This article addresses human papillomavirus (HPV) infection, one of the topics covered by the Clinical Protocol and Therapeutic Guidelines for Comprehensive Care for People with Sexually Transmitted Infections, published by the Brazilian Ministry of Health. The Protocol and Guidelines have been developed based on scientific evidence and validated in discussions with specialists. This article presents epidemiological and clinical aspects and guidelines for health service managers and health workers about diagnosing and treating people with papillomavirus infection. This theme is a significant public health issue since it is the most prevalent sexually transmitted infection globally, capable of triggering the oncogenic process of cervical cancer and the possibility of anogenital warts. Important information is presented for gaining knowledge about HPV and action strategies for infection prevention and control, provision of quality care, and effective treatment of the disease. Infection by the human papillomavirus is the sexually transmitted infection of the highest contagion, superior to genital herpes and HIV. The article addresses the infection by the human papillomavirus (HPV). This subject composes the Clinical Protocol and Therapeutic Guidelines (PCDT) for Comprehensive Care for People with Sexually Transmitted Infections (STI), published by the Health Surveillance Secretariat of the Ministry of Health. For the PCDT elaboration, a selection and analysis of the evidence available in the literature were performed, and examined by a panel of specialists. The National Committee approved the document for the Incorporation of Technologies in the Brazilian National Health System (Conitec)HPV is a double-stranded, non-encapsulated DNA virus, a member of the Papillomaviridae family. It infects the squamous epithelium and can induce the formation of a wide variety of cutaneomucous lesions, especially in the anogenital region. More than 200 types of HPV are identified, of which approximately 40 affect the anogenital tractThe main form of HPV transmission is sexual activity of any kind, including the deposition of the virus in the fingers through genital contact and autoinoculation. Exceptionally, during labor, the formation of cutaneomucous lesions in newborns or recurrent laryngeal papillomatosis may occur. Transmission by fomites is rare. It is the most transmissible STI, greater than genital herpes and human immunodeficiency virus (HIV) infections-Generally, the infections are asymptomatic. Approximately 1% to 2% of the infected population will develop anogenital warts, and about 2% to 5% of women will undergo alterations in oncotic colpocytology. The prevalence of infection is higher in women under 30 years of age. In contrast, the vast majority of HPV infections in women  have spontaneous resolution within an approximate period of up to 24 monthsThe types of HPV that infect the anogenital tract can be of low or high oncogenic risk. The types belonging to the low-risk group  often occur in benign lesions and low-grade squamous intraepithelial lesions. HPV types from the high-risk group  or oncogenic are often associated with high-grade squamous intraepithelial lesions and carcinomas. It is important to stress that about 85% of low-grade lesions contain HPV of the oncogenic groupIn most people, HPV infection does not produce any clinical or subclinical manifestation. The latency period can vary from months to years. The prevalence of low and high-risk HPV viral groups in the population is demonstrated in Subclinical manifestations of HPV can be detected by oncotic cytology, using magnifiers, coloring substances, and colposcopy, accompanied or not by biopsy. Lesions associated with low oncogenic risk viral types are generally low-grade squamous lesions, equivalent to the histopathological diagnosis of mild dysplasia or cervical intraepithelial neoplasia (CIN) grade 1. In contrast, lesions associated with high-risk oncogenic HPV infections are usually associated with high-grade squamous intraepithelial lesions and histopathological diagnosis of intraepithelial neoplasia grade 2 (CIN 2) or 3 (CIN 3). Besides the uterine cervix, other epithelia may suffer the oncogenic action of the virus, originating vaginal, vulvar, perianal, penile, and anal intraepithelial neoplasmsThe clinically detectable manifestations induced by HPV are polymorphic and can be pointed (condyloma acuminatum), spiculated, with circumvolutions, or even flat. Their size varies from one millimeter to several centimeters. They can be single or multiple, flattened or papular, although in most cases, they are papillomatous. The lesions’ surface is opaque, velvety, or similar to that of cauliflower, and they may be of skin color, erythematous, or hyperpigmented. Generally, the lesions are asymptomatic and may be pruritic, painful, friable, or hemorrhaging,In men, lesions occur most frequently on the inner fold of the foreskin, balanopreputial sulcus, or glans and may also be located on the skin of the penis and scrotum,People living with HIV report a higher frequency of multiple infections, anogenital warts, intraepithelial lesions, and anogenital neoplasms due to HPV infectionThe diagnosis of anogenital warts is clinical. The biopsy for histopathological study must be performed when there is any doubt in the diagnosis (suspected neoplasms or other conditions), in the presence of atypical lesions or lesions that do not respond appropriately to treatment, and in suspicious or very voluminous lesions in people with immunodeficiencies. In the case of women with anogenital warts, a gynecological examination including cervical cytology is required for cervical cancer screening and, in the presence of cytological abnormalities, colposcopy and biopsy if necessaryIn the presence of anal lesions, a proctological examination with anoscopy and rectal touch, and even high-resolution anoscopy, would be idealSerological tests demonstrating natural antibodies, or after vaccination against HPV, lack practical applicability and have been used only in studiesThe objective of the treatment of anogenital warts is to eradicate visible lesions (condylomata). However, there is no evidence that the treatments available so far modify the natural history of HPV infection. Even without treatment, the lesions can disappear, remain unchanged or increase in number and volume. Treatment of warts does not eliminate the HPV infection. The HPV DNA that remains in the infected cells can remain inactive (latent) for prolonged periods. The first episode or recurrence of symptoms may occur months or even years after the initial infection. Therefore, those who do not eliminate HPV can also transmit the virus, even after treatment or removal of the lesions. After initial elimination, the recurrence of warts is common: one year after treatment, approximately half of the cases develop new wartsSeveral clinical and surgical treatments for anogenital warts are available, among which the following topical medicines: 60-80% trichloroacetic acid; 5% imiquimod cream; 10%-25% podophyllin solution; 0.5% podophyllotoxin solution; and 0.15% podophyllotoxin cream. As for the 0.5% podophyllotoxin solution, although not available in Brazil, neither at SUS nor at private health services, it must be highlighted for its benefits. The treatment with imiquimod or podophyllotoxin presents the convenience of self-application, although it requires guidance and rigorous monitoring due to the possibility of side effects. In turn, trichloroacetic acid and podophyllin must always be applied by the assistant physician and, likewise, with strict monitoring, due to the potential caustic effects. There is consensus that ablative techniques, such as electrotherapy, cryotherapy, and laser, are highly effective in treating anogenital warts. However, the evidence basis on the clinical efficacy and cost-effectiveness of the treatments used on anogenital warts is limited. The treatment must be individualized, considering the characteristics of the lesions, the availability of resources, the adverse effects, and the health professional’s experience. As this is a generally self-limiting disease, treatments that generate disfiguring scars should be avoided. Warts with soft, moist, and non-keratinized characteristics usually respond well to treatment with topical application of, for example, 5% imiquimod, 0.15% podophyllotoxin cream, or 60-80% trichloroacetic acid, depending on the type of lesion and skin characteristics. Physical ablative methods are more effective in the treatment of keratinized lesionsThe therapeutic option change should be considered when there is no significant improvement after three sessions or if warts do not disappear after six to eight treatment sessions. It is also possible to combine treatments, especially in complicated cases associated with immunosuppression, always with strict control of the normal tissues’ inflammatory effects,,There is also the possibility that anogenital warts will disappear without any treatment, especially in immunocompetent individualsIt is also relevant to consider that the presence of genital warts acts as a “marker” for STI, as it is estimated that 20% of people with anogenital warts have a simultaneous STI, including chlamydia, HIV infection, or syphilis. Therefore, screening for other STIs should be performed. Chlamydia infection, when associated with HPV, not only complicates the treatment of the condylomata but also facilitates the progression of CIN lesions-Prophylactic vaccination is safe and effective in preventing HPV infection and its complications. There is evidence of the benefit of vaccination, both for individual and collective protection, with a reduction in benign and malignant lesions-The vaccine is most effective in adolescents vaccinated before the first sexual contact, with an antibody production ten times higher than that found in the naturally acquired infection within two years. HPV vaccination does not lead to changes in sexual behavior among adolescents. Health professionals should indicate vaccination and promote the increase of vaccination coverage in the countryThe HPV prophylactic vaccine must be a priority for SUS, considering its benefits related to immunogenicity, efficacy, and safety for the population’s health.


The introduction of primary HPV screening has doubled the number of colposcopy referrals because of the direct referral of HPV-positive women with a borderline or mild dyskaryosis (BMD) cytology (ASC-US/LSIL) triage test. Further risk-stratification is warranted to improve the efficiency of HPV-based screening.FAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping in HPV-positive women with BMD (n = 294) in two Dutch screening trials. Absolute CIN3+ risks and colposcopy referrals within one screening round were calculated.This study evaluated the discriminative power of FAM19A4/miR124-2 methylation, HPV16/18 and HPV16/18/31/33/45 genotyping, respectively. The CIN3+ risk after a negative result could be lowered to 2.8% by combining methylation and extended genotyping, at the expense of a higher referral percentage of 75.5%.Methylation analysis discriminated well, yielding a CIN3+ risk of 33.1% after a positive result and a CIN3+ risk of 9.8% after a negative result. HPV16/18 and HPV16/18/31/33/45 genotyping resulted in a 27.6% and 24.6% CIN3+ risk after a positive result, and a 13.2% and 9.1% CIN3+ risk after a negative result. Colposcopy referral percentages were 41.2%, 43.2%, and 66.3% for FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals.The use of  Human papillomavirus (HPV)-based cervical screening has an increased sensitivity for the detection of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer compared with cytology-based screening, but has a suboptimal specificity , 2 and tThe Netherlands was the first country that implemented primary HPV screening with cytology triage in the national cervical screening programme in 2017 . HPV-posFAM19A4/miR124-2 methylation analysis has shown a high sensitivity for cervical cancer and advanced high-grade CIN  in HPV-positive women [HPV16/18 genotyping  or HPV16/18/31/33/45 genotyping (extended genotyping) has been considered for triaging HPV-positive women with BMD cytology, because CIN3+ risks vary between individual high-risk HPV genotypes –15. An ave women , 23.FAM19A4/miR124-2 methylation, HPV16/18 genotyping, HPV16/18/31/33/45 genotyping and combinations thereof in HPV-positive women with BMD cytology in two large Dutch population-based screening cohorts.In this study, we evaluate the performance of n = 167 and POBASCAM n = 192) were selected. Fifty-two samples were excluded due to insufficient leftover material for methylation analysis (VUSA-Screen n = 41 and POBASCAM n = 11).This study is a post hoc analysis within the VUSA-Screen trial and POBASCAM trial. HPV-positive women with borderline or mild dyskaryosis (BMD)  and registered in the trial register . The study was designed to evaluate the effectiveness of combined HPV and cytology testing in the Dutch national screening programme. A detailed description of the VUSA-Screen trial has been published before . CervicaThe POBASCAM trial is a population-based cervical screening study within the Dutch screening programme with enrolment between January 1999 and September 2002. The POBASCAM trial was approved by the Medical Ethics Committee of the VU University Medical Center (no 96/103A) and by the Ministry of Public Health VWS no 328 650) and registered in the trial register . The study was designed to evaluate the effectiveness for CIN2/3 detection of combined HPV and cytology testing in the intervention group compared with sole cytology testing and blinded HPV testing in the control group. A detailed description of the POBASCAM trial has been published before , 27. Cer 650 and In both studies, histology results were classified as no dysplasia, CIN grade 1, 2, 3, or cervical cancer. Adenocarcinoma in situ was classified as CIN3. Histopathologic follow-up data were collected through the nationwide network and registry of histopathology and cytopathology (PALGA) .HC2-positive samples from the VUSA-Screen trial were tested with GP5+/6+ PCR-EIA. All EIA-positive samples from both trials were typed by reverse line blot (RLB) assay for identification of 14 high-risk HPV types  . SamplesFAM19A4/miR124-2 methylation analysis was performed as described previously [FAM19A4/miR124-2 methylation result (VUSA-Screen n = 3 and POBASCAM n = 10) and were excluded from further analysis. Methylation status was labelled positive if the QIAsure Methylation Test® result exceeded the preset ΔΔCt value threshold for methylation positivity according to the manufacturer’s instructions.eviously , 31, bliFAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping. The performance of five triage strategies was evaluated: (I) FAM19A4/miR124-2 methylation, (II) HPV16/18 genotyping, (III) HPV16/18/31/33/45 genotyping, (IV) HPV16/18 genotyping combined with FAM19A4/miR124-2 methylation and (V) HPV16/18/31/33/45 genotyping combined with FAM19A4/miR124-2 methylation. Strategy (I) was labelled positive if the methylation result was positive. Strategy (II) was labelled positive if HPV 16 and/or 18 was present. Strategy (III) was labelled positive if HPV 16, 18, 31, 33, and/or 45 was present. Strategy (IV) was labelled positive if HPV 16 and/or 18 was present or if the methylation result was positive. Strategy (V) was labelled positive if HPV 16, 18, 31, 33, and/or 45 was present or if the methylation result was positive. Sensitivity and specificity were estimated with Wald 95% confidence intervals (95% CI). Direct referral percentage was calculated as the percentage of positives from each screening strategy and the number of referrals needed to detect one CIN3+ was calculated by dividing the number of screen positives by the number of true positives. We constructed 95% confidence intervals (95% CI) for the risk differences in STATA . If the 95% CI did not contain the value 0, the difference was considered significant. All other statistical analysis were performed with SPSS Statistics .Data of the VUSA-Screen trial and POBASCAM trial were pooled and absolute CIN3+ risks were calculated. All CIN3+ detected within one screening round  were included. Separate estimates were retrieved for single and combined triage strategies using FAM19A4/miR124-2 methylation analysis , in whom 56 CIN3 and 1 cervical carcinoma were detected. The median age of women included was 35.0 years (range: 29–60 years) and median time to CIN3+ detection was 0.7 years (IQR: 0.2–1.3 years). In total, 121/294 women (41.2%) tested methylation positive, 127/294 women (43.2%) tested HPV16/18 positive and 195/294 women (66.3%) tested HPV16/18/31/33/45 positive.The final study population consisted of 294 HPV-positive women with BMD and valid results for HPV genotyping and FAM19A4/miR-124 methylation, HPV16/18 genotyping, HPV16/18/31/33/45 genotyping or combinations thereof. Fig. FAM19A4/miR124-2 methylation, HPV16/18 or HPV16/18/31/33/45 had a CIN3+ risk of >9%. Combinations of HPV genotyping and methylation lowered the CIN3+ risk among test-negatives with a CIN3+ risk of 6.5% (95% CI: 1.8–11.1%) among women who were both HPV16/18-negative and FAM19A4/miR124-2 methylation-negative and a CIN3+ risk of 2.8% (95% CI: 0–6.6%) among women who were both HPV16/18/31/33/45-negative and FAM19A4/miR124-2 methylation-negative. Supplementary Table Table Table FAM19A4/miR124-2 methylation, HPV16/18 genotyping  and HPV16/18/31/33/45 genotyping (extended genotyping) for additional risk-stratification of HPV-positive women with BMD cytology to reduce direct colposcopy referral rates, while maintaining high sensitivity for CIN3+ detection. The choice of additional triage tests depends on the CIN3+ risk, the number of repeat tests and the number of colposcopy referrals that are deemed acceptable.We evaluated FAM19A4/miR124-2 methylation resulted in the highest CIN3+ risk of 33.1% and the largest risk-difference between test-positives and test-negatives (23.2%). Recently Bonde et al. also showed in a large multicentre cohort that FAM19A4/miR124-2 methylation detected the very large majority of CIN3 in HPV-positive women with ASC-US/LSIL [FAM19A4/miR124-2 methylation led to a referral percentage of 41.2% and a sensitivity of 70.2%. Still, the CIN3+ risk after a negative test for any of the single strategies was >9%. Combined strategies lowered the CIN3+ risk among test-negatives substantially with the lowest CIN3+ risk of 2.8% among women who were both HPV16/18/31/33/45-negative and FAM19A4/miR124-2 methylation-negative. Despite a high referral percentage of 75.5%, a benefit of this combined strategy is that the CIN3+ risk among triage-negative women seems sufficiently low to recommend return to the regular cervical screening programme.Of the single triage strategies, -US/LSIL . In our FAM19A4/miR124-2 methylation has shown a very high sensitivity for cervical cancer [FAM19A4/miR124-2 methylation test is associated with high spontaneous regression [FAM19A4/miR124-2 methylation has also prognostic value for development of cervical cancer in the long term. De Strooper et al. found a significantly lower 14-year cervical cancer risk among HPV-positive, FAM19A4/miR124-2 methylation-negative women compared with HPV-positive, cytology-negative women. Collectively, these data indicate that FAM19A4/miR124-2 methylation-negative CIN2/3 have high regression rates and low cervical cancer progression risks. As a consequence, methylation analysis may be considered when deciding whether women can be returned to routine screening.l cancer  and adval cancer . Hence, l cancer . Furthergression  (Kremer,FAM19A4/miR124-2 methylation on the number of referrals and detection of CIN3+ will be considerable. Surely, the high colposcopy referral rates and low CIN3+ risk in the national screening programme makes the need for additional triaging of HPV-positive women with BMD even larger than anticipated based on our studies. For the implementation of methylation analysis, applicability and reproducibility of the test among different sample types and DNA extraction methods is essential. Floore et al. evaluated the intra- and inter-laboratory agreement of the FAM19A4/miR124-2 methylation test with the use of several cervical scrape collection media and several DNA extraction methods and showed a good to excellent intra- and inter-laboratory agreement on the assay and the full workflow [In the new HPV-based screening programme in the Netherlands, the proportion of HPV-positive women who were directly referred for colposcopy because of BMD cytology was 70.3% in year 2019 . The cumworkflow .A strength of our study is that we used data of two large population-based HPV DNA screening trials within the Dutch screening programme. As shown in Supplementary Table FAM19A4/miR124-2 methylation could reduce direct colposcopy referral rate with 60%, while retaining high CIN3+ sensitivity. The combination of FAM19A4/miR124-2 methylation with HPV16/18/31/33/45 genotyping would reduce colposcopy referrals with only 25%, but the low residual CIN3+ risk would obviate the need of short-term follow-up testing.To conclude, the implementation of primary HPV screening with cytology triage in the Netherlands has led to a two-fold increase in direct colposcopy referrals and ≤CIN1 diagnosis, mainly because of the direct referral of women with BMD. Additional risk-stratification of HPV-positive women with BMD by Supplementary Table 1


To determine the prognostic risk factors of patients with acute epidural hematoma (AEDH), a scoring system was established based on gray–white matter ratio (GWR) and internal verification was performed.All patients with AEDH who underwent surgical treatment in Qinghai Provincial People's Hospital from January 2013 to June 2019 were continuously collected. The clinical and imaging data of the patients were collected. According to Glasgow Outcome Scale at 3 months after operation, the patients were divided into poor and good prognosis groups, respectively. The GWR value of the nonhematoma side was measured at the inner capsule area. Univariate and multivariate analyses were used. Independent predictors significantly related to the prognosis of AEDH were screened out and a nomogram was established based on these factors.A total of 170 cases were included in this study, the Glasgow Coma Score (severe and moderate), cerebral hernia, midline shift, preoperative GWR, postoperative GWR, hematoma thickness/midline shift, time from coma to surgery, and decompression of bone flap were the independent risk factors for predicting the poor prognosis of AEDH. Moreover, the prediction ability of nomogram was higher than any other independent predictive factors.The nomogram model established represents the most effective factor to predict the prognosis of operated AEDH. The scoring system is characterized by high accuracy, simplicity and feasibility, with a wide range of clinical application prospects. The patients with large hematoma are prone to suffer from cerebral hernia in the early stage, and an emergency surgical intervention would be necessary. Most of the patients with AEDH recover well, but 10% would still die. Therefore, it is particularly important to evaluate the prognosis of AEDH patients at an early stage.Traumatic brain injury (TBI) is one of the main causes of accidental death among the under-45-year-old population. and Helsinki CT score have been often used to evaluate the prognosis of TBI in clinical practice. However, these scoring systems have some shortcomings. For example, the Rotterdam CT scoring system only describes the midline shift (MLS) and compression state of basal cistern, which would fail to take into account the specific characteristics of a single disease case. Guo et al have proposed the effect of swirl sign on prognosis in AEDH, while no other features of AEDH have been investigated. At present, there is only 1 scoring system for acute subdural hematoma, while there is no scoring system for AEDH. Therefore, in this study, based on previous findings, a scoring system for the prognosis of AEDH was proposed, and the internal verification was carried out.Rotterdam computed tomography (CT) score22.1This study was approved by the ethics committee of Qinghai Provincial People's Hospital. Informed consent was obtained from each patient.2.2All patients with AEDH who underwent surgical treatment in Qinghai Provincial People's Hospital from January 2013 to June 2019 were continuously collected.2.3Subjects more than 18 years old; diagnosed with unilateral AEDH by CT, located on the superior side of the cerebellar tentorium, representing the primary life-threatening cause; with abbreviated injury scale (AIS) (other than the head) less than 3 points; epidural hematoma was cleared, and decompression of bone flap was determined according to the situation during operation; and with no history of craniocerebral surgery or epilepsy.2.4Patients with cerebral infarction, cerebral hemorrhage or intracranial occupying lesion in the past; with cancer or other serious diseases; with incomplete clinical, image or follow-up data; with penetrating brain injuries; and with any disease that would affect the coagulation function, such as coagulopathy, renal dysfunction, anticoagulant therapy, antiplatelet therapy, etc.2.5The general data mainly included the sex, age, nationality, occupation, past medical history, treatment, cerebral hernia, AIS, Glasgow Coma Score (GCS), time from coma to surgery, and Glasgow Outcome Scale (GOS). The imaging data mainly included the hematoma volume, state of basal cistern, hematoma thickness (HT)/MLS, and gray matter (GM) and white matter (WM) Hounsfield values in nonhematoma side. Gray–white matter ratio (GWR) was obtained according to the preoperative and the first postoperative CT images, according to the following formulation: GWR = GM/WM.2.6The patients were divided into a poor prognosis group (GOS 1–2 points) and a good prognosis group (GOS 3–5 points), respectively, according to the postoperative 3-month GOS.2.7CT scanning was performed using a unified protocol, with the equipment from the General Electric Company (USA) . Based on the medical imaging information management system (version 2.0), the circular region of interest was placed independently in the measurement area by a radiologist and a neurosurgeon. The original CT values of GM and WM were measured in the region of interest.2.8 the putamen (PU) was selected as the GM measurement area and the posterior limb of internal capsule (PIC) was selected as the WM measurement area. However, the PU and pallidus (PA) were not clear, and therefore, the lentiform nucleus (LN) was used as the measurement area in this study , and 95% confidence intervals (CI). The Chi-square test was used for univariate analysis to preliminarily identify risk factors associated with poor prognosis. The significant risk factors in the univariate analysis were analyzed by the binary logistic regression analysis. P < .05 was considered statistically significant.The receiver operating characteristic (ROC) curve was plotted using the graphics printer. According to the ROC curve, the continuous variables were evaluated, and the cutoff value was determined. The SPSS software  was used for the univariate and multivariate logistic regression analyses. All factors associated with poor prognosis were assessed using the Using the R software version 3.4.3, the forest map and nomogram were drawn based on the results from the binary logistic regression analysis. The predictor with the highest beta coefficient was assigned 100 points based on the weight, and the other predictors were assigned corresponding points. In order to prove the net benefit of the established nomogram prediction score system, the decision curve was used to analyze the independent risk factors and the nomogram prediction scoring system. Clinical influence curve was used to verify the accuracy of the above decision curve analysis.33.1A total of 632 patients were enrolled in this study. Among these subjects, 170 cases met the inclusion criteria, while 462 cases were excluded .In the ROC analysis, the factors of age, difference of MLS (the difference between the preoperative and postoperative MLS), HT/MLS, preoperative GWR, postoperative GWR, and the time from coma to surgery were analyzed. Our results showed that the area under curve values of all the above factors was more than 50%, and the area under curve value of HT/MLS was the largest at 81.0% Fig. .3.23.2.1P = .05, the factors of age, state of basal cistern, GCS, cerebral hernia, difference of MLS, preoperative GWR, postoperative GWR, HT/MLS, time from coma to surgery, and decompression of bone flap were correlated with poor prognosis   was the most dangerous factor for poor prognosis, followed by the postoperative GWR ≤1.368 . However, the state of basal cistern and age were not independent predictors of poor prognosis , cerebral hernia, difference of midline shift, preoperative GWR, postoperative GWR, HT/MLS, time from coma to surgery, and decompression of bone flap. The forest map analysis of the prognostic risk was then carried out Fig. .3.4The cutoff point of 50% was adapted, that is, the patient's score was greater than 243 points, which indicated poor prognosis Fig. .In the clinical influence curve, the red curve represented the number of people who were classified as positive (high risk population) according to the simple model, and the blue curve represented the number of true positive people under each threshold probability. When the threshold probability was low, the distance between the red curve and the blue curve was large, indicating significant difference in the numbers between the high-risk and actual positive people; and the opposite results indicated small difference. When the threshold probability was more than 25%, the predicted positive number was basically consistent with the actual positive number Fig. .The threshold probability of 35% was used herein, which meant that the predicted positive subjects basically matched the actual positive ones. Therefore, the nomogram scoring system was selected to evaluate the prognosis of patients with high accuracy.3.5The data were exhibiting nonnormal distribution, and therefore the median was used for statistical analysis. Our results showed that there was no difference between the preoperative and postoperative GM in the nonhematoma side, and there was significant difference between the preoperative and postoperative WM Table .4 the PU was selected as the GM measurement area and the PIC was selected as the WM measurement area. However, the PU and PA were not clear due to the hematoma compression, and therefore the LN was used as the measurement area in this study. The method might be more feasible and rigorous to calculate the GM and WM.According to the GWR calculation method reported by Gentsch et al, However, herein, the measurement areas selected were LN and PIC, which did not affect the gray–white measurement, nor influence the prognostic score.The haemostatic agents were used within the surgical site, which could influence the CT values.P < .0001). In this study, our results showed that the mortality of patients was positively correlated with time from coma to operation. In addition, our results showed that the mortality was 35% in patients with time from coma to operation more than 4 hours. The difference might be related to the differences in the sample size. It might also be correlated with the fact that Qinghai province is located on the plateau, and the people living there have relatively stronger tolerance to cerebral ischemia and hypoxia. Therefore, a control study concerning the high altitude and plain areas should be conducted to clarify the influence of hypoxia environment on prognosis in the future.The time from coma to surgery was one of the most important factors affecting the disease prognosis. It has been previously shown that the fatality rate of patients who undergo surgery within 4 hours after coma is 30%, while that of patients who undergo surgery after 4 hours is 90% ( However, in this study, our results showed that there was no significant difference between the preoperative and postoperative CT values of GM. However, there was significant difference in the CT value between preoperative and postoperative WM, where the CT value of postoperative WM was lower than the preoperative WM. Moreover, our results showed that the WM was more easily to be affected by ischemia and hypoxia, even if the compression was relieved. It would continue to be influenced by ischemia and hypoxia, while GM was basically not impacted. This phenomenon might be related to the vascular densities of GM and WM. At the same time, this might be due to the significant decrease in the CT value of preoperative WM when the hematoma volume was large enough. However, the CT value of WM after removing hematoma exhibited no significant change or even be lowered, compared with before surgery.In the early stage of TBI, the cytotoxicity and angiogenic edema could co-exist, and the densities of GM and WM might be decreased.In this study, our results showed that the prognosis was poor with the preoperative GWR ≤1.307 and/or postoperative GWR ≤1.368. Moreover, smaller ratios would indicate worse prognosis; and vice versa. These findings suggest that the prognosis of patients was worse when the GM was affected by ischemia and hypoxia than when WM was affected. The GM was the region of the nucleus, and when the nucleus was damaged, it could cause serious dysfunction, thus affecting the disease prognosis.Since HT near the head top is larger while MLS is smaller, the HT/MLS value would be larger. Therefore, data analysis was conducted with the maximum value excluded. In this study, our results showed that the prognosis was poor with HT/MLS ≤ 2.52. Moreover, smaller ratios would indicate worse disease prognosis; and vice versa. These findings were consistent with those met in clinical practice. Because the brain tissue has certain compensation abilities, when the hematoma volume is larger, the hematoma spread around, making the HT increase not obvious. But if the amount of bleeding exceeded the compensation ability of brain tissue, it will lead to the greater MLS and the consequent smaller HT/MLS.When difference between preoperative and postoperative MLS was found to be greater than 2.9 mm this indicated poor prognosis. This is because the preoperative hematoma volume determined the MLS size. The larger hematoma volume was, the more obvious MLS was, and after the hematoma was cleared, MLS is reduced, and the difference of MLS became larger. Therefore, the larger MLS difference indicated worse prognosis.In this study, the need to perform decompression of bone flap indicated poor prognosis. As the decision to perform decompression of bone flap, depends on the patient's intracranial pressure, brain tissue edema, cerebral hernia, GCS, and other comprehensive factors. So it is logic to expect that the patients who underwent decompression of bone flap would have worse prognosis.There were some limitations of this study. First, this was only a single-center retrospective study. Second, the operation time and method might also have impacts on the prognosis. Further, in-depth multicenter prospective studies with larger sample sizes are still needed in the future.5Nomogram model is the most effective factor to predict the prognosis of AEDH. The GCS, preoperative GWR, and postoperative GWR are closely related to the poor prognosis.In this study, all authors have made crucial contributions and agreed to publish the article. In particular, Dr. Yang played the leading role in the investigation. Meanwhile, Dr. He was mainly responsible for writing articles, Dr. Du and Dr. Jin were mainly responsible for data collection, statistical analysis, and other sections.Conceptualization: Mingfei Yang.Data curation: Yunxing Luo, Chaonan Du, Xiaoqing Jin.Formal analysis: Yunxing Luo, Chaonan Du.Investigation: Xiaoqing Jin.Methodology: Xiwu He, Mingfei Yang.Project administration: Mingfei Yang.Resources: Yunxing Luo.Software: Yunxing Luo.Writing – original draft: Yunxing Luo, Xiwu He.Writing – review & editing: Mingfei Yang.


Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance.Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin (fzo-1). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1;pdr-1;+/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1;+/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes.We assessed heteroplasmy levels in three Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.The online version contains supplementary material available at 10.1186/s12915-022-01241-2. Unlike the nuclear genome, mitochondrial DNA (mtDNA) is present in multiple copies per animal cell. For instance, each human somatic cell contains an average of ~ 1000 mitochondria, with each mitochondrion harboring 1–10 mtDNA copies –5. Some Caenorhabditis elegans model system [Drosophila model systems, especially in germ cells [The repertoire of heteroplasmic mutations varies among cells and tissues, mainly due to replicative segregation (drift) of the mitochondria during cell division and mitochondrial bottlenecks that appear during embryo development . Howeverl system –17. Mitol system , 19. In l system . Furtherrm cells , 21, 22.rm cells , 16, 23.rm cells , 24. Altrm cells , 26. We,C. elegans harboring mitofusin mutant (fzo-1) to animals carrying either of three heteroplasmic mtDNA deletions, which differed in size and mtDNA positions. These experiments resulted in embryonic lethality and developmental delay, which were alleviated in subsequent generations concomitant with a complete loss of the truncated mtDNA molecules. Since the rate of truncated mtDNA loss diverged between the heteroplasmic strains, the sensitivity of the fusion machinery to different mtDNA mutations, in addition to its interaction with mitophagy and relevance to human diseases are discussed.Here, we took the first steps towards testing this hypothesis by crossing C. elegans strain uaDf5/+ harbors a mixture of intact (+mtDNA) and ~ 60% of a 3.1 kb mtDNA deletion (ΔmtDNA) [fzo-1, the C. elegans orthologue of MFN1/2, led to a slight reduction in the levels of the heteroplasmic ΔmtDNA, although without any phenotypic consequences [fzo-1(tm1133) deletion (hereafter designated as fzo-1(mut)) on the inheritance of the ΔmtDNA.The stable heteroplasmic (ΔmtDNA) . Althoug(ΔmtDNA) , 27, 28.(ΔmtDNA) . We show(ΔmtDNA) . In conjequences . We, theuaDf5/+ heteroplasmic hermaphrodites (+/ΔmtDNA) with fzo-1(mut) heterozygote males , 48.7% fzo-1 heterozygotes (ht), and 25.3% fzo-1 wild type (wt) ;+/ΔmtDNA worms hatched, as compared to fzo-1(mut) animals ;+/ΔmtDNA adults were similar to fzo-1(mut) ;+/ΔmtDNA animals were developmentally delayed, and none of them reached adulthood after six days as compared to fzo-1(mut) . This was in contrast to self-crossed fzo-1(wt);+/ΔmtDNA animals, all of which reached adulthood after six days , we monitored the development of progeny of the self-crossed fzo-1(ht);+/ΔmtDNA worms, followed by genotyping the resultant adult animals . We continued to follow the hatching and development of their progeny, i.e., fzo-1(mut);+/ΔmtDNA (G2m) and fzo-1(wt);+/ΔmtDNA (G2wt), across four generations , with 75% of the animals reaching adulthood only after 9 days. Surprisingly, the G3m animals showed significant improvement , with ~ 60% of this population reaching adulthood after six days. Moreover, G4m animals showed a full reversal of ΔmtDNA-associated adverse effects . The hatching percentage of the G4m generation was similar to that of G1 animals  and remained stable over subsequent generations  and ΔmtDNA were abrogated. We hypothesized that if the ΔmtDNA is not tolerated in the background of fzo-1(mut), then selection against the ΔmtDNA should occur. To test this prediction, we assessed the levels of ΔmtDNA by quantitative PCR  across the G1m-G4m generations (using gravid adults) in both the fzo-1(mut);+/ΔmtDNA and the fzo-1(wt);+/ΔmtDNA strains. We found that ΔmtDNA levels declined by 10-fold in the G2m fzo-1(mut) animals, as compared to +/ΔmtDNA parental strain . Values of ΔmtDNA reached below detection levels in most G3m (N = 18/21) and G4m (N = 20/21) animals ;+/ΔmtDNA animals  progeny (Gm→Gwt). Since traces of ΔmtDNA were neither detected in Gm->Gwt animals  and ΔmtDNA depend on the size or genomic location of mtDNA deletions. To achieve this goal, we characterized two additional mtDNA deletions obtained from the Million Mutation Project strain collection [C. elegans strains: bguDf1 (derived from strain VC40128), harboring a mixture of intact +mtDNA along with mtDNA molecules lacking ~ 1 kb (1kbΔmtDNA) encompassing two essential mtDNA genes  and three tRNAs ; the second strain, bguDf2 (derived from VC20469), harbors in addition to the +mtDNA, a ~4.2 kb mtDNA deletion (4kbΔmtDNA) encompassing four different essential genes  and five tRNAs . Notably, the levels of the 1kbΔmtDNA and 4kbΔmtDNA were stable over > 100 generations  in the presence of functional (wild type) fzo-1. Reanalysis of whole-genome sequencing data for the two mutant strains identified +mtDNA and deletion sequences, as previously described [uaDf5/+ [We next asked whether the deleterious interactions between llection . Specifiescribed , 30. The[uaDf5/+ , truncatfzo-1(mut) heterozygote males, followed by self-cross of F1 progeny ;+/1kbΔmtDNA did not deviate from the expected Mendelian ratio (23.5% homozygous fzo-1(mut), 43.2% fzo-1(ht) and 33.3% fzo-1(wt)  animals harboring +/4kbΔmtDNA (6.4% homozygous fzo-1(mut), 59.3% fzo-1(ht) and 34.3% fzo-1(wt)  differentially tolerates mtDNA deletions based on size and/or mtDNA position.Heteroplasmic hermaphrodites of both strains were separately crossed with fzo-1 progeny across four subsequent generations  animals harboring the 4kbΔmtDNA than in animals harboring either 1kbΔmtDNA or 3kbΔmtDNA in both G1m and G2m animals . It is worth noting that the levels of both types of ΔmtDNA did not significantly change across the G1wt-G4wt generations of the fzo-1(wt);+/ΔmtDNA animals  progeny (Gm→wt). qPCR analyses revealed no traces of the ΔmtDNA copies in subsequent generations  in association with oocyte production [C. elegans life cycle selection against ΔmtDNA occurred. Given that the relative levels of ΔmtDNA are maintained during normal development [P < 0.001; Fig. fzo-1(mut) worm development, in agreement with the observed adverse effect of heteroplasmy on the hatching and development of G2m animals.In oduction , 31. We,elopment , we compl stage L in assocl stage L in assocP < 0.05; Fig. To examine the possible association of ΔmtDNA levels with embryo lethality, we compared ΔmtDNA levels of unhatched embryos (unhatched > 48 h after being laid) to newly hatched larvae (L1). As expected, given that ~ 85% of G2 embryos did not hatch, ΔmtDNA levels of unhatched embryos were similar to the relative ΔmtDNA levels of newly laid embryos. In contrast, ΔmtDNA levels in L1 animals were reduced by 2-fold ;+/ΔmtDNA animals  (here named pdr-1(mut)) males and established a stable pdr-1(mut);+/ΔmtDNA strain. Consistent with previous findings, the ΔmtDNA levels were elevated in this strain (75%) [pdr-1 and fzo-1 in the context of +/ΔmtDNA heteroplasmy, we then crossed pdr-1(mut);+/ΔmtDNA heteroplasmic hermaphrodites with pdr-1;fzo-1 heterozygous males, let the F1 progeny self-cross, and isolated fzo-1(ht);pdr-1(mut) hermaphrodites that are harboring +/ΔmtDNA ;+/ΔmtDNA did not deviate from the expected Mendelian ratio, the genotype distribution of fzo-1(ht);pdr-1(mut);+/ΔmtDNA was strongly affected, as follows: 9% homozygous fzo-1(mut);pdr-1(mut), 55.8% fzo-1(ht);pdr-1(mut), and 35.2% fzo-1(wt);pdr-1(mut) ;pdr-1(mut) double mutant, as reflected in further reduction in fitness.Since Parkin mediates the turnover of mitofusins and hence impacts their activity , 34, we in 75%) –17. To e% –17. Tofzo-1(mut);pdr-1(mut);+/ΔmtDNA G1m, similar to the parental strain (11/11 were adults by day 7); and most of the G1m progeny (G2m eggs) hatched ;pdr-1(mut);+/ΔmtDNA hatching and development was unaffected  and ΔmtDNA on fecundity and developmental timing were aggravated by pdr-1(mut), supporting fzo-1-pdr-1 epistasis.We next monitored the development and fecundity of these animals. Phenotypic characterization revealed a developmental delay in the fzo-1(mut);pdr-1(mut);+/ΔmtDNA. To directly examine this, we compared the levels of ΔmtDNA molecules in fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals ;+/ΔmtDNA ;pdr-1(mut);+/ΔmtDNA as compared to the single mutant strain fzo-1(mut);+/ΔmtDNA. Specifically, we found lower ΔmtDNA levels in G1m and G2m fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals ;pdr-1(mut);+/ΔmtDNA animals ;pdr-1(mut);+/ΔmtDNA animals, where only one genomic copy of fzo-1 remained functional, ΔmtDNA levels declined and in some individuals were lost across ~ 25 generations ;pdr-1(mut);+/ΔmtDNA animals ;+/ΔmtDNA  animals ;pdr-1(mut);+/ΔmtDNA animals displayed similar mtDNA levels in the gonad compared to soma, for both truncated and intact mtDNA molecules ;pdr-1(mut);+/ΔmtDNA double mutant displayed a reduction in total mtDNA levels, again supporting epistasis. Taken together, these data suggest that disrupting parkin-mediated mitophagy increased the organismal selection in fzo-1(mut);pdr-1(mut);+/ΔmtDNA individuals, which is associated with reduced fitness.Finally, we asked whether impaired mitophagy affects selection against ΔmtDNA in the worm germline. To this end, we examined the ratio of ΔmtDNA and +mtDNA between the gonad and soma in double mutant als Fig. E, F and DNA Fig. G, H. Whifzo-1 mutation impacted the developmental pace of the worms [Our cross-generational analyses revealed complete loss of heteroplasmic deleterious mtDNA deletions when mitochondrial fusion is compromised. This demonstrated that heteroplasmy of deleterious mtDNA molecules could not be tolerated unless in the presence of a functional compensatory mechanism inherent to the mitochondrial network and the mitochondrial quality control machinery , 22, 23.he worms –37. NeveDrosophila, germline selection of heteroplasmic mutations was directly observed in response to compromised fusion and quality control machinery [fzo-1(mut);pdr-1(mut)) in the presence of ΔmtDNA sharply increased lethality already in G1 animals (reflected by the deviation from Mendelian ratios). Likewise, we found that disrupting Parkin-mediated mitophagy did not block germline selection, affecting the specific removal of mtDNA. These findings agree with germline selection in Drosophila, where downregulation of mitophagy factors, BNIP3 and Atg1, significantly blocked selection against fragmented mitochondria in germline cysts [In parallel, our discovery that the levels of heteroplasmic deletions are specifically reduced in the gonad when the fusion machinery is impaired Fig. B is consachinery , 21, 22.fzo-1(mut) animals. Firstly, increased apoptosis was observed upon disruption of mitochondrial transition from a globular to a tubular organization in C. elegans oogenesis [fzo-1(mut) animals  patients, will affect patterns of heteroplasmy. Our deep mtDNA sequencing analysis of three CMT2A pedigrees lends first clues that this might be the case . WhereasC. elegans generations underlines the fusion machinery as an attractive candidate target for future treatment of mitochondrial disorders. For example, the activity of protein quality control systems, including this machinery, declines during the aging of the individual [The complete loss of ΔmtDNA across dividual , 48, anddividual . It woulC. elegans, we demonstrated that fzo-1 (mitofusin) is a key modulator of mtDNA heteroplasmy while showing its impact on the transmission of heteroplasmic deletions across generations in living animals. Firstly, we discovered that a fzo-1(mut) led to the complete loss of three different mtDNA deletions, separately. These findings provide experimental support for the hypothesis that functional complementation among mitochondria in the intracellular network likely enables the survival and prevalence of deleterious heteroplasmic mtDNA mutations in the population [fzo-1(mut) was differentially sensitive to the size/nucleotide positions of the different mtDNA deletions. Third, fzo-1(mut);pdr-1(mut) double mutants selected against the survival of animals with heteroplasmic ΔmtDNA deletion and accelerated the loss of ΔmtDNA molecules across generations. Taken together, our results demonstrate the importance of cross-talk between mitochondrial fusion and the mitochondrial quality control machinery in protecting living animals from the adverse impact of inheriting deleterious mtDNA molecules.Here, by manipulating the fusion machinery in pulation , 26. SecEscherichia coli OP50-1 strain at 15 °C.A list of strains used in this work and name abbreviations are found in Additional file fzo-1(mut) ;pdr-1(mut);  animals, we used one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test. We used the same test to compare the levels of ΔmtDNA, +mtDNA, and TMRE staining in fzo-1(mut) and fzo-1(wt) strains  and individuals (n) in each condition tested are noted in the figure legends  Fig. C or fzo-nt Figs. B or wildant Fig. C, we usefzo-1 or pdr-1 deletions. The list of PCR primers is found in Additional file Animal genotype was determined using a single worm PCR Phire Animal Tissue Direct PCR Kit (Thermo Scientific) with primers to detect fzo-1(tm1133) animals (strain CU5991) are very poor in mating and, therefore, were first crossed with males expressing a yellow fluorescent protein marker (unc-54p::YFP). Heteroplasmic hermaphrodites  were then crossed with fzo-1(tm1133);unc-54p::YFP heterozygote males to ensure maternal inheritance of mtDNA deletions  animals that were heterozygotes for fzo-1(tm1133), fzo-1(ht), were identified and maintained using single worm genotyping, establishing independent heteroplasmic lines carrying fzo-1(ht).Mutant pdr-1(gk448) animals (strain VC1024) were first crossed with heteroplasmic hermaphrodites (uaDf5/+) and with fzo-1(tm1133) to establish double mutant strains. fzo-1(tm1133);pdr-1(gk448) double mutants were crossed with unc-54p::YFP (marker); then, the F1 heterozygote males, fzo-1(tm1133);pdr-1(gk448);unc-54p::YFP, were crossed with heteroplasmic hermaphrodites, ΔmtDNA;pdr-1(gk448) to ensure maternal inheritance of uaDf5  animals that were homozygous to pdr-1(gk448), pdr-1(mut), and heterozygotes for fzo-1(tm1133), were identified using PCR genotyping. fzo-1(ht) were then maintained using single worm genotyping to establish a heteroplasmic line carrying fzo-1(ht);pdr-1(mut).Mutant ites uaDf/+ and wiuaDf5/+, bguDf1/+, or bguDf2/+) were isolated, allowed to lay eggs, and genotyped using a single worm PCR for fzo-1. The progeny of fzo-1(ht) was again isolated, allowed to lay eggs, and screened to identify mutant or wild type fzo-1 animals . Heterozygous progeny was maintained to generate G1. The progeny of mutant or wild type animals  was then monitored and/or isolated and allowed to lay eggs. This was repeated over several generations , G4m hermaphrodites were crossed with wild type males; the progeny was allowed to self-propagate, isolated, allowed to lay eggs, and genotyped. Heteroplasmy levels in fzo-1(wt) animals (Gm→Gwt) were then examined.To test for residual ΔmtDNA (N) and individuals examined (n) in each condition tested are noted in the figure legends  and individuals examined (n) in each condition tested are noted in the Figure legends  or 100 μM TMRE  (Biotum). The animals were kept on the plates for 24 h in the dark and then recovered on regular plates for 2 h. Animals were then fixed with 4% paraformaldehyde and imaged using a LEICA DM5500 B epifluorescence microscope. MitoTracker was imaged using a × 40 or a × 60 numerical aperture objective with a 633-nm line for excitation. TMRE was imaged using a × 10 numerical aperture objective with a 549-nm line for excitation. TMRE staining was quantified using CellProfiler cell image analysis software. The numbers of individuals examined (n) in each condition tested are noted in the figure legend of Additional file Age-synchronized adults were placed on NGM plates seeded with the Total DNA was extracted using a QuickExtract kit (Lucigen). Unless otherwise indicated, DNA was extracted from a single worm. When populations were examined, ~ 5 animals were collected. For embryos, DNA was extracted from 15 to 30 embryos. For gonad-soma analysis, gonads were dissected from 5 to 10 animals per biological repeat. DNA was extracted separately from the gonads and soma.CT (threshold cycle) of triplicate values obtained for these mtDNA molecules was normalized to a nuclear DNA marker using the 2−ΔΔCT method [CT values of truncated to total mtDNA for a given animal or strain. The numbers of biological repeats (N) and individuals examined (n) in each condition tested are noted in the figure legends  with KAPA SYBRFAST qPCR Master Mix (KAPA Biosystems). Analysis of the results was performed using CFX Manager software (Bio-Rad). To quantify the different mtDNA molecules, three sets of primers were used for truncated (ΔmtDNA), intact (+mtDNA), and total mtDNA molecules for each of the three deletions examined  database, corresponding to two of the strains used in this study: SRR801606 - SRR801609 for strain VC40128, and SRR793379 - SRR793382 for strain VC20469 . Reads wG2 wild type or mutant animals were placed in a drop of ultra-pure water on a coverslip slide, and a 25-gauge needle was used to remove the gonads from the body of the animals. Gonads or the remaining carcasses were then transferred to a DNA extraction buffer.Additional file 1: Table S1. Genotypes distribution of F2 progeny in different heteroplasmic strains. Table S2. Cox proportional-hazards regression analyses. Table S3. Fractional regression analyses. Table S4. A list of C. elegans strains used in this study. Table S5. A list of primers used in this study. Figure S1. Characterization of +/ΔmtDNA animals. Figure S2. Characterization of fzo-1(wt);+/ΔmtDNA; animals. Figure S3. Characterization of the 1kbΔmtDNA and 4kbΔmtDNA animals. Figure S4. Characterization of fzo-1(wt);pdr-1(mut);+/ΔmtDNA animals.Additional file 2. Individual data values. Spreadsheets of numerical data for Figures


In Japan, a significant number of adolescent females noted unusual symptoms after receiving the human papillomavirus (HPV) vaccination, of which the vast majority of them were initially diagnosed with psychiatric illnesses because of the absence of pathologic radiological images and specific abnormalities in laboratory test results. Later these symptoms were thought to be adverse effects of HPV vaccination. However, a causal link between HPV vaccination and the development of these symptoms has not been demonstrated. Between June 2013 and March 2021, we examined 200 patients who noted various symptoms after HPV vaccination. In total, 87 were diagnosed with HPV vaccination-related symptoms based on our proposed diagnostic criteria. The clinical histories of these 87 patients were analyzed. The age at initial vaccination ranged from 11 to 19 years old (mean ± SD: 13.5 ± 1.5 years old), and the age at the first appearance of symptoms ranged from 12 to 20 years old (mean ± SD: 14.3 ± 1.6 years old). The patients received an initial HPV vaccine injection between May 2010 and May 2013, but the first affected patient developed symptoms in October 2010, and the last affected developed symptoms in October 2015. A cluster of patients with a post-HPV vaccination disorder has not appeared in Japan during the last five years. Our study shows that, in Japan, the period of HPV vaccination considerably overlapped with that of a unique post-HPV vaccination disorder development. This disorder appears as a combination of orthostatic intolerance, chronic regional pain syndrome, and cognitive dysfunction, but its exact pathogenesis remains unclear. Therefoeenagers ,3. Begineenagers . SimultaIn our previous two reports ,6, we deBetween June 2013 and March 2021, we examined the symptoms and objective findings of 200 HPV vaccinated female patients. According to our proposed diagnostic criteria , we obtaDuring the past eight years, 200 female patients visited our hospital with the suspicion of HPV vaccine-related adverse effects . Of these, we excluded 19 patients who had symptoms before vaccination and 5 who received the HPV vaccine after 30 years of age. An additional 28 patients whose symptoms or disorders were explained by known diseases or who had abnormal laboratory data were also excluded, specifically, eight with epilepsy, six with psychiatric or anxiety disorders, three with systemic lupus erythematosus, one with juvenile idiopathic arthritis, one with anti-SGPS antibody-positive polymyositis, and nine with other diseases. For the remaining 148 patients, the clinical manifestations and objective findings were analyzed. The results showed that 32 patients were diagnosed with definite vaccine-related symptoms, and 55 were diagnosed with probable vaccine-related symptoms. The patient’s symptoms and signs of the 87 patients diagnosed are summarized in The temporal distribution of the period of initial vaccination and the appearance of the first symptom in the diagnosed 87 patients is shown in The temporal relationship between the HPV vaccination and the development of the symptoms was as follows: the first HPV vaccine injection was in May 2010 and the last was in May 2013 a. The fiHPV vaccine safety has been reported in HPV vaccination-predominant countries ,11,12. EAccording to the reports of a Japanese special committee ,22, 3.39Variable clinical manifestations in the post-HPV vaccination disorders can be explained by a combination of orthostatic dysregulation, mainly appearing as POTS, CRPS, and/or cognitive dysfunction ,6,23. ReIn our previous report , we descNevertheless, while there is a possible occurrence of adverse effects after HPV vaccination, these results do not necessarily signal the negation of the usefulness of this vaccine for the prevention of uterine cervical cancer . If the


Genotypes were analyzed for18 high-risk HPV (hrHPV) and 6low-risk HPV(lrHPV) types. A total of 1075 women were included with a mean age of 49.1±10.5 years. HPV prevalence was 27.6% (297/1075) with 19.4% (209/1075) women with only hrHPV, 5.3% (57/1075) with only lrHPV. Multiple infections (hrHPV/lrHPV) were detected in 31/240 cases of hrHPV (12.9%). A total of 353 hrHPV genotypes were analyzed; the most common HPV types were HPV51 (11.0%), HPV68 (10.8%), HPV53 (9.1%) and HPV 52 (7.1%). HPV16 and HPV18 represented respectively 4.8% and 4.0% of hrHPV genotypes. Abnormal cytology was observed in 34 cases (3.2%), with 14 ASCUS (1.3%), 10 LSIL (0.9%), 5 HSIL (0.5%), 3 ASC-H (0.3%) and 2 AGC (0.2%). Fifteen (44.1%) were hrHPV and 4 (14.7%) lrHPV; 7 cases of hrPHV were in the age-group 25–34 years. Among 1041cases of normal cytology, 225 had positive hrHPV detection (21.6%). This is the first population-based study of HPV profiles in our country, and we found a high prevalence of hrHPV. The most common genotypes were HPV51, 68, 53. These results could serve for cancer vaccination strategies and HPV surveillance in Martinique.The Caribbean ranks seventh among the world regions most affected by cervical cancer. HPV-prevalence and genotype distributions also differ from regions. Knowledge of HPV genotype profiles is important for patients care and HPV vaccination implementation. The objective of this study was to describe HPV genotype distribution and risk factors in a population-based cohort of women in Martinique. In this study, 1312 women were included and underwent cervical cancer screening with successful sample collection between 2009 and 2014. Sociodemographic and clinical variables were recorded. Cytological examination of cervical vaginal smear was performed and classified(Bethesda). Detection of HPV DNA was performed with the PapilloCheck Cervical cancer is a global public health problem caused by persistent human papillomaviruses (HPV) infection leading to malignancy. In women, it ranks fourth in terms of cancer incidence and mortality worldwide, with respectively rates of 13.1 and 6.9 per 100 000 women. In the Caribbean, cervical cancer ranks in third position in terms of incidence and mortality (15.5/100 000 women and 8.5/100 000 women).In France, cervical cancer is ranked twelfth in terms of incidence (6.7/100 000 women) and tenth (2.3/100 000) in terms of mortality [Martinique is a French department; in total, there were 380,877 inhabitants in Martinique as of 1 January 2015, and 3,057 deaths were recorded that year, with a rising trend since 1990. The mortality rate increased from 6.9 per thousand in 2000 to 8.1 per thousand in 2015, largely related to population aging .The natural history of cervical cancer develops over a period of 15 to 20 years, along a continuum of pre-cancerous histological lesions  that result from persisting infection with high-oncogenic-risk serotypes of the human papillomavirus (HPV). HPV infection is generally transitory and the majority of low grade lesions associated with infection heal spontaneously and do not systematically progress towards the stage of pre-cancerous or invasive lesions. In France, cervical cancer screening primarily consists in regular and recommended  screening by smear test among women aged 25 to 65 years on a three-yearly schedule, after 2 normal smear tests performed at one year’s interval. If the smear test result shows Atypical Squamous Cell of Unknowned Significance (ASC-US), it is recommended to search for high-risk HPV (hrHPV) types.When considering cancer prevention strategies, knowledge of HPV genotype profiles is important for HPV vaccination implementation and screening of cervical cancer. There is a huge need to get access on HPV genotype distribution in Martinique, due to the higher incidence of cervical cancer in our region. To perform a monitoring of the HPV evolution in the next years, we need to first describe the epidemiological context in Martinique, to better improve the cervical screening and the HPV vaccination in the future. The objective of this study was thus to describe HPV genotype distribution in a cohort of women in Martinique. We also studied clearance of HPV infection among women who presented normal cervical cytology and performed follow-up to assess outcomes developed cancer in situ or invasive cervical cancer.We performed a prospective study among women aged 25 to 65 years who had complementary health insurance with a single company (Mutuelle UFR), were resident in Martinique, and underwent a smear test for cervical cancer screening. Women who were included had not received HPV vaccination. The exclusion criteria were a history of total hysterectomy, or a history of cervical lesions of grade CIN2 or higher. Our study was based on a sample of 14,333 women who were invited to participate to this study in Martinique.Training and information sessions were organised for general practitioners, specialists and midwives regarding the use of liquid medium for combined screening. Women participating in the study could attend the physician of their choice to undergo screening, as long as the physician was participating in the study. Information leaflets provided details of cervical cancer screening, the study procedures, follow-up after the smear test and HPV test performed, and an informed consent document was also included, to receive a collection kit with the liquid medium vial.The screening strategy comprised both a smear test and a HPV test. Invitations were sent out from July 2009 to February 2011 with reminders by the insurance company to all its eligible members. Women aged 25 to 65 were invited to participate in age groups, starting with women aged 50 to 65, then women aged 40 to 49, then 25 to 39 years. Women were included between 1stJuly 2009 and 28thFebruary 2014. The validated liquid media chosen for this study were NovaPrep (NovaCyt France) and Easyfix (Labonord).Samples for cytological analysis of smear tests were prepared using the automated NOVACYT system. The interpretation of smear test results was done according to the 2014 Bethesda classification .Samples were classified as atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), or high-grade squamous intraepithelial lesion (HSIL), Atypical Squamous Cell evocating High grade lesion (ASC-H), Atypical Glandular Cells (AGC). In case of abnormal cytology, a histological sample was obtained by colposcopy-directed biopsy or endocervical curettage. Confirmation of pre-cancerous lesions of the cervix and invasive cancers was done by histological examination of biopsy samples. Pathology charts were analysed using the electronic database DIAMIC software.© Kit by the Virology Laboratory of the University Hospital of Martinique. Genotypes were analyzed for high-risk HPV (hrHPV) and low-risk HPV (lrHPV). The PapilloCheck® test allows the identification of DNA from eighteen hrHPV types, namely: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 and six lrHPV: types 6, 11, 40, 42, 43, 44/55. Genotypes were also classified according to the IARC Monograph Working Group in Alpha HPV types [Extraction of HPV DNA was performed using Easymag from Biomérieux. Detection of HPV DNA was performed with the PapilloCheckAppropriate management was provided for women in whom anomalies were found. French recommendations regarding the management of patients with abnormal smear test are those of the French national cancer institute (INCa) .For positive HPV tests, a one-year follow-up was performed with both smear test and HPV test. If both tests were negative, a smear test was performed at 3 years. If the smear test was still negative with a positive HPV test, a one-year follow-up was performed once again. If both the smear and HPV tests were positive, a colposcopy was performed. If the colposcopy was negative, a one-year follow-up was performed. Follow-up of the women in the study made it possible to identify cases of cancer recorded in the database of the General Cancer Registry of Martinique. Cancer cases occurring between 2009 and 2016 were identified after data extraction.The prevalence rate of HPV infection in the cohort was calculated with 95% confidence intervals (CIs). Prevalence was obtained by dividing the number of women with normal cytology infected by at least one HPV genotype by the total number of women. Age is described using the mean, median and interquartile range (Q1–Q3). Stratification according to age was performed within each age group: 25–34, 35–44, 45–54, 55–64, and 65 years or more.Sociodemographic and clinical variables were recorded, namely marital status, profession, number of children and age at first intercourse (<18year or ≥ 18year). Socio-professional categories were noted in four groups: group 1 ; group 2 ; group 3 (i.e. retired people) and group 4 . Analyses were performed using SAS version 9.4 . Results are presented as mean ± standard deviation or number and percentage. Cross-tabulations were analyzed by Chi-square test or Fisher’s exact test as appropriate. Comparisons of prevalence rates with other countries were based on p-values. For all analyses, two-sided p-values of less than 0.05 were considered statistically significant.The study was approved by the Ethics Committee (CPP Bordeaux) on 25 March 2009 under the number 2008-A01658-47. All women provided written informed consent.Overall, 1312 women were included and underwent cervical cancer screening with successful sample collection. A total of 237 women were excluded . HPV prevalence was 27.6% (297/1075) with a total of 240 (22.3%) women with at least one hrHPV genotype detected. A total of 19.4% (209/1075) of women had hrHPV, 5.3% (57/1075) having only lrHPV, and multiple infections (hrHPV/lrHPV) detected in 31/240 cases of hrHPV (12.9%). Among these, 164 had a single viral genotype (68.3%), 51 (21.2%) had 2 genotypes identified, and 25 (10.4%) had more than 2 types of hrHPV identified.Socio-demographic characteristics of the study population are presented in A total of 449 HPV genotypes were analyzed and 353 were hrHPV. We analysed the number of co-infections with both hrHPV and lrHPV genotypes; the results are shown in In total, 31/297 women were co-infected with at least one hrHPV and one lrHPV genotype, corresponding to 10.4% of all women with HPV-positive results.Among the 1,075 participants, 1,041 (96.8%) had normal cytology, and 225 had positive hrHPV detection (21.6%). Abnormal cytology was observed in 34 cases (3.2%), with 14 ASCUS (1.3%), 10 LSIL (0.9%), 5 HSIL (0.5%), 3 ASC-H (0.3%) and 2 AGC (0.2%). Fifteen (44.1%) were hrHPV and 4 (14.7%) lrHPV; 7 cases of hrPHV were in the 25-34-year age-group.Among these women, the genotypes detected in the abnormal cytology samples are detailed in Among the 225 women with normal cytology and positive hrHPV test, only 107 attended the study follow-up.Of these 107 women with an initial diagnosis of hrHPV, 68 (63.5%) lost at least one hrHPV genotype (50 lost one and 18 lost 2 or more), while 13/107 (12.2%) acquired at least one new hrHPV genotype. The average time to follow-up visit for HPV 18, 35 and 52 genotypes was 3.5±1.6 years, while the average time to follow-up for all 107 women was 3.2±1.6 years.Among the 1,075 women in the study cohort, one woman developed invasive cervical cancer, and 7 women had CIN3. The characteristics of these 8 women are presented in Few studies are available in the French West-indies on the distribution of genotypes in cervical malignancy, and this is the first study performed in Martinique in a general population-based setting. The novelty of this study is that it provides, population-based data about HPV genotype profiles in a French region of the Caribbean where there is an excess of cervical cancer.In this study, a total of 1075 women were included, and HPV prevalence was 27.6%. The most common HPV types were HPV51 (11.0%), HPV68 (10.8%), HPV53 (9.1%) and HPV 52 (7.1%), while HPV16 and HPV 18 represented respectively 4.8% and 4.0% of hrHPV genotypes.There is wide disparity between studies of HPV genotypes in the Caribbean, with most being carried out in samples and/or cohorts in the hospital setting. Few data are available at population level. Epidemiological studies have been performed in French Guiana –10 and sA recent population-based study was performed in Curacao on 1075 women. Overall HPV prevalence was 19.7%. The most frequent genotypes were HPV16 (2.3%), 35 (2.1%) and 52 (1.8%); a total of 27 (2.6%) women were detected with abnormal cytology .In our study, 1,041 women (96.8%) had normal cytology, and among these, 225 had positive hrHPV detection (21.6%); abnormal cytology was observed in 34 cases (3.2%).A cross-sectional study performed in Jamaica in 2010 among 852 women reported a HPV prevalence of 34.9% (297/852). This study showed that HPV 16 or 18 was detected in 10.1% (86/852) of women. Among the 75 women with abnormal cytology results, the majority (84.0%) were HPV-positive. The HPV types were 16 (6.2%), 35 (6.0%), 58 (5.4%), 18 (4.3%),and 66 (4.2%) .Another study was designed in 2014 to characterize high-risk human papillomavirus (HPV) infections in women in Saint Kitts and Nevis. High-risk HPV was detected in 102 of 404 (25.2%) in Saint Kitts and Nevis. HrHPV genotypes 52 (14.7%), 35 13.7%), 51 (10.8%), 45 (10.8%), and 31 (10.8%) were the most common high-risk types. The majority of women in this study (94%) in Saint Kitts and Nevis who were infected with high-risk HPV had normal cytology [.7%, 51  was developed to expand coverage of the previous quadrivalent (HPV6/11/16/18) vaccine.According to data on HPV genotype distribution of our study, the percentage of HPV infections which can be prevented by using a 9-valent vaccine was 47.9%. The distribution of genotypes observed in our study provides new insights into the circulating genotypes in Martinique. Among studies published heretofore from Martinique, none had provided epidemiological data at population level. Given the selection of our participants from among the clientele of a large health insurance company, it is likely that our results are not representative of the complete genotype distribution among the population of women with complementary health insurance in Martinique, due to the size of our study; this could generate a selection-bias in our study. Our results show that the distribution of genotypes in Martinique is different to that habitually observed in Europe. Of particular note is the fact that HPV 16 and 18 were not the most prevalent high-risk types in our study.The estimated crude HPV prevalence among women with normal cytological findings worldwide was 7.2%, with the highest prevalence observed in Latin America and the Caribbean (16.1%) . In our We studied clearance of HPV infection among the 107 women who presented normal cytology but a hrHPV-positive test. Of these 107 women who initially had hrHPV detected, 12.2% acquired at least one new HPV genotype. The study of virus clearance is key to understanding the mechanisms mediating the development of cervical cancer after HPV infection. Our results showed the utility of implementing genotyping for the detection and monitoring of HPV infection in our population.About 90% of HPV infections are no longer detectable after 2 years. If the HPV infection persists, it can cause cervical cancer. The persistence of a high-risk HPV infection is a necessary risk factor but other co-factors  would also play a role in the genesis and development of cancer . The novData from the Martinique Cancer registry showed that only one cervical cancer occurred with initial hrHPV 51 and 66 in our study.Nonetheless, one must take into account the detection capacity of the tests, which may lead to under- or over-estimation of the prevalence of different HPV genotypes. The variability between test kits is an important point to keep in mind when comparing results between studies.Regarding cytology, the techniques used (slides or liquid medium) may yield different results. Indeed, each commercial kit is different and sensitivity varies. Thanks to the present study, a circuit for diagnosis based on liquid medium smear test with biomolecular HPV testing was put in place, simplifying the healthcare pathway of women undergoing screening. Indeed, up to the time when this study was performed, samples were sent to mainland France for analysis.During the period from 2015 to 2017, the standardized national coverage for 3-yearly cervical cancer screening was 58.7%. Data by geographical area reveal marked disparities, with the lowest regional coverage rates observed in Guadeloupe, French Guiana, Martinique and Mayotte (<50%) [In terms of future perspectives, analysis of HPV genotype data should make it possible in the future to cross-reference with population data such as the cancer registry database, or the social security system, to assess the healthcare pathway of women with cervical cancer. Our study does not make it possible to investigate differences that may exist in access to care or socio-economic status. Our study is useful to understand the factors explaining differences in genotype prevalence according to age and socio-economic status.In the literature, some studies showed that there was a link between HPV infection and socioeconomic position ,17; in oThe next steps are to provide sufficient data to get an overview of the assessment of HPV tests in clinical practice. In fact, since this study has been performed, the French National Health Authorities developed new algorithms for cervical cancer screening including HPV test ; it is rThere is wide variation between studies of HPV genotypes in the Caribbean, underlining the compelling need for multicentre comparative epidemiological studies in our region. This analysis of HPV profiles showed that the most frequent HPV genotypes were HPV51, 68, 53, namely different from those most commonly found elsewhere . HPV vaccination, in addition to screening by smear test, is the most effective means of preventing cancerous lesions of the cervix. Given the specificities of HPV distribution in Martinique, the implementation of a surveillance programme for HPV infection is a key public health issue for our region. Finally, these preliminary findings, from a cohort of 1075 patients, underline the necessity of accounting for heterogeneity in genotypic profiles in the surveillance and prevention of HPV-related infection in the Caribbean.


Detection and genotyping for high-risk HPV were done by an automated system (Abbott® m2000sp). Six percent of MSM in this cohort had high-risk HPV present in the mouth/oropharynx. This cohort averages 29 years of age, more than half were unemployed (53.3%), and 66.8% were human immunodeficiency virus (HIV) seropositive. The most common sexual practice was anal sex (69.4%) followed by oral sex (28.6%), and by rimming (9.6%). A significant association between oral insertive sex and oral/oropharyngeal HPV status was demonstrated . An incidental but significant association between rimming and HIV status was found , and HIV seropositive participants had higher oral/oropharyngeal HPV presence. The HPV prevalence of 6% reported in this study is in alignment with global reports. The prevalence of oral/oropharyngeal HPV in this MSM cohort was influenced by sexual practices. MSM participants who practiced rimming appear to be at higher risk of HIV acquisition. Given the transmission routes of HPV in this vulnerable population, vaccination must be urgently studied as an intervention for prevention.Data lag is evident when observing studies focussing on human papillomavirus (HPV) prevalence in the head and neck of men who have sex with men (MSM) in Southern Africa. Sexual behaviours other than anal intercourse, and associated factors are similarly underreported. HPV vaccination has not yet commenced for this population group. One hundred and ninety-nine MSM were enrolled in this study. Participants completed a questionnaire followed by a clinical oral examination, and a rinse-and-gargle specimen in Thinprep The virus is responsible for a variety of benign lesions, potentially malignant lesions, and malignancies in the form of squamous cell carcinoma (SCC) of different anatomic sites lined by mucosa Information Centre on HPV and Cancer (ICO) ReportStudies on head and neck HPV infection involving MSM populations are very limited. The 2019 MSM are a high-risk vulnerable population and are more prone to acquiring oral/oropharyngeal HPV infection due to the practice of high-risk sexual behaviours. These individuals are also at a higher risk to acquire HIV infection. This study investigated the presence of oral/oropharyngeal HPV in a group of 199 MSM and forms an important baseline contribution to the Southern-African database.2.This cross-sectional descriptive study was performed in the Clinical Research Unit (MeCRU) and Department of Virology at Sefako Makgatho Health Sciences University in Pretoria, South Africa. This unit falls within a 28 km radius of four townships . The villages of Winderveldt and Mmakau, and the North-Western suburbs of Pretoria form part of the catchment area served by the unit.2.1.Men who self-declared to have sex with other men, who were 18 years or older, resided in targeted areas, and who consented to participate in this study were included. A recruitment team comprising of a community liaison officer (CLO) and 2 other recruiters was formed. During the initial phase of the study they established links with community based organisations to discuss and educate communities about the realities that needs to be faced concerning the MSM population. Local health care facilities were consulted to reinforce the links that the recruitment team had already established. Consultations with other men's health organizations, enabled training of the health care workers at the facilities to educate and equip them, to create a MSM-safe and friendly environment at the health care facility. Contact with the MSM occurred in environments where they felt comfortable to interact with staff/data collection teams.2.2.Participants received an information leaflet upon arrival at the research unit. Thereafter a group discussion took place and informed consent was obtained. Participants completed a self-administered questionnaire. Demographic data were collected followed by intra-oral examination and rinse specimen collection. Current sex practice was defined as any of the described forms of sexual contact having occurred in the 6 months leading up to participant enrolment. The examination was performed in a fluorescent lighted room with a LED headlamp using standard infection control procedures. Patients were referred to the dental hospital for diagnosis and management of any abnormalities seen during their examination.® vial containing the Preservcyt® solution. Rinse specimens were then delivered to the laboratory on the same day for processing which was uneventful. The specimens were processed according to standard operating procedures of the Department of Virology, Health Science Laboratory, Sefako Makagatho Health Sciences University.After the clinical oral examination, participants rinsed their mouth with saline for 15 seconds and gargled for another 15 seconds, then spit the contents into a Thinprep2.3.High-risk (HR)-HPV detection and genotyping was done using the Abbott® m2000TM RealTime system which is a qualitative assay used to detect 14 HR-HPV types , and to genotype HPV 16 and HPV 18. This real-time, multiplex PCR detection kit was designed to provide clinically meaningful results through the multiplex PCR, and through the clinically-based assay cut off. The assay is clinically optimised for cervical cancer screening and also optimized for high reliability through the probe specificity design and Human-Beta Globin internal control. The clinically-based assay cut-off is utilized in this study for the detection of HR-HPV in oral washes.2.4.It was calculated that at least 123 MSM should be recruited for an expected frequency of 25% MSM in the population and taking into account globally and locally reported prevalence rates for oral/oropharyngeal HPV . A sample size of 200 participants was finally decided on, but one participant was deemed a screen failure. The screen failure was defined as an enrolled participant that subsequently withdrew consent for sample collection.® Excel® database. Laboratory results were recorded on a data collection sheet and included data on the detection of the 14 HR-HPV types. Data from the intra-oral examinations were noted in each participant's file and entered into the spreadsheet.The data were extracted from the questionnaire to protect the data integrity and anonymity and entered into a Microsoft® (version 9.4 for Windows®) and the 5% significance level was used.Descriptive data were summarized as categorical values by frequency and percentage tabulation. Continuous variables were summarized by the mean, standard deviation where applicable and median. The chi-squared test was used to assess the relationship between HPV status and race, HIV status, current sex practices, number of partners and Socio-economic status. The relationship between HPV status and age was assessed by the Wilcoxon rank sum test, since the data did not meet the assumption of the independent samples t-test. The strength of associations was measured by the Cohen's d for parametric tests and the r-value for the non-parametric tests and reported where applicable. Data analysis was carried out using SAS2.5.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent was informed and obtained in writing from all individual participants included in this study.This project was specifically approved by the Sefako Makgatho Health Sciences University Research Ethics Committee, clearance number SMUREC/D/300/2016.3.Twelve participants (6%) had detectable oral/oropharyngeal HR-HPV, with 1 of these being HR-HPV-16. HIV serological tests revealed that 66.8% of the participants were infected with HIV and of these 10 (7.5%) were HPV positive compared to only 2 (3.1%) of those who were HIV negative. The majority (105) of the participants were unemployed (53.3%) compared to 36.0% who were employed and there was a number of students enrolled in the study (10.7%). The mean age of participants was 29 years and the median age 27 years . The majority of participants where younger than 35 years of age and there was a decline in the age demographic just after 27 years of age. Most of the participants self-identified as black (98.5%) and the remainder as dual heritage (1.5%). Of those who identified themselves as black 6.2% were HPV positive while none of the dual heritage individuals had HPV infection . One parThe mean age in years of the oral HPV-positive participants was 30.3; the standard deviation was 6.2. The median age for oral HPV positive participants was 29. The age range between the oral HPV positive and negative was the same; positive being between 20–42 years and negative being between 18–47 years .The most commonly reported ‘current sex practice’ was anal sex (69.4%) followed by oral sex (28.6%), and by rimming (9.6%) . FourteeMany participants (80.9%) have practiced oral receptive sex (participant's mouth), 60.8% have performed oral insertive sex (partner's mouth), while 52.8% have performed both. There has been a significant association between oral insertive sex and oral/oropharyngeal HPV status . Of those who have ever practiced oral insertive sex, 10.0% were HPV-positive (n = 12), compared to 0.0% of those who have never practiced oral insertive sex specifically (n = 78) . These dThe results further indicate that more participants (46.2%) performed rimming bottom (having anus licked by partner) compared to rimming top (licking a partner's anus) (19.6%).A significant association between rimming and HIV status  was observed. Of those who practiced rimming in the preceding 6 months, 94.7% were HIV-positive, compared to 63.9% HIV positivity in those who did not practice rimming (n = 180) during the same time .None of the study participants presented with any intra-oral HPV-associated or HPV-like lesions when examined. Other intra-oral abnormalities (non-HPV related) were observed in 26.1% (n = 52) of the participants and recorded as incidental findings. Within this group of 52 participants, the most commonly recorded oral/dental abnormality was dental caries (51.9%).4.Studies on HPV in MSM populations are generally limited to vaccination studies that focus on anal and/or penile infections as measurements of outcomes. Six percent of the 199 participants presented in this study had oral/oropharyngeal HR-HPV, and four of these were positive for HPV-31, and one for HPV-16. General reports on HPV detection in the mouth and oropharynx of South African populations and on oral/oropharyngeal tissue specimens are very limited and are reflected in This study's prevalence of 6.1% falls within the lower end of the global prevalence range and is lower than the only other South African study, the Cape Town study focussed on MSM which was at 11.5% Individual studies report a varying range of oral/oropharyngeal prevalence rates: In the UK King and colleagues Data on the oral lesions or oral abnormalities found in the population described as MSM is sparse This study found a much younger median participant age (27 years) than that of the Cape Town study (32 years) This report's MSM cohort mostly self-identified as black (98.5%). The Cape Town study In this study, 66.8% of the MSM participants were HIV seropositive and this group showed an HPV prevalence of 7.5% (n = 10) . Oral/orOral HPV has been reported with higher frequency in HIV-positive MSM greater than 40 years of age. A comparative study shows the majority of participants as Caucasian 96.5% with a median age of 39 years for HIV negative participants, and 44 years for HIV-positive participants An incidental finding from this study was that rimming was significantly associated with HIV-positivity. This could suggest that in this MSM population group, rimming is a risk factor to acquire HIV, and it is recommended that patients who identify as MSM, who have HPV infection, and report the practice of rimming, should be screened for HIV infection. The Cape Town study Despite the literature demonstrating an association between oral and oropharyngeal HPV infection and higher numbers of genital or oral sex partners Infection by HPV is associated with an increased risk for HIV acquisition One meta-analysis showing a higher oral/oropharyngeal HPV prevalence in HIV-positive than in HIV-negative MSM also revealed a pooled prevalence for HPV 16 of 3.0% in HIV-negative MSM and 4.7% in HIV-positive MSM; a pooled prevalence of all HR-HPV types of 9.1% in HIV-negative MSM and 16.5% in HIV-positive MSM; and a pooled prevalence for the presence of any HPV type of 17.1% in HIV-negative and of 28.9% in HIV-positive MSM. The analysis confirmed that oral/oropharyngeal HPV presence was higher in HIV-positive MSM This study demonstrates that oral insertive sex is a significant risk factor in the acquisition of oral/oropharyngeal HPV infection in this population group (p = 0.0057). In contrast, Muller et al. In this study, HPV infection has a statistically significant presence in participants who performed oral insertive sex as opposed to those who performed oral receptive sex (p = 0.0038) . The resData from Davidson et al. 5.Data from South African, but specifically from a younger black MSM group, is very rare. The prevalence data points in this report cannot be extrapolated to the general South African MSM population but provides an important component to the baseline for research into oral/oropharyngeal HPV-infection of vulnerable MSM in South Africa and in Africa.Although lower for MSM cohorts, the 6% HR-HPV oral/oropharyngeal prevalence in this study is in alignment with global reports. The prevalence of oral/oropharyngeal HPV in this MSM cohort was influenced by sexual practices. Different detection methods of the HPV, the difference in specimen collection methods, processing, storage, method of DNA extraction, sensitivity and specificity amongst the various global and local studies makes correlation challenging.The significant associations found between oral insertive sex and oral/oropharyngeal HPV status , and between rimming and HIV status  supports the ano-oral-genital/genital-oral-anal HPV transmission routes in this population group, showing how HR-HPV gains access to the mouth and oropharynx, keeping in mind the HIV-seropositive rate of 66.8% in this group. Further studies can be designed to determine the natural history and burden of HPV-associated diseases in the South African MSM community, and also for the effective HPV prevention and public health strategies in these population groups. The current targeted interventions and HPV vaccination of boys to reduce the burden of HPV related diseases amongst MSM in South Africa requires further controlled prospective studies to provide the supporting evidence.This lower HPV-positivity indicates the need for a much larger sample size and for a prospective approach which will be valuable to fully understand the dynamics of oral/oropharyngeal HPV infection in this local MSM population.This population is almost exclusively black, and this is because of the geographic locality of the population. These data cannot be extrapolated based on race, and any race-based or ethnicity-based conclusions must be extremely carefully considered as it has immense potential for wrongful and inappropriate racial profiling. Even then, the authors strongly urge against this.Although not quite a limitation, the lower HPV prevalence in this MSM which has a considerable HIV seropositive burden is curious and reinforces that further investigation into oral/oropharyngeal HPV transmission is needed in this population group.


There has been concern about the risk of suicide among veterans returning from deployment to Afghanistan and Iraq as part of Operation Enduring Freedom, Operation Iraqi Freedom, and Operation New Dawn (OEF/OIF/OND). This study assessed suicide risk among OEF/OIF/OND veterans by gender and unit component. Firearm related suicide was also briefly examined.The study cohort was identified from records of the US Department of Defense. Vital status and cause of death through 2016 was obtained from the Mortality Data Repository, which obtains data from the National Death Index. Suicide risk was first assessed using standardized mortality ratios (SMR)s, comparing the rate of suicide among all veterans, both collectively and separately by gender and unit component  to the expected based on the US population adjusted for age, race, sex, and calendar year. Risk of suicide among active duty compared to reserve/National Guard veterans and male compared to female veterans was assessed with hazard ratios (HR) s, generated by Cox proportional hazards models, that included the covariates race, age, marital status, rank, and branch of service. There was an increased risk of suicide when all OEF/OIF/OND Veterans were compared to the US population, . Both male and female veterans had an increased risk of suicide when compared to their gender specific non-veteran counterparts, , respectively. Active duty veterans had an increased risk of suicide compared to reserve/National Guard veterans, . Male veterans had an almost 3-fold increased risk compared to female veterans, . Among all veteran suicides 68.3% involved a firearm, including 68.7% among males and 59.5% among females.All OEF/OIF/OND veterans have an increased risk of suicide compared to non-veterans. Veterans will benefit from enhanced access to mental health services and initiatives to promote suicide prevention. Strategies that emphasize lethal means safety, an evidence based suicide prevention strategy which includes increasing safe storage practices  can help address this increased risk of veteran suicide. Following a 2007 CBS news report about high rates of suicide among veterans , Defense Manpower Power Data Center, which is a data repository of all US military personnel records.Mortality data was obtained by matching all cohort veterans against data included in the VA/DOD Mortality Data Repository (MDR). The MDR contains all-cause mortality for all military service members who separated from military service since 1979. At the time this study was initiated MDR had cause of death data through 2016. Cause of death and fact of death in the MDR are obtained from the National Death Index (NDI). Suicides were all deaths identified with one of the following International Classification of Disease, 10th revision (ICD-10) codes; X60-X84, Y87.0 s to approximate suicide risk among subgroups of this study’s OEF/OIF/OND veterans. CRs per 100,000 PYR for all OEF/OIF/OND veterans collectively and then by strata of interest were compared. Beginning of follow-up for veterans who served in active duty components was the date they separated or retired from the military through the end of May 2015, while beginning of follow-up for reserve/National Guard component veterans was the end date of their last OEF/OIF/OND deployment through May 2015. End of follow-up for all veterans was the earlier of date of death or December 31, 2016.Suicide risk was also assessed by comparing the observed number of suicides among OEF/OIF/OND veterans to the expected based on the number of suicides in the US population, adjusted for age at entry to follow-up, race, sex, and calendar year. This comparison is expressed as a standardized mortality ratio (SMR) and was generated by the National Cancer Institute software, known as SEERSTAT®  and active duty veterans had an increased risk of suicide compared to reserve/National Guard veterans . Whites had an increased risk of suicide compared to non-whites  and not married had an increased suicide risk compared to married . Enlisted veterans had an almost 2-fold increased risk of suicide compared to officers, , and ground troops, i.e. served in Army or Marines, had an increased risk of suicide compared to non-ground troops, .− 3.0; 95% C.I., − 4.4,− 1.6); for active duty veteransFigure − 1.9; 95%, C.I., − 3.6,− 0.2); and for reserve/National Guard veterans . Male OEF/OIF/OND veterans, similar to the total cohort of veterans, had their largest risk of suicide after one year of follow-up, HR = 41.4, whereas female OEF/OIF/OND veterans had their largest suicide risk after ten years of follow-up, HR = 18.3. Like the other subgroups examined, males had a statistically significant trend of decreased risk of suicide over 13 years, , whereas female veterans have a trend of increased risk of suicide over time, although it was not statistically significant, ..