation (supporting); methodology . Trevor A. Branch: Formal analysis (supporting); investigation (supporting); methodology ; writing – review and editing (supporting). Leigh G. Torres: Conceptualization (lead); funding acquisition (lead); investigation ; methodology ; project administration (lead); supervision (lead); writing – review and editing (lead).The authors declare that they have no competing interests.