Pipes are commonly used to transport fuels, air, water, gas, hydraulic power, and other fluid-like materials in engine rooms, houses, factories, airplanes, and ships. Thus, pipe routing is essential in many industrial applications, including ship construction, machinery manufacturing, house building, laying out engine rooms, etc. To be functional, a pipe system should be economical while satisfying spatial constraints and safety regulations. Numerous routing algorithms have been published to optimize the pipe length and the number of elbows. However, relatively few methods have been designed to lay out pipes which strictly meet the spatial constraints and safety regulations. This article proposes a distance-field-based piping algorithm to remedy this problem. The proposed method converts the workspace into a 3D image and computes a distance field upon the workspace first. It then creates a feasible space out of the workspace by peeling the distance field and segmenting the 3D image. The resultant feasible space is collision-free and satisfies the spatial constraints and safety regulations. In the following step, a path-finding process, subjected to a cost function, is triggered to arrange the pipe inside the feasible space. Consequently, the cost of the pipe is optimized, and the pipe path rigidly meets the spatial constraints and safety regulations. The proposed method works effectively even if the workspace is narrow and complicated. In three experiments, the proposed method is employed to lay out pipes inside an underwater vehicle, a machinery room, and a two-story house, respectively. Not only do the resultant pipes possess minimal costs, but they also meet the spatial constraints and safety regulations, as predicted. In addition to developing the routing procedure, we also design a visualization subsystem to reveal the progression of the piping process and the variation of the workspace in the run time. Based on the displayed images, users can therefore evaluate the quality of the pipes on the fly and tune the piping parameters if necessary. Pipes, hoses, electric circuits, and cables are commonly used to transport fuels, water, air, hydraulic power, control signals, and electric currents in ships, factories, engine rooms, houses, etc. . Pipe roThe pipe path must be collision-free and connect the sources and destinations.The pipe length and number of elbows should be minimized. The pipe path should be straight or rectangular.The pipe path should be close to the walls, floors, ceilings, obstacles, and other pipes such that pipe racks are easier to build.The gaps between the pipe and other pipes and the space separating it from obstacles should be wide enough to allow for accessibility.The pipe must be away from hazards for safety concerns. The pipe should not cross or occupy free spaces reserved for humans and machinery.The geometric shapes, complexities, and attributes of workspaces vary on a case-by-case basis, and these factors influence pipe routing in many ways. There is rarely a generic rule for designers to follow when conceiving their pipe designs. However, the safety regulations and spatial constraints which should be met by a pipe have been well studied and published in the literature. They can be briefly summarized as follows ,7,8:The Conditions 1 and 2 are set for producing a functionable and low-cost pipe. These two conditions can be met if Dijkstra’s shortest path method or the A* algorithm is used to lay out the pipe ,8,9. OptConditions 3–6 are mutually conflicting and are hard to be explicitly satisfied by the pipe. Under condition 3, the spatial gaps between the pipe and the obstacles should be small such that creating the pipe racks is easier. However, these metrics are required to be wide in conditions 4 and 5 for the sake of accessibility and maintenance. Condition 6 prohibits the usage of a free space by the pipe, even though the free space contains no equipment. To meet conditions 3–6, many piping algorithms rely on specialized cost functions to guide the direction and position of the pipe. Under the supervision of these cost functions, the pipe will receive penalties if it is close to hazards and win advantages if it is attaching to unharmful obstacles and pipes ,13,14,15In this article, a distance-field-based method is proposed for arranging pipes in narrow and complex workspaces such that not only are the costs of the resultant pipes minimized but also the spatial constraints and safety regulations are strictly satisfied by the pipes. The key steps of the proposed method can be briefly described as follows: First, by carrying out a voxelization computation, the proposed method splits the problem domain into a 3D image of voxels. A distance field ,17 is thRecently, a researcher proposed building immersive virtual reality tools  and expePiping algorithms can be categorized into two major groups, the automatic methods and the semi-automatic ones . The forSince Lee published his piping algorithm in , many auIn , Jiang aThe problem domains of pipe routing vary on a case-by-case basis, and the applications of the pipes are very different ,15,18,19Some piping problems are so domain-specific that experts’ knowledge has dominant influences on the topology of the underlying pipes. For example, in , Yue et Deterministic algorithms produce the optimum solution when routing a single pipe. However, the optimization of multiple pipe routing is NP-hard and is intractable for deterministic algorithms. Many researchers have proposed to employ heuristic methods for scheduling the orders of the pipes and branches first and then use deterministic approaches to lay out individual pipes later. Other researchers propose to employ heuristic algorithms not only to schedule the piping process but also to route individual pipes. In , Jian etIn addition to ACO methods, genetic algorithms (GA) are commonly used for laying out multiple pipes too. In , Kim et Using fit functions, objective functions, and cost functions to guide pipe paths is very common in pipe-routing algorithms. In , Dong anThe rest of this paper is organized as follows. In In this section, the proposed piping method will be presented, including its flowchart and key computational steps. Each computational step is described in detail separately. The pseudo-codes of the algorithms dedicated to routing the pipes and computing the distance field are contained in The flowchart of the proposed piping method is shown in The key steps of the proposed piping method will be presented in the rest of this section, including the voxelization process, distance field computation, feasible space extraction, and pipe-path searching. How these computations influence the efficiency and quality of the pipe-routing process are also discussed. Conventional piping methods lack the capability to reveal the progression of the piping process. In the proposed piping procedure, a graphical module is developed for displaying the obstacles, distance field, feasible space, and pipe paths on the fly. Hence, the users can evaluate the quality of the pipe-routing process and modify the controlling parameters if necessary. This graphic module is described in this section too.As shown in In the voxelization task, we enclose the problem domain by using a bounding box first. The bounding box is then split into voxels by using a regular grid . The voxu is the distance function to be solved, and S represents the surfaces of the obstacles, for which the distance function is set to zero.Following the voxelization process, we invoke a numerical procedure to compute the shortest distance from each free-space voxel to the obstacles. This distance function is governed by the following Eikonal equation ,17:(1)(∂u for Equation (1). The procedure of the RFMM can be described as follows: First, we find the voxels in the interiors of the obstacles and give them a negative distance to assert that these voxels are excluded from the pipe-routing computations. In the next step, the voxels of the free space are grouped into a set, which is called FAR. Initially, their distances are set to infinity. Following that, the voxels on the obstacles’ surfaces are searched and kept in a set called DONE. These voxels are labeled as visited, and their distances are set to zero, as shown in In this research, we use the revised fast marching method (RFMM) proposed in  to compuAfter creating these three sets, the voxel that is in CLOSE and possesses the shortest distance is removed from CLOSE and inserted into DONE. Hence, DONE is enlarged. After that, those voxels which are in FAR and are adjacent to this newly visited voxel are removed from FAR and inserted into CLOSE, and their distances are updated by solving Equation (1) numerically. Thus, the scope of FAR is shrunk. These set operations and distance calculations are repeated until all voxels are moved from FAR to CLOSE and then from CLOSE to DONE. The example in u stands for a possible distance of the current voxel, and a, b, and c are the coefficients of the polynomial. These coefficients result from approximating the partial derivatives of Equation (1) by using forward and backward difference methods. The methods for determining a, b, and c and solving for u are given in To compute the distance of a voxel in CLOSE, the RFMM converts the non-linear differential equation of Equation (1) into a quadratic polynomial by first using the distances of its adjacent neighbors in DONE. The polynomial can be formulated as follows:a, b, and c, can be found in [The two roots of this polynomial are then computed by using the quadratic formula of Equation (2). Once the roots have been calculated, the voxel’s original distance is compared with the maximum root. If the maximum root is smaller, the voxel’s distance is set to the maximum root. Otherwise, the voxel keeps its original distance. The detailed steps, pseudo-code, and formal proof of the RFMM, including the manipulation of the sets DONE, CLOSE, and FAR and the calculations of the coefficients found in . We omitThe distance field forms a level set. We can divide it into a series of layers as if we were peeling an onion. The voxelization process converts the problem domain into a 3D image, and thus we can manipulate it by using erosion, dilation, and breadth-first search. Based on these features, we extract the feasible space from the problem domain and confine the pipe routing there to ensure that the spatial constraints and safety regulations are automatically satisfied.When building a pipe, we must first ensure that the pipe connects the terminals and is collision-free from the obstacles. We should then minimize its length as well as the number of bends. In addition, the pipe has to meet the spatial constraints and safety regulations such that its construction, usability, maintenance, and operational safety can be guaranteed. Taking so many factors into consideration simultaneously while arranging the pipe is challenging. In this research, we propose to generate a feasible space in the free space which contains no obstacles and intrinsically satisfies the spatial constraints and safety regulations first. The pipe path is then routed inside this space only, and thus detecting collisions and checking for violations of spatial constraints and safety regulations becomes unnecessary. Therefore, the complexity of the piping process is greatly simplified.There are two major purposes of the spatial constraints. The first one is to force the pipe to be close to the obstacles  such that the construction of the pipe racks is easier and less free space is consumed. The second purpose is to preserve reasonable gaps between the pipe and the obstacles so that the installation, operation, and maintenance of this pipe will be convenient. Obviously, these two purposes conflict and are hard to achieve at the same time.u satisfy the following constraints:To resolve this conflict, we extract a feasible space from the free space by using the distance information kept in the free-space voxels. First, starting from the surfaces of the obstacles, we perform a breath-first search to collect those free-space voxels whose distances mind and maxd are the lower and upper bounds of the distance values of the feasible space, respectively. They are determined as follows.r is the radius of the pipe, and 1ε and 2ε are the minimum and maximum allowable gaps between the pipe and the obstacles, respectively. These two parameters are adjustable, based on the attributes of the pipe. By tuning these two parameters, the designers can ensure that the feasible space is wide enough for arranging the pipe and that the pipe is neither too close to the obstacles nor too far from the obstacles.The variables a and b of 1ε and 2ε are set to 0.5 and 1.5, the bounds mind and maxd are set to 1 and 2 based on Equation (4). Thus, those voxels with distances within this range are included in the feasible space (the green and yellow voxels), as shown in An example is displayed in Parts By following the rules of Equation (3), the resultant feasible space meets the spatial constraints. However, it may fail to satisfy the safety regulations and may exclude some of the pipe terminals. For example, as shown in The dilation starts at all the dangling terminals, which are not connected to the feasible space. We search those free-space voxels adjacent to the dangling terminals and label them as potential feasible voxels. These potential feasible voxels form small regions surrounding the dangling terminals. In the following steps, we gradually expand these regions by using breadth-first search until they touch the feasible space. Finally, we relabel all the potential voxels as feasible-space voxels and stop the dilation process.To keep the feasible space away from the hazards, we erode those voxels which are too close to the hazards from the feasible space. First, we search the voxels next to the hazards. If they are feasible-space voxels, they are removed from the feasible space such that they will not be used in the following pipe-routing task. The erosion then continues in a breadth-first-search manner until the distance (number of layers) of the breath-first search exceeds a predefined limit. As the erosion ends, the feasible space is guaranteed to be at a safe distance from the hazards.In G = {V, E} such that we can employ Dijkstra’s method to compute the pipe path. The conversion procedure is as follows: The voxels in the feasible space form the vertex set V, and the adjacency relations between these voxels constitute the edge set E. Because the feasible space is a subset of a 3D regular grid, each vertex has at most six neighbors, residing in the ±x, ±y, and ±z directions. Thus, the pipe path will consist of only straight lines and rectangular bends.In the following step, we convert the feasible space into a graph G and before routing the pipe, we calculate the Manhattan distances between all the pairs of terminals and find the pair of terminals that possesses the longest Manhattan distance. One of these two vertices is designated as the source, while the other one serves as the destination. In the subsequent step, the shortest path connecting these two vertices is computed by using Dijkstra’s algorithm.After creating Once the path-finding task has been completed, the vertices residing in the computed path are regarded as destinations, and one of the remaining terminals is treated as the new source. The path-finding procedure is then carried out to build a branch connecting this terminal to the existing path. The above computations are repeated until all the terminals have been connected to the pipe path. The pseudo-code of the pipe-routing procedure can be found in G is derived from a 3D regular grid, the resultant pipe path would contain many elbows if we merely employed Dijkstra’s algorithm to find the shortest pipe path. To reduce the bends, the following cost function is used to measure the distance between the source and the vertex which is to be visited in the next step:C(v) is the accumulated distance from the source to vertex v, and i, vi+p(v1) represents the cost of the edge connecting vertices iv and i+v1. Vertices i−v1, iv, and i+v1 stand for the previous, current, and next visited vertices in the path-finding calculation, respectively, and ω is a penalty selected by the users to punish the pipe if it makes a 90-degree turn. This cost function increases the path length by 1 + ω, instead of 1, if the path makes a bend. Hence, straight paths are preferred over elbows.Because Compared with conventional piping algorithms, the proposed method employs a very simple cost function. This is because the feasible space created in the previous stage already meets the spatial constraints and safety regulations. There is no need to include any penalty or benefit terms in the cost function to repel the pipe path from the hazards or to attract the pipe path to the obstacles. The image shown in r units of the centerline (r is the radius of the pipe) and label them by using the pipe’s ID. Therefore, these voxels will be owned by the pipe and will be regarded as obstacle voxels in future computations. Following that, cylinders with radii equal to r are generated along the centerline to produce the surface of the pipe. Finally, the pipe surface is decomposed into triangular patches and kept in an STL (STereoLithography) file.The pipe path constructed by the above algorithm forms only the centerline of the pipe. To complete the work, we search for the voxels which are within To reveal the progression of the piping process, the proposed pipe system contains a graphical module to display the problem domain, distance fields, and pipes. The graphical module depicts pipes by using surface rendering first. It then displays the obstacles, free space, and feasible space by using volume rendering such that the internal structures of the problem domain as well as the related positions of the pipes inside the workspace can be comprehended. By using this strategy, both the depths of the pipes and their adaptation to the obstacle surfaces are illustrated.In the volume-rendering computations, our graphical module assigns high opacities to the voxels belonging to the boundaries of the free space, feasible space, and obstacles and gives low opacities to the voxels inside these regions. Therefore, these regions are revealed, and the pipes are not occluded or blurred in the resultant images. To achieve real-time visualization, the rendering process is accelerated by using the graphics processing unit (GPU). Detailed steps of the real-time volume rendering can be found in .Based on the proposed pipe-routing method, we implemented a software system dedicated to routing pipes. Three experimental workspaces had been created to test the capabilities of the piping program. These workspaces are the chamber of an underwater vehicle, a machinery room, and a house. Each workspace is divided into layers and rooms by using floors, ceilings, and walls. To connect these rooms, there are holes in the walls, floors, and ceilings. Thus, pipes must go through these holes to connect their terminals located in different rooms. Multiple pipes are arranged in each workspace, and each individual pipe is composed of several terminals residing in different rooms. Thus, the pipes not only have to penetrate the holes but also have to grow branches to connect their terminals. Furthermore, the pipe diameters are varied. This makes the pipe-routing process more challenging.Pipe routing is difficult in surface ships ,4,5,8,10To test the capability of the proposed piping algorithm, we purposely create a domain mimicking the internal space of the pressure hull of an underwater vehicle. The layout of the domain is shown in Two pipes are to be routed through this workspace. The positions of the terminals are specified by using the indices of the voxels. The first pipe consists of seven terminals, and its radius is 4 cm. The coordinates of the terminals are listed in Following that, the Manhattan distances between all the terminals are computed. The Manhattan distance between terminals 1 and 2 is the longest, and thus the first branch of the pipe is aiming to connect terminals 1 and 2. The branches connecting terminals 3, 4, 5, 6, and 7 to the main pipe path are then computed. The pipe path is constructed by using Dijkstra’s algorithm and employing Equation (4) to calculate the cost of the pipe path. The penalty for generating a bend in a path is 0.5 voxel lengths.The results are depicted in Part d of After routing the first pipe, we arrange the second one. The locations of its terminals are listed in a of b of c of d of The first pipe occupies some portions of the original free space. The revised workspace is shown in Part As the results show, the second pipe penetrates many bulkheads, walls, and floors, as its terminals are far apart from each other and located in different rooms. Secondly, the second pipe does not cross the first pipe, even though the free space is reduced. This is because the first pipe is regarded as an obstacle and excluded from the feasible space once it has been constructed. Thirdly, the second pipe attaches to the walls, ceilings, and floors, as the first pipe does. This phenomenon is caused by the scope of the feasible space, which is at most 7 cm from the obstacles. The paths of these two pipes are collision-free and meet the predefined regulations and constraints. Thus, this example verifies the effectiveness of the proposed method for creating the feasible space.a of Pipes are commonly used in machinery rooms to transport air, cooling water, and fuels. They are the veins and arteries of facilities. Hence, pipe routing is important for the layout of machinery rooms. In another test, we artificially built a machinery room as shown in Part a of Three pipes are to be constructed. Each pipe has multiple terminals, located in different regions. The radii of the pipes are 5, 4, and 2 cm, respectively. The pipes must connect their terminals and attach to the floors, the walls, and the surfaces of the machines to save space. The three pipes are shaded in red, green, and blue colors. The red pipe is the thickest, while the blue pipe is the thinnest. We route the red pipe first because it is the thickest pipe and needs more space. The green pipe is then routed, followed by the blue pipe. The routing results are shown in Part As the image shows, all the pipes successfully connect their terminals even though the geometrical complexity of the workspace is high, and the terminals are separated by the walls. Their paths are collision-free and are attached to the walls, floors, and facility surfaces. Only a small portion of the free space is occupied by the pipes. Thus, they strictly meet the spatial constraints.These pipes share the big hole in the first wall, as there is no alternative gateway. However, they use different holes in the second wall because of their sizes and orders. The red pipe is the first pipe to be constructed. It occupies the best positions. Its path contains a few bends, but these bends are caused by the shapes of the facilities and the geometry of the workspace. On the other hand, the green pipe contains many bends. This is because that it can only utilize the space and holes left by the red one, and it must avoid colliding with the red pipe. Therefore, it makes some turns. Though the blue pipe is the last pipe to be built, it contains fewer bends than the green pipe. This can be explained as follows: The blue pipe is a thin pipe and can go through the small holes and narrow gaps left by the previous two pipes, and hence it has fewer bends. Its bends are mostly caused by the shapes of the facilities and the geometry of the room.Pipes are commonly utilized in houses to transport tap water, gas, sewage, and air to maintain good living conditions. In addition to spatial constraints and safety regulations, the pipes in a house must satisfy aesthetic conditions too. Otherwise, they will degrade the values of houses. In the last test, we create a two-story house and use it as the workspace for routing pipes. Two pipes are routed to connect terminals located in all the rooms of the house to serve as water and ventilation pipes. We create holes in the walls such that the pipes can travel out of the house and then re-enter the house if necessary. However, to make the routing harder, each hole allows only one pipe to pass through.b of The results are shown in Part The spatial gaps between the pipe and the obstacles, hazards, and other pipes are deterministically decided. Conventional piping algorithms use fit functions to direct the pipe path and hope that the spatial constraints and safety regulations are met by the pipe. As mentioned before, their fit functions may not produce the desired results because they are heuristic. Even if the pipe satisfies the spatial constraints and safety regulations, the resultant spatial gaps are not bounded or predictable.The feasible space is collision-free and satisfies the spatial constraints and safety regulations. Thus, it is unnecessary to detect collisions or to calculate the distances between the pipe and the obstacles, hazards, and other pipes at the run time.3) voxels, the time complexity of creating the feasible space is bounded by O(2kn), where k is the number of distance levels in the feasible space, and 2n stands for the number of voxels in a level of the distance field. Conventional methods use polygonal facets to represent the problem domain, making extracting a region from the workspace and modifying the workspace very expensive.Our problem domain is composed of voxels. Modification of the workspace and creation of the feasible space are carried out in the 3D image space. They utilize mature image-processing techniques. Assuming that the image space is composed of O voxels. By using A* or Dijkstra’s method, the cost of routing a pipe inside the workspace is bounded by O(n3). However, our piping procedure confines the pipe routing inside the feasible space which contains only O(2kn) voxels. Thus, the time complexity of the pipe routing is bounded by O(2kn). The time complexity is reduced by an order.The workspace is composed of O algorithms ,25 to seThough our method is a deterministic one, the designers can adjust the feasible space by tuning the parameters of Equation 4), and hence the spatial gaps can be modified according to the intrinsic properties of the pipes. Based on a similar approach, we can adjust the distances between pipes and hazards by controlling the distance of the erosion to assure the safety of pipes. The ordering of the pipes has a great influence on the resultant pipe paths, as shown in the example of , and henThe proposed piping method divides the workspace into voxels. The accuracy of the pipe routing is greatly influenced by the voxel size, including pipe lengths, spatial gaps, and distances to hazards. The accuracy is linearly proportional to the resolution of the underlying grid. Increasing the grid resolution improves the precision. However, the memory usage is enlarged too. If the main memory space is limited, we proposed to adopt an out-of-core strategy to solve the problem. First, we represent each room as an independent domain and set intermediate points in the holes. Each room is then kept in a disk file. The pipe routing is performed independently in each room. Therefore, we have to fetch the digital representation (a 3D image) of only one room into the main memory at the run time. We then create the feasible space for the current room and compute the all-pair shortest paths between the intermediate points and the terminals. Following that, the pipe paths of the current room are exported to a disk file. After completing the routing tasks for all the rooms, we fetch the pipe paths of all the rooms into the main memory and merge them according to the intermediate points to complete the pipe-routing process.Recently, some researchers published state-searching algorithms based on the IDA* method ,25. ThesIn this article, we present a pipe-routing method. The major capabilities of the proposed method can be summarized as follows: First, the costs of the resultant pipes are optimized. Second, spatial constraints and safety regulations are strictly obeyed by the pipes. Third, the progression of the piping process is revealed in real time. To accomplish these results, the proposed method decomposes the problem domain in a 3D image and computes a distance field upon the free space of the problem domain. A free space is then extracted from the distance field such that the spatial constraints and safety regulations are automatically satisfied. In the following step, a revised Dijkstra’s method is employed to compute the pipe path such that the pipe’s cost is reduced. In addition, the piping procedure contains a graphical module which is capable of displaying the workspace, distance field, and pipes in real time by using surface and volume rendering. Three experiments were conducted to test the proposed piping method. In the experiments, the proposed piping procedure produced decent results in different workspace, and all the goals of piping were met. Its efficacy has been verified by these experiments.The scheduling of the pipes plays an important role in the quality of the pipe system. We propose to utilize experts’ experience and AI methods for scheduling pipe routing while employing our piping algorithm to route individual pipes. Memory usage is another concern for the proposed piping method. If the main memory space is not sufficient to carry out our high-precision pipe-routing process, we suggest that the piping process should be carried out in an out-of-core manner. In the out-of-core piping process, we divide the problem domain into rooms and lay out pipes in each room first. The pipes are then merged to form the final pipe system. IDA* algorithms require less memory than A* and Dijkstra’s methods in path finding. In future work, we intend to employ an IDA* searching algorithm for calculating pipe paths to further reduce memory usage in pipe routing.


Oropharyngeal cancer (OPC) is an important cause of cancer‐related mortality. Early detection of OPC results in a favorable prognosis and higher survival rates. Infection by high‐risk types of human papillomavirus (HPV) is a risk factor for OPC with an upward trend globally. Medical students' knowledge and awareness of HPV‐related OPC can be crucial in the preventive efforts.To assess HPV knowledge among medical students at the University of Jordan, with particular focus on its relation to different cancers.This paper‐based survey study was conducted in November 2019. The survey items were based on previously validated surveys used to evaluate HPV‐related OPC knowledge among dental students and professionals. To assess HPV knowledge and students' confidence in personal history taking and physical examination, we developed a knowledge and confidence scores that showed acceptable reliability.n = 697, 58.2%). Among the participants, 93.3% heard of HPV prior to this survey (n = 1118). Higher levels of knowledge regarding cervical cancer, OPC and HPV vaccination was seen among clinical students compared to their preclinical counterparts, but their overall HPV knowledge was low. Only 18.4% and 21.0% of the clinical students correctly identified the association of HPV with penile and oropharyngeal cancers, respectively. Additionally, 34.5% of the clinical students were not aware of the availability of HPV vaccines. The majority of students (92.0%) reported that the university courses were their major source of knowledge about HPV.The total number of participants was 1198 students, with a median age of 21 and female predominance (A profound lack of knowledge regarding HPV role in OPC was found among medical students. This insufficiency included several aspects of the virus and its associated diseases. Such gaps in knowledge could have negative consequences in early detection and prevention of OPC and should be addressed by evaluation of the current curriculum. This survey was partly based on our previous study that was used to assess UJ dental students' awareness and attitudes towards HPV‐related oropharyngeal cancer.Since English is the official teaching language at the Medical School/UJ, the questionnaire was developed and distributed in English.2.2The study was approved by the Deanship of Scientific Research at UJ and by the Institutional Review Board (IRB) at Jordan University Hospital (JUH) on 21/10/2019 . The study was conducted anonymously, and all data were treated with confidentiality. To keep anonymity of the students in this study, verbal consent was obtained from the participants as approved by JUH IRB.2.3The survey comprised 21 items as follows: a section on demographic characteristics of each participant , a defining question on whether the participant has heard of HPV before the survey. For those who answered (Yes) to the previous question, a request to complete the survey followed with a 6‐item section on HPV knowledge, involving (Yes/No/I do not know) questions, and a 9‐item section on the transmission routes and HPV‐associated clinical disease , and an item to assess the main source of knowledge about HPV. Finally, a section was included using a 5‐point Likert scale to assess how comfortable does the participant feel during history taking that involves discussing personal topics with patients and attitude towards physical examination.2.4The content validity of the current questionnaire involving relevance, and representativeness was conducted by three authors  who are involved in medical and dental education at UJ. To assess the overall knowledge of the participants regarding HPV, we used an HPV Knowledge Score (HPV K‐score), consisting of the following 15 points: 1) ubiquity of HPV infections; 2) existence of more than 100 HPV types; 3) only a group of HPV types are associated with cancer; 4) inapparent nature of most HPV infections; 5) existence of HPV vaccines; 6) failure of Pap smears to detect all HPV‐induced premalignant lesions; 7) HPV association with cervical cancer; 8) HPV association with oropharyngeal cancer; 9) HPV association with penile cancer; 10) lack of HPV association with nasopharyngeal cancer; 11) lack of HPV transmission through blood transfusion; 12) sexual transmission of HPV; 13) cold sores are not caused by HPV; 14) genital warts are caused by HPV; 15) common warts are caused by HPV. Each correct response for any of the previous points was scored as a single point, while incorrect response/non‐response (implying lack of knowledge) or I don't know response (for the first six points) was scored as zero. Thus; the maximum possible HPV K‐score was 15 while the minimum was zero. The Cronbach's alpha value of 0.737 ensured an acceptable internal consistency for the proposed HPV K‐score.2.5The evaluation of clinical students' attitude towards history taking that involves discussing personal topics with the patients and towards physical examination was done using personal history taking and physical examination confidence scale . The participants were asked to report how comfortable they feel with each of the following five points: 1) asking patients questions regarding their lifestyle; 2) asking patients about sexually transmitted infections; 3) asking patients about sexual abuse; 4) physically examining the patient; 5) physically examining a patient of the opposite sex. The scale ranged from ‘not comfortable at all’, which was given a minimum score of 1, to ‘most comfortable’, which was given a maximum score of 5. Higher Confidence scale values indicated greater comfort in attitude towards discussing personal topics with patients and towards physical examination. The Cronbach's alpha value of 0.774 ensured an acceptable internal consistency for the proposed Confidence scale.2.6p < 0.050. For scale variables , mean and standard deviation (SD) were calculated. Chi‐squared test (χ2 test) was used to assess the statistical difference between categorical variables while Mann–Whitney U (M–W) and Kruskal–Wallis (K–W) tests were used to assess the statistical difference between categorical and scale variables. Univariate analysis of variance was used to assess the potential effect of confounding variables  in relation to Knowledge and Confidence scores.All statistical analyses and data description were done using IBM Statistical Package for the Social Sciences (SPSS) 22. Statistical significance was considered for 33.1n = 697) of the study participants and the median age for participants was 21 years . Jordanian students predominated the study population , while students of non‐Jordanian citizenship formed 27.6% of all participants (n = 331). Students of non‐Jordanian citizenship were distributed among 23 different nationalities with Kuwait, Iraq, Palestine and Syria forming the most common countries of origin (n = 232). The highest response rate was found among the second‐year students , while the lowest was for the sixth‐year students . Students with GPA higher than 3.0 (very good and excellent) formed 73.4% of all participants who stated their GPAs . Also, clinical students were more likely to have heard of HPV compared to pre‐clinical students . Males showed a higher knowledge that Pap smears may not detect all HPV‐induced cervical premalignant changes . Also, males showed higher knowledge of HPV association with oral and penile cancers compared to females . Correct identification that HPV cannot be transmitted through blood transfusion was seen more frequently among females . For non‐Jordanian students, lower level of knowledge regarding HPV association with cervical cancer was seen compared to Jordanian students . Also, non‐Jordanian students had a lower knowledge about sexual transmission of HPV compared to Jordanian students .Comparisons of HPV knowledge per item in relation to sex and nationality among the whole study participants, showed that females had higher knowledge of the existence of HPV vaccines compared to males . The overall HPV knowledge which was deduced through HPV K‐score, was higher among Jordanian students  compared to their non‐Jordanian counterparts .The HPV K‐score was used to evaluate the overall knowledge per curriculum year, sex and nationality. The main improvement in HPV knowledge was seen moving from the fourth curriculum year  into the fifth curriculum year . Among the covariates, the latest GPA was found as a significant predictor of HPV K‐score (p < 0.001).Univariate analysis with HPV K‐score as the dependent variable, curriculum year as the fixed factor, and the following as covariates: sex; nationality; and the latest GPA, showed that the improvement in HPV knowledge was associated with progression through curriculum years .The total number of the clinical students with a valid confidence scale was 509/539 (94.4%). Upon comparing the Confidence scale among the different study variables , we did not find any statistically significant differences. For clinical students, no significant improvement in the Confidence scale was found moving from 4th curriculum year , to 5th curriculum year  and finally among 6th curriculum year students , followed by history taking that involves asking the patients about history of STI. For the five items, no statistically significant differences were found in relation to gender, nationality and curriculum year except for asking the patients about their lifestyle (Table p = 0.570), with no significant results for the covariates.Univariate analysis with the confidence scale as the dependent variable, curriculum year as the fixed factor, and the following as covariates: sex; nationality; and the latest GPA, showed the lack of improvement in students' confidence in history taking that involves discussing personal topics with the patients and towards physical examination with progression through curriculum clinical years , followed by the internet/social media outlets , and school courses . The participants who relied on university courses as the source of knowledge about HPV had a higher HPV K‐score compared to those who did not .The vast majority of the study participants reported that the university courses were their major source of knowledge about HPV  at Jordan University Hospital (JUH) on 21/10/2019 . To keep anonymity of the students, verbal consent was obtained. This approach was approved by JUH institutional review board.


Its presence was more frequent in non-malignant alterations than in malignant neoplasias. The most frequent genotypes in the HPV-positive samples were low-risk (LR) HPV-42 followed by high-risk (HR) HPV-31.Breast cancer is the leading cause of cancer death among women worldwide. Multiple extrinsic and intrinsic factors are associated with this disease’s development. Various research groups worldwide have reported the presence of human papillomavirus (HPV) DNA in samples of malignant breast tumors. Although its role in mammary carcinogenesis is not fully understood, it is known that the HPV genome, once inserted into host cells, has oncogenic capabilities. The present study aimed to detect the presence of HPV DNA in 116 breast tissue biopsies and classify them according to their histology. It was found that 50.9% of the breast biopsies analyzed were malignant neoplasms, of which 74.6% were histologically classified as infiltrating ductal carcinoma. In biopsies with non-malignant breast disease, fibroadenoma was the most common benign neoplasm (39.1%). Detection of HPV DNA was performed through nested PCR using the external primer MY09/11 and the internal primer GP5+/6+. A hybridization assay genotyped HPV. HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% non-malignant breast disease (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. However, there are no statistically significant differences between these groups and the existence of HPV DNA ( Breast cancer is the most common and fatal cancer among women in developed and developing countries. According to data from the World Health Organization, 2,261,419 new breast cancer cases were reported worldwide in 2020, as well as 684,996 deaths [It is known that many factors are involved in the development of breast cancer, such as the environment, age, hormones, alcohol consumption, fat in the diet, a diet poor in fruits and vegetables, family history, obesity, tabaquism, alcoholism, number of offspring, breastfeeding, estrogen levels, estrogen receptors ,3, amongViruses are considered a controversial etiological risk factor for breast cancer. Viral DNA from human papillomaviruses (HPV), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), and human herpesvirus type 8. (HHV-8) has been found in healthy and breast cancer samples ,5. Howevenv gene sequence, which codes for the MMTV envelope protein, in 38% of 314 breast neoplasms [Since Bittner, in 1943, identified the mouse mammary tumor virus (MMTV) as the etiological agent of breast cancer in mice , severalIn 2017, Islam et al. reported a pattern in the presence of HPV in normal and benign tumors and a markedly increased presence in malignant breast tumors, indicating its pathological importance in breast cancer. HPV was also associated with poor hygienic conditions and patient malnutrition, together with ethnicity .Integrating the HPV genome into the host genome may cause chromosomal instability and trigger carcinogenesis ,11,12. IPersistent infection with HR-HPV is considered one of the main causative biological factors in developing cervical cancer (CC). HR-HPV 16 and 18 are responsible for more than 65–75% of precancerous cervical lesions and CC. Furthermore, HPV is associated with carcinomas such as head and neck, anal, vulva, oral, vagina, and penile cancer . Cervical cancer is the third most common type of malignant tumor and the fourth cause of cancer death among women worldwide. It is also one of the deadliest cancers among women in underdeveloped countries . In MexiHowever, the oncogenic role of HPV in the development of breast cancer has not yet been clarified, so this study aimed to determine the prevalence of high- and low-risk HPV in breast biopsies diagnosed with benign-alteration and malignant-alteration neoplasms from Mexican women. A total of 116 formalin-fixed, paraffin-embedded breast samples from 2009 to 2019 were used for the present study. The remaining tissues were donated to and collected by the Mexican Social Security Institute in Zacatecas. The diagnosis associated with each sample was confirmed by histopathological diagnosis using hematoxylin-eosin (HE) staining and classified according to the World Health Organization (WHO) classification system [Fresh biopsies were treated with formaldehyde immediately after surgical removal and processed for inclusion in paraffin. Tissue sections were cut and stained with hematoxylin-eosin (HE) for observation under an optical microscope. The pathology specialist performed the analysis and made the histological and clinical diagnoses.® FFPE Tissue kit . Ten 5 µm tissue sections of FFPE breast samples were cut, deparaffinized by incubation with xylene, and washed and rehydrated with ethanol. After complete deparaffinization, the samples were digested with proteinase K at 56 °C for one hour and inactivated at 90 °C. The amount and quality of the DNA were evaluated using a UV-VIS spectrophotometer Q500 (Quawell®) at 260–280 nm. The integrity of the extracted DNA and the absence of PCR inhibitors were assessed by polymerase chain reaction (PCR) amplification of the β-globin gene using 5 µM of primers KM29/PCO4 (2 (25 mM), 1 U Taq DNA polymerase , 0.5 µL of dNTP (10 mM), and water. The amplification of the β-globin gene was performed under the following conditions: initial activation of the enzyme at 95 °C for 2 min, followed by 40 cycles under the following conditions: 95 °C for 30 s, 55.4 °C for 30 s, and 72 °C for 30 s, with a final elongation step at 72 °C for 5 min. The amplicon was visualized in agarose gel (1.5%) stained with ethidium bromide. The images were digitally processed using the Electrophoresis Documentation and Analysis System 120 .DNA purification was performed using a QIAmpM29/PCO4  and 50 nThe detection of HPV DNA was first carried out by screening all the samples by end-point PCR using the primers GP5+/6+, which generated a 150 pb fragment. Subsequently, a nested PCR was performed on the samples that tested negative for HPV to increase sensitivity.Genomic DNA samples from the cervical cancer cell lines SiHa and Caski were used as positive controls for MY09/11 and GP5+/6+ amplification. A paraffin block without tissue and a PCR mix without DNA were negative controls. The primers used are reported in ®, EPO402), and water. The amplification was performed under the following conditions: initial activation of the enzyme at 95 °C for 3 min, followed by 39 cycles under the following conditions: 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s with a final elongation step at 72 °C for 5 min. The second PCR reaction was performed with 5 µL of the first amplicon and the GP5+/6+ primers. The initial activation of the enzyme was performed at 95 °C for 3 min, followed by 39 cycles under the following conditions: 95 °C for 30 s, 48 °C for 30 s, and 72 °C for 30 s with an elongation step at 72 °C for 5 min. The amplicon products were visualized in agarose gel (2%) stained with ethidium bromide.For the nested PCR, MY09/11 primers were used to obtain the first amplicon of 450 bp. Subsequently, GP5+/GP6+ primers were used on the first amplicon. The first PCR reaction was carried out with 100 ng of DNA in a total reaction volume of 25 μL containing 2.5 μL of buffer (10×), 1.5 μL of MgCl2 (25 mM), 0.5 μL of each MY09/MY11 primer (10 μM) , 0.5 μL Quantitative PCR was performed when the samples had a DNA concentration lower than 10 ng/ul. The first amplicon was amplified using primers MY09/11 followed by qPCR. The qPCR was carried out in a 7500 Fast Real-Time PCR System  in a total reaction volume of 25 µL containing 5 µL of the first amplicon, Platinum SYBR Green qPCR SuperMix-VGD , 0.5 µL of each the GP5+/6+ primers (10 µM), 0.1 ul of ROX Reference Dye Solution (25 µM), and water. The amplification was performed under the following conditions: 50 °C for 120 s, 95 °C for 120 s, followed by 40 cycles under the following conditions: 95 °C for 15 s, 48.4 °C for 30 s, 60 °C for 30 s. The data were analyzed using Applied Biosystems 7500 Software v2.0.6, Foster City, California.Samples positive for amplified HPV L1 gene DNA were subjected to genotyping using the LCD-Array HPV-Type 3.5 kit , which allows for the identification of 32 low-, intermediate-, and high-risk HPV types .2 (25 mM), 1 μL primer mix MY09/MY11 or 2 μL primer mix 125, 1 μL dNTPs (10 mM), 0.3 μL of Taq polymerase (5 U/μL) , 100 ng of template, and water. Genomic DNA from the CaSki cell line was used as a positive control. The run was conducted as follows: 3 min at 95 °C, followed by 41 cycles of 1 min at 94 °C in denaturation, 1.5 min at 45 °C in alignment, and 1.5 min at 72 °C in extension, and a final extension of 3 min at 72 °C.The amplicons generated previously from the biotinylated primers were used for hybridization on the chip: (1) MY09/MY11, which generated a fragment of approximately 450 bp. (2) “125” primers, an internal sequence of the 450 bp fragment (the kit’s own). Both amplicons were combined before hybridization. The PCR was performed according to the manufacturer’s protocol. The reaction mixture was prepared using 2.5 μL buffer (10×), 2 μL MgClHybridization was carried out according to the manufacturer’s protocol. Biotin-labeled PCR products were hybridized with HPV subtype-specific capture probes immobilized on the surface of the LCD chip. After washing, each field was incubated with a secondary solution (enzyme conjugate). The PCR fragments were then hybridized with capture probes, and the place where they joined was revealed with an enzyme substrate that generated a blue precipitate. Data reading was performed using the LCD SlideReader V9 software.p-value was less than 0.05.The breast samples were grouped according to the histological diagnosis. The group measured the presence/absence distribution of HPV DNA by simple counting. Chi-squared tests and Fisher’s exact test were used to compare the presence/absence of HPV DNA between histological diagnosis, sex of patients, tumor size (TMN), and clinical stage. SBR scales were compared using Mann–Whitney tests. The age of patients and tumor size (in cm) between HPV-positive and -negative samples were compared using t-student and Mann–Whitney tests, respectively. All statistical tests were performed in GraphPad Prism version 6. Differences were considered significant when the The histopathological diagnosis results from 116 breast samples were classified as shown in Of all the samples analyzed, 50.9% (59/116) were malignant neoplasms, 4.3% (5/116) were in situ neoplasms, 0.9% (1/116) were borderline neoplasms, and 17.2% (20/116) were benign neoplasms. Benign or non-neoplastic alterations of the mammary gland were also diagnosed, 21.6% (25/116) of the analyzed tissue samples. Breast tissue samples and axillary lymph nodes without alterations were also found, 9.5%  of the total.The distribution of the types of malignant neoplasms diagnosed was as follows: 74.6% (44/59) infiltrating ductal carcinoma, 13.6% (8/59) infiltrating lobular carcinoma, 1.7% (1/59) mucinous carcinoma, and 1.7% (1/59) metaplastic carcinoma. The distribution of benign neoplasms was as follows: 90% (18/20) fibroadenoma, 5% (1/20) adenomyoepithelioma, and 5% (1/20) intraductal papilloma. Regarding non-neoplastic alterations, 60% (15/25) of the samples were diagnosed with cystic fibrous mastopathy, 24% (6/25) corresponded to mastitis, and 16% (4/25) with hyperplasia.HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% of benign neoplasms (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. It was not detected in in situ neoplasms or borderline neoplasms. Regarding malignant neoplasms, HPV was detected in 18.2% (8/44) of the diagnosed biopsies as infiltrating ductal carcinoma, in 25% (2/8) of the biopsies of infiltrating lobular carcinoma, and was in the only sample diagnosed as mucinous and metaplastic carcinoma. Among benign neoplasms, HPV DNA was only identified in 38.9% (7/18) of the fibroadenoma samples. Regarding benign alterations, HPV was detected in 40% (6/15) of the biopsies with cystic fibrous mastopathy and 50% (3/6) of the biopsies of mastitis . No relaThe most frequent HPV genotype in the samples was LR-HPV 42, which was identified in 19% (6/31) of the analyzed samples, followed by HR-HPV 31  and HR-HPV 59 . LR-HPV 44 and HR-HPV 58 were identified in only 6% (2/31) of samples, and HR-HPV 51 in 3% (1/31). Ten percent (3/31) of all HPV-positive samples had mono-infection, while 16% (5/31) had co-infections. Two samples were positive for two virus genotypes, HPV 58/51 (1/31) and HPV 31/42 (1/31) . Three sBreast and cervical cancer are the leading cause of death for women worldwide, mainly in developing countries . HPV is In breast cancer, HPV has been proposed in several studies as a probable causative agent of breast cancer carcinogenesis ,6.A controversial fact is that HPV DNA has been reported in healthy breast samples. It would be very interesting to make a follow-up study on samples donated by women with healthy breast tissue but positive for HPV DNA to observe if, over the years, they developed some mammary carcinoma, which would support the hypothesis that HPV is an oncogenic factor in breast cancer .The role of HPV in breast cancer carcinogenesis remains controversial due to inconsistent data on the presence of HPV DNA in tumor samples from patients with breast cancer and a lack of clarity regarding the route of HPV transmission from one organ to the other.The variability of the reported results within the same country could be explained by the quantity and quality of the samples analyzed, considering that breast cancer samples have a lower viral load, making HPV challenging to detect. Other factors that may introduce noise in the study of this subject include the preprocessing of the examined samples, the HPV DNA detection method, and the distribution of HPV among women in each country.Our results show the presence of HPV DNA in 26.7% (31/116) of the samples, which is in accordance with the findings of other authors in different Latin American countries, whose detection rate ranges from 0 to 49% and the average frequency is 25% ,44,54,64Classified according to their oncogenic characteristics, the prevalence of high-risk HPV types was higher than those with low risk. However, none of these genotypes were identified in this study, and the prevalence of low-risk genotypes was higher than high-risk genotypes. Methodological diversity may partly explain the differences in HPV positivity between studies. However, more importantly, it has been suggested that the viral load of HPV in breast cancer is low . Once ceThe low prevalence of HPV reported by some studies results from low sensitivity. Therefore, the present study used two variants of the PCR technique to increase the sensitivity and reduce the risk of false negatives. HPV-specific amplicons were detected in 13.8% (16/116) of samples when analyzed by one-step PCR, while the real-time PCR approach increased the positivity rate to 26.7% (31/116).The differences in HPV prevalence between studies can also be explained by false-positive results, in which contamination is a crucial factor. The present study followed a strict quality control procedure, and the results showed no signs of cross-contamination.The use of broad-spectrum primers versus specific primers is somewhat controversial since broad-range primers target the HPV L1 gene sequence that could be lost during the integration of the virus into the host genome .It has been suggested that HPV virions present in paraffin-embedded tissue samples may be destroyed during fixation and sample processing. Therefore, HPV may be difficult to detect in tissues preserved for long periods of storage . Some auIn 1999, Henning et al. reported that 46% of women with a history of HPV-16-positive high-grade cervical intraepithelial neoplasia (CIN III) lesions were correlated with both ductal and lobular breast carcinomas . WidschwYasmeen et al. made an important observation about breast cancer behavior and HPV. They reported that HPV16 is frequently present in invasive and metastatic breast cancer and less frequently in in situ breast cancer .In a retrospective study, Atique et al. (2017) reported that the incidence of breast cancer was higher among 800,000 HPV-infected patients than among the non-HPV-infected population .The mechanism by which HPV can infect mammary gland cells is still unknown. However, two main hypotheses have been proposed; they are summarized in Once they have managed to approach the mammary gland cells, the next question is how do HPV viruses penetrate the cells? One hypothesis explains that oncogenic HPV types of the alpha genus use a complex network of proteins for endocytosis and cellular transport, the latter organized by a specific subset of tetraspanins, annexins, and associated proteins such as integrins and EGFRs . IntegriIn 2019, de Carolis et al. detected the same HPV genotype in the same patient’s extracellular vesicles from serum, breast, and cervical tissue. Therefore, the authors suggested that HPV DNA was associated with mammary malignancies and was transferred to the stromal cells of the gland by extracellular vesicles .In an in vitro study, the MCF-10A cell line was transfected with HPV-18 to determine whether HPV may be the starting point of carcinogenesis in breast cancer through APOBEC3B (A3B) overexpression. Their results demonstrated that HPV infection induces upregulation of A3B mRNA and that infected cells exhibited a more malignant phenotype than parental cells since A3B overexpression caused γH2AX foci formation and DNA breakage. The expression of these malignant phenotypes was restricted by shRNA to HPV E6, E7, and A3B. These results suggest an active involvement of HPV in the early stage of breast cancer carcinogenesis through the induction of A3B . A3B expThe presence of HPV in breast tissue is an important finding, but it is not a sufficient condition to establish an etiological role for this virus in developing breast cancer or any other breast pathology. However, the results suggest a possible role of HPV in breast pathologies as a co-participant in molecular pathogenesis processes that differ from other HPV-associated neoplasms.According to the results reported in this work, it was possible to detect HPV in breast tissue with malignant neoplasms but also normal tissue. The possible mechanisms by which HPV is present in the breast tissue that would respond have been proposed for its presence in healthy tissue. However, still, there is a pending question. Is it possible that persistent HPV infections in the mammary gland could achieve carcinogenic processes as in the case of cervical cancer?Since 1992, HPV infection has been proposed as a possible risk factor for the development of breast cancer. Several authors have suggested that the increased incidence of HPV infection may be linked to environmental factors. These observations support the hypothesis of a possible infectious etiology in the development of sporadic breast cancer based on studies that report the presence of sequences of different types of high-risk HPV (oncogenic) in breast carcinoma tissues. However, to date, the results have been controversial and inconclusive. Further studies are required to demonstrate an association between HPV and breast cancer.


Patients with chronic lymphocytic leukemia (CLL), a B‐cell malignancy characterized by impaired humoral and cellular immunity, are at increased risk of developing cutaneous squamous cell carcinoma (cSCC). Human papilloma virus (HPV) is the most common sexually transmitted infection worldwide and it has been associated with various malignancies, including cSCC. Impaired cell‐mediated immunity is considered a primary risk factor in HPV‐induced cSCC. We examined cSCC lesions from CLL patients with consensus review and HPV genetic analysis to further characterize the relationship between HPV and prevalence of cutaneous malignancy in this population. Eleven patients with CLL contributed 35 cSCCs. Treatment with chemotherapy shortened the latency time to first cSCC. HPV was detected in 54% of the lesions. Among the HPV‐positive cSCC lesions, 84% of the lesions contained alpha‐genus HPV, 42% contained beta‐genus HPV, and 26% of the lesions contained both genera. There was a significant association between HPV‐containing lesions and peritumoral lymphocytic inflammation, suggesting this as a future area for further characterization. The majority of the lesions, including those with alpha‐genus HPV, occurred in sun‐exposed areas, such as the scalp and face. These findings may lead to practice‐changing recommendations for skin cancer, including the use of vaccinations to reduce HPV‐associated skin cancer. CLL is Although the incidence of cSCC among CLL patients is well established, the clinical features predisposing this population to higher risk of NMSC remain poorly characterized. A recent study from Mayo Clinic examined a large CLL cohort and established that the strongest risk factor for developing SCC was a prior history of any skin cancer . CLL‐intIn cervical carcinoma and squamous cell carcinoma (SCC) of the oropharynx and larynx, human papilloma viruses (HPVs) of the alpha‐genus, most frequently, HPV‐16, ‐18, ‐31, and ‐33, drive carcinogenesis and are categorized as HR‐HPV subtypes. HPVs found in cutaneous epithelial surfaces are most often of the beta and gamma genera. There is strong evidence demonstrating a central role for beta genus HPV, specifically HPV‐5 and HPV‐8, in skin cancer risk of immunosuppressed individuals, specifically via coincident mutation burden, including UV‐signature mutations . This paThe purpose of this study was to further characterize the role of HPV in the incidence of cSCC in CLL patients. We hypothesized that CLL patients with cSCC would harbor cutaneous HPV and the lesions would be morphologically unique to those of immunocompetent patients.22.1A retrospective cohort study was conducted at the Michael DeBakey Veterans Administration Medical Center (MEDVAMC) in Houston, TX, in accordance with institutional review board (H‐24175 Baylor College of Medicine and MEDVAMC Research Committee). We queried both VISTA pathology and CPRS clinical records systems from 2013 to 2018. The search included patients with specimens for diagnosis of cSCC as well as CLL/small lymphocytic leukemia (SLL). The clinical diagnosis of CLL identified by SNOMED search was confirmed by board‐certified hematologists and included medical chart review of clinical parameters, laboratory data, and treatment history. The original cohort of CLL patients included 40 subjects. In total, 11 subjects with both CLL/SLL, and one or more cSCC, for which there was sufficient tissue to perform molecular studies, were identified. Further analysis was performed on previously procured diagnostic tissue samples that met the predefined enrollment criteria as described above.2.2Histopathologic review of conventional hematoxylin & eosin (H&E) stained sections of formalin‐fixed paraffin‐embedded (FFPE) sections, 4–6 micrometers thickness, was performed under conventional light microscopy.Each case was independently reviewed, and findings were tabulated by two pathologists (A & B) who were blinded to each other's review as well as previously rendered clinical and pathologic diagnoses. The two reviews were compared by a project manager and if results were concordant, the first interpretation was utilized. If the reviews were discordant, a third pathologist reviewed sample while blinded to the original two interpretations. The third interpretation was used with consensus review to achieve the final study interpretation.During review, cSCC cases were examined for the specific morphologic characteristic of cSCC, presence of features of previously described HPV infection in cSCC , as well2.3oC; annealing, 1 min at 63oC; extension, 1‐minmin at 72oC, and 40 cycles were applied. The PCR products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining and UV light. The obtained putative PCR products were purified, cloned, and sequenced. The sequencing data were evaluated by computer‐assisted analysis using NCBI‐BLAST program.DNA was extracted from the FFPE samples utilizing Gentra Puregene Tissue Kit (Qiagen). Polymerase chain reactions (PCRs) for HPV , beta‐gl2.4Tumor and viral features associated with cSCC in CLL patients were examined using Chi Square and Fisher's exact test, as appropriate. Analyses were conducted using SAS software, version 9.4 .3Among the 11 CLL patients included in the study, the median age at diagnosis was 64 (range 38–87 years old); 100% were Caucasian, 100% were male, and 18% had advanced stage disease as determined by Rai criteria. Mean latency time from CLL diagnosis to cSCC was 5.02 years (range 0.3–16.5 years), after exclusion of a single patient whose latency was 33 years (> 3 SD from median of remaining cohort). Patients who received chemotherapy exhibited a shorter latency to first cSCC, approximately 1.98 years , with 8% of these displaying keratoacanothomatous cSCC features (cSCC‐KA). Briefly, the conv‐cSCCs Figure  were comTumor samples from 11 CLL patients (corresponding to 35 cSCC tumors) were submitted for viral DNA genotyping. Fifty‐four percent of the submitted samples were positive for at least one HPV virus subtype. Of these, 84% of samples were positive for alpha‐genus, which include HR‐HPV‐16/18 and verrucae‐associated HPV‐27. Forty‐two percent of the HPV‐positive lesions expressed beta‐genus HPV, including epidermodysplasia verruciformis (EV)‐associated HPV‐5. Twenty‐six percent of HPV‐positive lesions contained both alpha and beta genus subtypes  or HPV status (p = 0.45). Interestingly, 21% of the conv‐cSCC were associated with HR‐alpha‐genus viral subtypes, followed by verrucae‐associated HPV‐27 in 17% of samples examined. Cutaneous SCCs with wart‐like features were more likely to display virus association, with 67% of the samples analyzed showing HPV positivity. In contrast to conv‐cSCC, the cSCC‐wf lesions were equally likely to display alpha or beta genus specificity. HR‐HPV and HPV‐27 virus associations were less likely in cSCC‐wf  belonged to patients who did not receive chemotherapy, while 12 lesions (34% of the tumor samples) belonged to patients treated with chemotherapy. A majority of the lesions in both groups were positive for HPV, 52% in the nontreatment group and 58% in the treatment group. One of the patients had both HPV‐positive and HPV‐negative lesions, with a majority of the HPV‐positive lesions being noted after treatment with chemotherapy. One of the chemotherapy‐treated patients displayed HPV‐negative lesions; this patient was treated with anti‐CD20 therapy, while the remaining patients received purine analogues, combination therapy with anti‐CD20/purine analogue, or a BTK inhibitor.There was a preponderance of HPV‐16/18 alpha genus infections found in our study cohort but interestingly, these were not found in the anogenital regions where they are typically described. In our CLL cohort, the bulk of the SCC lesions were predominantly found in sun‐exposed areas (Figure 4It is well established that patients with CLL have a higher risk of secondary malignancies than the general population, predominantly skin cancers of keratinocyte origin. Studies have reported atypical clinical presentation of cutaneous malignancy in patients with CLL . We descIn immunosuppressed patients, HPV viruses of the beta genus contribute to the pathogenesis of cSCC , 19 and In our cohort, more than 50% of the lesions contained HPV DNA, suggesting viral contribution to cSCC pathology in CLL. We found that a majority of both conv‐cSCC and cSCC‐wf were involved by HPV, 58% and 67%, respectively. In previous studies, beta genus HPV‐15 and 38 comprised 50% of the virus infection found in skin lesions in the CLL population . We expeAll the cSCC lesions described in this study occurred in sun‐exposed areas. This was of particular interest since several of the lesions contained HR‐HPV, specifically HPV‐16 and ‐18, both of which are characteristically described as occurring in anogenital skin. It is well accepted that in immunocompetent patients, UV damage constitutes the main pathogenic driver for cSCC. Despite HPV infection and immunocompromised state, UV damage remains an important contributor of cSCC pathogenicity in CLL patients.Lymphocytic infiltrates have been previously described in the cSCC of CLL patients . In our Taken together, we propose that a diagnosis of CLL leads to a dysfunctional immune system, with both elements of humoral and cellular immunity affected , 10, 23.A major strength of this study is the detailed histomorphologic description of cSCC in CLL, as well as characterization of virus genus and subtypes. Although the incidence of cSCC and prevalence of HPV in cSCC has been described in CLL patients, histomorphologic characterization has been limited. Moreover, we performed highly sensitive methodology to characterize HPV association. Although we report greater variability in genus subtypes than other studies, we also report that 43% of the lesions examined did not contain any virus, supporting a robust, high‐yield methodology with low cross contamination. Like other studies of its kind, a limitation of this study is the small sample size, comprised exclusively by Caucasian males, making generalizability of our findings difficult. An additional limitation was our inability to phenotype the lymphocytic infiltration to determine whether a characteristic signature of inflammatory cells predicts the prevalence of cSCC or genus‐specific HPV association. Despite these limitations, it is important to note that some of the trends described in prior studies with larger sample size were also observed here; specifically, reduced time to cSCC after chemotherapy treatment and lymphocytic infiltration of CLL lesions. These limitations can be mitigated in future studies by drawing from a non‐VA cohort that would include more females and a more ethnically diverse population, as well as revising the scope of pathology review to include flow cytometry.Newer therapies have improved progression free‐ and overall survival in the CLL population. As a result, secondary malignancies, such as cSCC, are likely to become important comorbidities in long‐term outcomes of CLL patients. A better understanding of the risk factors contributing to cSCC in this population would be important as we manage long‐term complications of disease and therapy. A confirmation of the role of HPV, particularly HR‐HPV, could lead to practice changing recommendations, such as a newly defined role for vaccinations in reducing HPV‐associated malignancies.PH collected and annotated the clinical data. MH, SM, BK, and DNC performed pathology. QH, ST, and PR conducted HPV DNA assays. MH and PH performed analysis of data. GR and IRS conducted statistical analysis. GR, DNC, and IRS designed and supervised the research study and interpretation of data. IRS wrote the manuscript. All authors reviewed and approved the final text.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.IRS was supported by the American Heart Association FTF‐17400011.


This study aimed to evaluate the correlation between HPV VL and PAX1m and its effectiveness in predicting cervical lesions. (2) Methods: A total of 476 women referred to colposcopy for abnormal cervical screening at the Peking University People’s Hospital between November 2020 and November 2021 were enrolled. PAX1m and HPV VL were determined by QMSP and BMRT-HPV reports type-specific VL/10,000 cells, respectively. (3) Results: PAX1m was significantly increased in HSIL, especially in cervical cancer, but there was no significant difference between cervical intraepithelial neoplasms 1(CIN1) and CIN2. However, HPV VL significantly differed between CIN1 and CIN2 but not between CIN3 and cervical cancer. In general, PAX1m positively correlated with all hrHPV VL, mainly in the HPV16/18 VL (p < 0.001), but had no relationship with the other 12 types of hrHPV VL. PAX1m had the highest specificity in diagnosing CIN2+, followed by HPV16/18 VL, which are higher than cytology ≥ASCUS. (4) Conclusions: Hypermethylation of PAX1 is associated with high HPV VL, especially HPV16/18, and both present advantageous specificity in detecting CIN2+.Background: PAX1 methylation (PAX1 Persistent infection with a high-risk type of human papillomavirus (hrHPV) can lead to cervical precancerous lesions and invasive cancer of the uterine cervix. Primary hrHPV testing is currently accepted for cervical screening over cytology based on its superior sensitivity and greater reassurance of negative predictive value for high-grade squamous intraepithelial lesions (HSIL) . HoweverINK4a  copies/10,000 cells, where Cn HPV is the quantity of HPV DNA, and Cn TOP3 is the number of human cells. The experimental procedure was conducted according to the kit manufacturer’s instructions. The detailed process was described by Dong and Duan , HPV16/18 VL[Log(HPV16/18)], other 12 types of hrHPV VL[Log(other-hrHPV)] and PAX1enring W , and difenring W . Two-taiThe median age of the 476 women was 40 years (range 34–49 years). The BMRT assay revealed that 285 (59.9%) of the women had a single HPV infection, 106 (22.3%) had multiple infections, 193 (40.5%) of the women were HPV16/18+, and 198 (41.6%) had other 12 types of hrHPV type. As for the pathological results, there were 100 cases of chronic cervicitis, 57 cases of CIN1, 118 cases of CIN2, 171 cases of CIN3, and 30 cases of cervical cancer. m levels were statistically different in different degrees of cervical lesions. PAX1m was significantly increased in HSIL and above, especially in cervical cancer (p < 0.001), although there was no significant difference between CIN1 and CIN2(p = 0.334). HPV VL also increased with CIN progression, and there was a significant difference between CIN1 and CIN2 but not between CIN3 and cervical cancer. The HPV16/18 VL was significantly higher in women with CIN3 than in those with CIN2 (p < 0.05), but there was no significant increase in the other 12 types of hrHPV VL . It is suggested that PAX1 hypermethylation has a high VL of all hrHPV, and HPV16/18 in particular, but had no significant difference in the other 12 types of hrHPV VLs (p = 0.948)  .m, when the interval with 5.69 (1.09–7.72), OR for CIN3+ reach up to 15.74 (8.11–30.55). With regards to the VL of all hrHPV and HPV16/18, when the moderate intervals was [4.75 (4.10–5.29)] and [4.76 (4.06–5.25)], the OR of CIN2+ were 10.08 (5.25–19.35) and 7.54 (3.15–18.04), which was higher than that with low intervals [3.24 (1.83–5.73) and 2.16 (1.18–3.94)]. While in the high intervals with [6.02(5.31–8.53)] and 5.93 (5.26–8.21), the OR of CIN2+ decreased to 5.03 (2.79–9.07) and 4.29 (2.10–8.76), and the same trend with other 12 types of hrHPV VLs was observed  > HPV31/33/52/58+ (OR: 5.25) > Other HPV+ (OR: 1.44). The OR for CIN2+ increased at Low [11.49 (10.45–16.26)], Moderate [9.21 (7.74–10.41)], and High [5.69 (1.09–7.72)] intervals of PAX1observed .m for identifying CIN2+ was 10.63, with an AUC of 0.64 , respectively. Regarding HPV VLs, all hrHPV VL had an optimal cutoff of 3.32 copies/10,000 cells (log10-transformed) for detecting CIN2+ . HPV16/18 VLs had an AUC of 0.66  and an optimal cutoff of 2.34 copies/10,000 cells for identifying CIN2+, see According to the ROC curves, the optimal cutoff of PAX1m (cut off ≤ 10.63) (Se: 46.4%) is lower than cytology (ASCUS+) (Se: 54.5%), hrHPV test (Se: 90.6%), and hr-HPV VL  (Se: 78.1%), but comparable to the HPV16/18 genotype (Se: 50.2%) and HPV16/18 VL  (Se: 48%). However, PAX1m (cut off ≤ 10.63) had the highest specificity (Sp: 86.6%) in diagnosing CIN2+, followed by HPV16/18 VL  (Sp: 84.1%), both of them higher than cytology ≥ASCUS(Sp: 72.6%)  .m and HPV16/18 VL exhibited higher specificity than cytology in detecting CIN2+.In our study, we found that hypermethylation of PAX1 is associated with a high viral load of hrHPV, especially HPV16/18. Women with hypermethylation of PAX1 had a significantly higher incidence of CIN3, especially cervical cancer, compared with CIN2 and below, but there was no significant difference between CIN1 and CIN2, indicating its potential role in the late status of cervical lesions. On the other hand, HPV VL had a significantly higher incidence of CIN2+ compared with CIN1 and below, but once processed to CIN3, HPV VL(except HPV16/18) did not increase with disease progression anymore, indicating that the replication and release of HPV particles was replaced by active proliferation of the disease at advanced stages of the cervical lesion. We also demonstrated that PAX1An association between hrHPV VL and the severity of cervical disease was first described in 1999 . Since tp < 0.001), but not between high-grade lesions and cervical cancer. We found that HPV16/18 VL was positively correlated with the grade of cervical lesions  but had no significant difference between CIN3 and cervical cancer. In addition, no significant correlation was found between the other 12 types of hrHPV VL and cervical lesions, but still of great significance was differentiating CIN2+ from CIN1 and below lesions, just as Wang M et al. reported . There was no statistically significant difference of specificity in the diagnosis of CIN2+ between PAX1m, HPV16/18 genotype, and HPV16/18 VL (p > 0.05), indicating both of them present advantageous specificity in detecting CIN2+.In 2020, the American Cancer Society (ACS) proposed updated screening guidelines that recommended women initiating cervical cancer screening at the age of 25 years and undergoing primary HPV testing every 5 years until the age of 65 . DespiteThis study has limitations. It was performed at a single center, and the sample size was relatively small, mainly because the participants were all from those referred to colposcopy with abnormal cervical screening. The correlation between PAX1 methylation and HPV VL and the relationship with the disease severity was not analyzed for each HPV genotype, as the number of cases involving each type of HPV would be smaller. In addition, HPV integration has not been explored. Some researchers believe that samples containing integrated HPV tend to have lower HPV VLs than that in purely episomal or mixed forms ,29, althHypermethylation of PAX1 is associated with high HPV VL, especially HPV16/18, and both of them present advantageous specificity in detecting CIN2+. HPV16/18 VL appears to have an overall specificity of 84.1% (95%CI: 77.4–89.4%) and PAX1 methylation with a specificity of 86.6% (95%CI: 80.3–91.5%) for detecting CIN2+.


Among sub-Saharan African women, cervical cancer is steadily increasing with more than 75,000 new cases and 50,000 deaths annually. Due to the vast ethno geography variation, Africa harbors heterogeneous genotypes of HPV. High-risk HPV [hr HPV] genotypes such as hr HPV-16,−18,-35, and−52 are abundantly reported in sub-Saharan Africa. The purpose of this systematic review and meta-analysis is to generate an evidence on the prevalence and the genotype distribution of hr HPV among sub-Saharan African countries.The review was conducted by following the preferred reporting items for systematic reviews and Meta-analysis. PubMed/Medline, Embase, Scopus, Google Scholar, Heath Technology assessment and Cochrane Library databases were used to retrieve published original studies between 2001 and 2021. It included studies that used PCR-based or hybrid testing to assess the presence of HPV DNA in a cervical biopsy, cervical swelling, and vaginal swelling. Statistical software for data science (STATA V16) software using a random-effects model was used to determine the pooled prevalence and type-specific distribution of HPV with 95% confidence intervals (CI). The I-squared statistic was used to describe the level of heterogeneity. The study protocol is registered on PROSPERO with reference number CRD42022311157.The review included 27 studies conducted in 19 sub-Saharan countries. A total of 16,506 study participants from 27 studies were included in a systematic review and 5,303 of them were infected with the hr HPV infection. Out these, only 3,075 of them were eligible for meta-analysis. The incidence proportion of estimatesof hr HPV infection among study participants with different health conditions ranges from 10.7 to 97.2% while the pooled incidence proportion of estimates is 34% (95%CI: 29–39). Among 3,075 women, 424 (13.8%), 305 (9.9%) and 279 (9%) were infected with HPV-16,−52 and−18, respectively. HPV-16 and−52 are the main genotypes causing the hr HPV infection in the Eastern and Southern African sub-contents, whereas HPV-16 and−35 are the main genotypes in the Western African countries.Depending on several factors, especially women's health conditions, the high rate of hr HPV infection with inconsistent genotype distribution shows that it is a growing public health challenge in sub-Saharan African countries. Therefore, to implement a vaccination-based prevention strategy and be effective, considering factors associated with hr HPV infection is crucial. Human papillomavirus (HPV) infection is a sexually transmittable disease caused by Human papillomavirus . The virCervical cancer (CC) is a malignant neoplasm arising from cells originating in the cervix. It is usually a slow-growing cancer that may not have clear symptoms. Consistent with research, 99% of CC cases are directly related to the hr HPV genotypes, and about 70% of them are caused by HPV-16 and HPV-18, worldwide . But thiCC caused by hr HPV covers 5.2% of all cancers worldwide. This means that out of the total cancer patients in high income and low income countries, 2.2 and 7.7% are CC patients, respectively , 7. In sStudies in the southern African countries have shown that the hr HPV genotypes that mainly cause CC vary from country to country. For example, HPV-16 and−52 in South Africa and Tanzania , HPV-16 Although hr HPV infection is a global public health problem, there is a genotype-based effective vaccine . There aThis means that to buy a vaccine and ensure its effectiveness, having valid evidence regarding the genotype distribution of the virus in a given locality is critical. Therefore, this systematic review and meta-analysis have prepared to fill the current information gaps regarding the prevalence and genotype distribution of hr HPV infection in sub-Saharan countries.The review protocol was prepared according to a statement recommendation made by the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) in 2015  22). Be. Be22). We identified publications by systematic searches of PubMed/Medline, Embase, Scopus, Google Scholar, Heath Technology assessment and Cochrane Library, from August 1 to September 15, 2021. The identified records were downloaded with an appropriate format and linked to the Endnote. The Boolean operators “AND” and “OR” were used to link keywords/terms and fetch publications from PubMed / MEDLINE (NCBI) databases. The Medical subject heading (MeSH) terms that we used are indicated in We applied several inclusion and exclusion criteria that were defined a priori to the records identified. Publications eligible for inclusion were those studies that reported the prevalence of hr HPV infection among sub-Saharan African countries as a primary outcome. We used the following criteria to include the texts in this analysis. These are: If the full article and its related data are accessible: If at least 25 women participated in the study; If cervical histology results are confirmed by exfoliated cervical cells or fixed/fresh biopsy results; If the hr HPV prevalence is calculated and at least five genotypes identified. In addition to that, we included studies that used either the amplified polymerase chain reaction (PCR) or non-amplified genotyping methods as a method of detection. There were no restrictions on PCR primers' utilization. We also included studies conducted on the HPV DNA tissue sources including fixed or fresh biopsies and/or exfoliated cells. Similarly, we excluded abstracts with unrelated data, non-English language publications, and published without original data . We also excluded studies that reported results from a case series or case report, and qualitative data.First of all, we identified relevant articles. Then, we removed duplicated publications using Endnote, version x7 , and manual screening. The first two authors (AS and NA) independently appraised the titles and abstracts of the retained publications and selected the relevant articles for possible inclusion in the review. Accordingly, studies conducted to determine the prevalence of hr HPV and genotype distribution were kept for review and further analysis. Correspondingly, other authors  reviewed the full text to assess the methodological quality of studies using the National Institute of Health (NIH) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies guideline . FinallyThe NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies checklist was used to evaluate the methodological quality of the retained publications. Accordingly, all studies that fulfilled at least 50% of the quality requirement as per the average positive score of the appraisers were considered for this review .We used the data abstraction format prepared in a Microsoft excel spreadsheet to extract all the necessary information for systematic review and meta-analysis. Two non-blinded investigators (AS and NA) extracted the data independently, reviewed it for discrepancies, and finally reached a consensus through discussion. The following variables were extracted: study design, year of publication, sample size, continental category of the study (according to WHO classification), study location, detection methods, clinical sample type, prevalence of hr HPV and type specific prevalence (genotypes) .Statistical pooling for incidence proportion of estimates was performed according to the random-effects model using a Statistical software for data science (STATA V16). The random-effects model of analysis was assumed since the studies identified were observational in nature and they had both clinical and methodological variabilities.Q and I2 tests as well as Q/df (degree of freedom) ratio results. Thus, the Cochrane Q test (p = 0.1), Q/df = 1, and I2 = 50% were considered as cutoff points to mark heterogeneity and to select the effective model of analysis. The forest plots were employed to present the pooled prevalence of hr HPV infection and genotypes. In line with this, subgroup analyses were carried out to explain patient features in sub-groups with the potential to account for the differences in the effect sizes of the hr HPV genotypes. Publication bias  was assessed by a graphical inspection of funnel plot  Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies guideline were retrieved .For systematic review, 27 studies from 19 sub-Saharan African countries, totaling 16,506 study participants were included. Out of these, 5,303 were infected with hr HPV genotypes. Similarly, among the selected 27 studies for systematic review, only 15 studies involving 7,924 study participants had complete information on the genotype distribution of HPV. Of these, 2,188 women were from East Africa and 1,660 were from southern Africa. The remaining 4,076 women were from the West African countries .Our systematic review reports hr HPV prevalence that varies by geographic location. Indeed, we also report that the prevalence and genotype differences have been seen among studies conducted in a country .Among the sub-Saharan African women who visited the health facilities for gynecological problems, the lowest prevalence of hr HPV infection was 10.7% (95% CI: 5.4–19.9) . HoweverOut of 5,303 study participants infected with hr HPV, we included 3,075 women from 15 studies who met the inclusion criteria (data on more than 5 genotypes). Accordingly, 424 (13.8%) women were primarily infected by HPV-16. As well as, 305 (9.9%) and 279 (9%) women were infected by HPV-52 and HPV-18 . Full deQ = 1,180.2, p < 0.1, df = 22, I2 = 98.3%). The heterogeneity might be observed as a result of combining different studies that used different laboratory methods of diagnosis  and study participants who had different levels of health condition during the time of enrollment.The collective analysis of 23 studies indicated that the pooled prevalence of hr HPV in sub-Saharan African countries was 34% (95%CI: 29–39) . The anap = 0.100). Additionally, Begg's correlation test revealed no evidence of the publication bias .Graphical visualization of the funnel plot found its symmetrical appearance which gave us hint about absence of the publication bias . This viWe conducted the subgroup analysis of the prevalence of hr HPV infection by dividing the countries into East Africa, West Africa, and Southern Africa sub-groups. As compared to the total sub-group prevalence of the hr HPV infection 34% (95% CI: 23–46); the highest prevalence, 53% (95% CI: 37–69) was found among studies in Southern African countries. However, contrary to this, the lowest sub-group prevalence of hr HPV, 28% (95% CI: 20–36) was observed among the East African countries .The type-specific sub-group prevalence of hr HPV from meta-analysis was done by sub-grouping the sub-Saharan African countries into the Eastern, Southern, and Western sub-Saharan African regions.Studies conducted in Ethiopia (3), Tanzania (2), Kenya (2), Uganda (2), and Botswana (1) were considered for meta-analysis to determine the type-specific sub-group prevalence of hr HPV in East Africa. Accordingly, 830 (27%) out of 3,075 women who were infected with hr HPV. Moreover, more than a quarter (224) of these 830 women are infected with HPV-16 and HPV-52 .In the meantime, a total of 1,115 (36.2%) out of 3,075 women were from Southern African countries. As seen in the eastern part of Africa, similarly, studies show that about one-fifth [226 (20.3%)] of women infected with HPV-16 and HPV-52 . In contIn general, the distribution of hr HPV genotypes from one sub-group to another is inconsistent with the exception of the primary and tertiary genotypes of the virus . TherefoGetting up-to-date and organized information about the pooled and type-specific prevalence of hr HPV helps to identify the underlying burden of the disease, evaluate the role of HPV vaccines in the prevention of the disease, and develop home-based diagnostic tests. With all these points in mind, this systematic review and meta-analysis have calculated the representative estimations of the type-specific prevalence and genotype distribution of hr HPV in sub-Saharan African countries.According to this systematic review, the prevalence of hr HPV varies from 10.7%  to 97.2%The pooled prevalence of hr HPV in the sub-Saharan African countries is 32.3% which is lower than the previously reported 50.5% pooled prevalence in 2015 . In a reThe type-specific prevalence of hr HPV among the sub-groups of sub-Saharan African countries was not uniformly distributed. According to this systematic review and meta-analysis, HPV genotypes HPV-16,−52,−18,−39, and−31 are widely distributed in the eastern part of Africa. When we look at studies to ensure the consistency of the genotype distribution, for example, in a study in Ethiopia, the most prevalent genotypes were hr-HPV- 16,−52,−18,−58, and−45 , which iThis meta-analysis has revealed that, in the Southern African region, the pooled distribution of HPV-16,−52,−18,−56, and−58 genotypes have been slightly different from that of East Africa. However, at the country level, in a study involving high school students in South Africa, the genotype distribution was significantly different. HPV-58 was the most commonly detected type, followed by HPV-52,−45,−16, and−18 . SimilarOn the other hand, the pooled HPV genotype distribution in the West African countries has been also characterized in a different form from the East and southern African sub- regions. That is, HPV-16,−35,−18,−31, and−45. In Benin, among Female Sexual Workers (FSWs) the three most common genotypes are HPV-58,−16, and−52. Thus, we have seen that the distribution of HPV genotypes differs between key vulnerable populations and the general population (The study had the following limitations. The study reviewed and analyzed studies conducted using an observational study design i.e., cross-sectional study design. This type of study design is less likely to predict causal-effect correlation. As a result, we are often exposed to bias, which leads to wrong conclusions. Authors were reviewed and realized that studies included in the review have highly variable sample size. This might potentially leads to interfere the nature of the study's representativeness. It also included studies that were conducted using different diagnostic tools. This may cause differences in sensitivity and specificity.HPV is currently increasing in sub-Saharan Africa countries and it is becoming a major public health problem. Similarly, the distribution of genotypes is inconsistent at the continental level and even varies from place to place within a country.HPV infection has a known and effective vaccine. But the production of the vaccine and its effectiveness will be measured when a vaccination program based on the type of virus is launched in a country. Thus, taking into account the key factors mentioned above, each country should have its own information on the prevalence of HPV infection and the type of virus. Doing so will help to develop and implement an effective vaccine-based disease prevention program.Conceptualization: AS. Data curation and formal analysis: AS and NA. Investigation, methodology, and validation: AS, NA, TG, BS, AnM, and AdM. Software and writing—original draft: AS. Supervision and writing—review and editing: NA, TG, BS, AnM, and AdM. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.