Rural Veterans at risk of fracture due to osteoporosis remain underdiagnosed and undertreated, in part due to location-related barriers to accessing care. Despite lowered cost and travel barriers to osteoporosis care through implementation of a telehealth model directed at rural at-risk Veterans that took advantage of many strengths of the VA’s healthcare system, only 30% of eligible Veterans accepted care. To understand low acceptance, we conducted 39 semi-structured telephone interviews with Veterans eligible for the clinic, including 19 who accepted screening and treatment, 12 who completed screening but declined treatment, and 8 who declined screening and treatment. Veterans who opted to be screened and/or treated for osteoporosis did so because: it was recommended by the VA; they were interested in learning more about their health; thought they may be at risk of osteoporosis; or believed screening would not cause them harm. Conversely, Veterans refused screening or treatment because of past negative experiences with medications, both bone and non-bone; a wish to not put anything else into their bodies; or the belief that their bone loss is not severe enough to warrant treatment. Outside medical professionals and peers influenced Veterans’ decisions to not take or alter their treatment. Cost and travel distance remained a barrier for Veterans who did not live near a VA facility with the necessary screening and treatment infrastructure. Many barriers to osteoporosis care remain despite efforts to remove them. Delivery systems must account for both instrumental and social access to care to reduce fracture risk.


While most HPV infections are asymptomatic and clear spontaneously, persistent infection with high-risk HPVs is associated with cervical cancer and with increased risk of HIV acquisition. Although several hypotheses have been proposed to explain this phenomenon, none has been confirmed. Our aim was to investigate the expression of host factors involved in the susceptibility to HIV infection among HPV-infected women. Cervical samples were collected to characterize the expression levels of HIV susceptibility markers in the mucosa of HPV-infected compared with HPV-uninfected women. No differences in the frequency of CCR5+, integrin α4β7+, activated and memory CD4+ T-cell were detected between the groups. We additionally evaluated the expression levels of genes involved in innate immune responses and in cell adhesion. HPV infected patients expressed higher levels of TLR9 and lower levels of pattern recognition receptors that recognize RNA . We also detected an impaired IFN pathway, with an increased Type I IFN and a decreased IFNα2 receptor expression. HPV+ samples displayed reduced expression of genes for adherens and tight junctions. Taken together, these results suggest that although HPV infection does not result in the recruitment/activation of susceptible CD4+ T-cell in the female genital tract, it leads to changes in the innate antiviral immune responses and in cell adhesion that are likely to favor HIV infection. Human papillomavirus (HPV) is the agent of a sexually transmitted infection (STI) occurring in ~12% of the world's population , and a rHaemophilus ducreyi and bacterial vaginosis (BV) cause an increase in the number of CCR5+, activated and α4β7+ integrin CD4+ T-cells in FGT  [such as Toll-like receptors (TLRs) and RIG-like receptors (RLRs) ] expressed by FGT cells initiate inflammatory responses through production of type I interferon (IFN-I) and other cytokines/chemokines , and thiTo better understand the relationship between HPV infection and HIV susceptibility, we analyzed immune responses at the FGT during HPV infection in order to identify mechanisms that could favor HIV acquisition. We assessed the frequency of HIV susceptible cells in cervical mucosa of HPV-infected women. Additionally, we evaluated the expression levels of innate immunity receptors (TLRs and RLRs), pathway mediators, cytokines, IFN-I, and IFN receptor in cervical cells from these patients, and observed changes in the innate immune responses in HPV-infected cervical tissues that may be responsible for HPV immune escape and an increased HIV susceptibility. We also evaluated mRNA expression of cell adhesion markers in the presence of HPV, observing a reduction, albeit not significant, in some of these such as E-cadherin and ZO-1, which suggest changes in FGT's capacity to function as a strong barrier against microorganisms.The study was approved by the Committee of Ethics in Research from the Instituto de Pediatria e Puericultura Martagão Gesteira  (acceptance number: 49035215.4.0000.5264). A total of 62 women were enrolled from the Instituto de Ginecologia of Universidade Federal do Rio de Janeiro  during their medical consultation in the Cervical Pathology and Colposcopy outpatient  and in the gynecology ambulatory. All of the participants provided an informed written consent. Exclusion criteria were: <18 years-old; pregnancy; menstruation at the day of the collection; abnormal cervical cytology but undetectable HPV by PCR; insufficient amount of sample; presence of chlamydia, trichomoniasis, syphilis, gonorrhea, HIV  (26). FrInitially, fresh cervical cells from seven HPV- and six HPV+ women were stained with CD3-FITC (UCHT-1), CCR5-PercP Cy5.5 (2A9) and CD4-APC (RPA-T4) antibodies , processed using BD Accuri C6 flow cytometer and data were analyzed using BD CSampler Software v.1.0.264.21. This equipment is set with a pre-optimized voltage and it is able to detect simultaneously epithelial cells and lymphocytes, allowing us to determine the percentage of lymphocytes among all cells collected from the cervix . A FACS Part of the collected cervical cells stored at −80°C in RNA Later (Invitrogen) were used for mRNA extraction using MasterPure Complete DNA & RNA Purification Kit  following manufacturer's instructions. The cDNA was synthesized from 1 μg of RNA using High Capacity cDNA Reverse Transcript Kit .Ct−Δ method.We designed customized Taqman array plates using the following probes: GAPDH = Hs02758991_g1; RNF114 = Hs00218782_m1; RNF125 = Hs00215201_m1; UCHL1 = Hs00985157_m1 (7 HPV+/HSIL and 7 HPV- patients were assessed); TLR3 = Hs01551078_m1; TLR4 = Hs00152939_m1; TLR7 = Hs00152971_m1; TLR9 = Hs00152973_m1; DDX58 (RIG-I) = Hs00204833_m1; IFIH1 (MDA5) = Hs01070332_m1; IFNA2 = Hs00265051_s1; IFNB1 = Hs01077958_s1; IFNAR2 = Hs01022060_m1; TRIM25 = Hs01116121_m1 (11 HPV+/HSIL and 15 HPV- patients were assessed); IL-8 (CXCL8) = Hs00174103_m1; IP-10 (CXCL10) = Hs00171042_m1; MIP-1α (CCL3) = Hs00234142_m1; MIP-1β (CCL4) = Hs99999148_m1; IL-6 = Hs00174131_m1; IL-1β = Hs01555410_m1; IL-10 = Hs00961622_m1; MCP-1 (CCL2) = Hs00234140_m1; ZO-1 (zonula occludens-1) = Hs01551867_m1; Occludin = Hs01049883_m1; Claudin 1 = Hs00221623_m1; Claudin 2 = Hs01549234_m1; Claudin 4 = Hs00976831_s1; E-caderin = Hs01023895_m1 (9 HPV+/HSIL and 12 HPV- patients were assessed); TNFα = Hs00174128_m1 (4 HPV+/HSIL and 8 HPV- patients were assessed). IFNγ expression level was measured with SYBR Green using target primers: F – 5′-GGAAAGAGGAGAGTGACAGAAA-3′; R – 5′-TTGGATGCTCTGGTCATCTTTA-3′ and GAPDH primers: F – 5′-ACTGTGGATGGCCCCTCCGG-3′; R – 5′-GGGGACACGGAAGGCCATGC-3′ (10 HPV+/HSIL and 10 HPV- patients). Since we did not have enough cDNA from all patients, the number of patients used for the different analyses was variable. Reactions were performed in triplicates using a 7500 Fast Real-Time PCR System (Applied Biosystems). Target genes were normalized to the housekeeping GAPDH gene and the relative expression was calculated converting the difference in cycle thresholds (ΔCt) using the 2https://string-db.org/], which includes 24,584,628 proteins from 5,090 species. STRING interaction maps are based on published direct  and indirect  associations. Genes were organized using the interaction graph viewer Medusa v.3.0 [https://sites.google.com/site/medusa3visualization/]  of each gene. nα assumes values between 0 and 1 and is defined as the ratio between HPV+ median expression and the sum between HPV+ and HPV- median expression. Thus, nα <0.5 implies that expression in HPV+ group is lower than the HPV-, while nα > 0.5 implies that HPV+ expression is higher than HPV- , ASC (n = 3), LSIL (n = 3), HSIL (n = 12), or tumor (n = 1). The predominant HPV subtype was HPV16 (7/24), followed by HPV31 (6/24) and HPV66 (3/24).Clinical and demographic information of the patients enrolled in the study are shown in p = 0.222, p = 0.626, Since there is evidence that HPV infection facilitates HIV acquisition, we assessed the proportion of CD4+ T-cells in the cervical samples . In this context, although the median percentage of CD4+ T-cells in all cell sample appeared higher in HPV+ samples, this difference was not statistically significant  and those with high-grade squamous intraepithelial lesion (HSIL) (n = 7), and we observed no significant differences (data not shown).To further characterize cervical CD4+ T lymphocyte subsets, in addition to CCR5, we assessed CD38, CD45RA, integrin α4β7, and integrin αEβ7 surface expression. Our results indicate that HPV infection did not significantly affect the frequency of these lymphocyte subsets in the mucosa . Activatp = 0.037, In addition to cervical samples, we characterized CD4+ T-cell subsets from peripheral blood and observed no significant differences between groups in cell subset frequencies , or in tNext, we decided to evaluate the expression levels of genes involved in pathogen recognition and innate immunity of cervical cells by qPCR, in order to assess if any differences detected could account for the higher HIV susceptibility in HPV-infected women. In this context, TLR3, which recognizes dsRNA, was not expressed in nine out of 11 HPV+ women (81.8%), while only two healthy controls did not express this receptor (13%) . TLR7 t. On averp = 0.9596)  . We also 0.9596) . Finally 0.9596) . However 0.9596) . AltogetHPV is known to modulate cytokine production, impairing pro-inflammatory response, possibly as a strategy to evade the host immune system, ultimately resulting in development of lesions in patients in which this modulation is successful . FocusinIt has been described in the literature that inflammation leads to increased tissue permeability , 32, 33.in silico information, it helps us to visualize the interconnections among all of these targets. In the heatmap, built with expression levels of these genes, we can observe clusters that are differentially expressed in HPV-infected mucosa. A cluster of upregulated genes in HPV+ women contained IFNs-I, IFNγ, TNFα, and TLR9, while genes involved in RNA recognition  and adhesion molecules formed a cluster of downregulated transcripts  on total and memory CD4+ T-cells from the cervix, no differences were detected between HPV infected and uninfected patients, similar to previous studies . HoweverWhen we examined the blood samples, as expected, we observed no major differences in CD4+ T-cell phenotypes between HPV infected and uninfected samples. However, patients with HSIL had significantly higher expression of β7 on systemic, activated, memory CD4+ T-cells compared to HPV uninfected samples. Due to the nature of HPV infection we do not expect it to have a significant effect on systemic immune response ; howeverWe also evaluated the expression levels of PRRs that play a key role in innate immune response. For TLR3 and TLR7, that recognize dsRNA and ssRNA, respectively, both were downregulated or not expressed in HPV-infected women. Our results are corroborated by a case-control study comparing cervical biopsies from healthy and carcinoma samples, where a reduction of TLR3 expression—both at mRNA and protein levels—was found in the presence of HPV . HoweverRIG-I/DDX58 and MDA5/IFIH1 are cytoplasmic receptors that recognize RNA of different structures and lengths . There iWhen we looked at the antiviral Type I IFNs, IFNα2, and IFNβ1, their expression was significantly upregulated in hr-HPV infected samples. These results were not expected since it is known that HPV reduces IFN-I production mainly via viral proteins E6 and E7 , 53. We In addition to type I IFN, we analyzed the expression levels of cytokines previously associated to HIV susceptibility and/or differentially expressed upon HPV infection  , 58, 59,in vitro HIV infection assay using patients mucosal cells. Despite these, we analyzed four aspects of HPV infection which could render the host more susceptible to HIV: (1) presence of HIV target cells in the HPV-infected mucosa; (2) expression of cytokines; (3) expression of innate immunity genes; (4) expression of cell adhesion molecules by mucosal cells. As far as we are aware, this is the first study that aimed at these four aspects simultaneously in this population. Our data indicate no differences in amounts or phenotypes of T-cells in HPV-infected mucosa, but characterizes the effect of hr-HPV infection on mucosal immune responses and permeability. Furthermore, the joint analysis of gene interaction and expression revealed clusters of HPV-upregulated—antiviral and inflammatory cytokines—and downregulated genes—PRRs that recognize RNA, IFNαR2 and adhesion proteins—suggesting that HPV either directly or indirectly modulates genes with similar functions. Altogether, mucosal inflammation, reduced ability to respond to immune stimulation and decreased expression of cell adhesion markers could facilitate HIV establishment in the female genital tract. Considering that the effects we observed are specific to hr-HPV infection, this further highlights the need for better HPV vaccination coverage, especially in the areas of high HIV incidence.Our study has several limitations including a small number of patients, the lack of histological staining, cytokine protein measurements and All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Comitê de Ética em Pesquisa do Instituto de Puericultura e Pediatria Martagão Gesteira da Universidade Federal do Rio de Janeiro (CEP/IPPMG). The patients/participants provided their written informed consent to participate in this study.AB, AG, and EM: conceptualization and writing original draft. AB, CP, ÂM, YF, and GA: sample collection. AB, LG, AS, AG, and EM: flow cytometer experiment and analysis. AB, CP, AG, and EM: gene expression experiment and analysis. JA, CC, MS, AG, and EM: funding acquisition. AB, AG, EM, CC, LG, AS, and MS: writing, reviewing, and editing. 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.


The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness ‘myosinopathy’ patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered. Titin is considered an integrator of muscle cell proteins but direct evidence is limited. Here, titin is inactivated in adult mouse muscles, which causes sarcomere disassembly, protein mis-expression and force impairment, recapitulating key alterations in critical illness myopathy patient muscles. Several different alternative transcripts are generated by the TTN gene, including full-length isoforms (3–3.8 MDa) termed N2B, N2BA (in the heart) and N2A , as well as the small, Z-disc-anchored Novex-3 isoform (~0.7 MDa)4. Moreover, the recently discovered Cronos isoform arises from an alternative transcription start site >150 kilobases (kb) downstream of the canonical promoter, in Ttn intron 2395. Ever since its discovery, TTN has been thought to hold the sarcomere together and provide it with stability and elasticity7. The role of TTN for the elasticity and stretch-dependent passive tension of myocytes is now well established9. Titin-based elastic force also complements and regulates the contractile force generated by actin and myosin11. Furthermore, TTN determines the length of the sarcomeric thick filaments12 and is important for de-novo sarcomere assembly17. It has also been frequently suggested that TTN is relevant for the maintenance of the mature sarcomere. This function has been more difficult to assess and details have remained unclear. However, TTN is necessary for cardiac development and its absence leads to early embryonic lethality18.Skeletal muscles are required for gait, posture, and many additional functions. As much as 80–90% of the volume of an adult skeletal muscle cell is occupied by the contractile units known as the sarcomeres. These structures are multi-protein assemblies of paracrystalline order, which essentially consist of three filament systems bordered by Z-discs: actin-based thin filaments, myosin-based thick filaments, and titin filaments. The giant titin proteins (TTN) span half-sarcomeres from the Z-disc to the M-bandTtn promoter in adult skeletal muscles. Our approach differs from previous strategies, because earlier TTN-deficient rodent models either preserved the structural continuity of the protein, e.g., by deleting only the cardiac N2B-element19, certain I-band immunoglobulin (Ig) domains21, the PEVK-segment23, or the distal M-band titin24, or they were designed to partially or completely eliminate TTN at early stages of development25. In our model, we find that inactivation of N2A and Novex-3 TTN in adult skeletal muscles reduces muscle mass, contractile strength, and myocyte stiffness, causes sarcomeric disintegration along with Z-disc aggregation and streaming, and results in preferential loss of thick-filament proteins. TTN-depletion also causes upregulation of titin-binding proteins involved in mechanotransduction and activates components of the protein quality control (PQC) machinery, providing additional insight into the role of TTN in sarcomere maintenance and protein turnover. Strikingly, expression of Cronos is unable to prevent cardiac embryonic lethality or disassembly of adult skeletal muscle sarcomeres. Our findings prove the long-hypothesized crucial function of TTN for protein homeostasis in mature muscle sarcomeres.Here, we report an inducible, conditional TTN-knockout (KO) mouse model targeting the TTN isoforms expressed from the canonical 26. A hallmark of CIM is the preferential loss of myosin and myosin-associated proteins but not thin-filament proteins in the sarcomeres of proximal and distal skeletal muscles27. We find that CIM patient muscles show not only preferential loss of thick filaments but also suffer from reduced TTN content, while sarcomeric actin and the major thin-filament protein nebulin are largely preserved. Our results suggest that downregulation of TTN is a contributing factor in the pathogenesis of CIM, and presumably also in other types of muscle atrophy.Interestingly, our conditional TTN KO-mouse model recapitulates key changes observed in the skeletal muscles of critically ill intensive care unit (ICU) patients with acute quadriplegic myopathy  or myosinopathy), which is seen in up to ~30% of ICU patientstm1a/+ mice with conditional potential, targeting the N-terminus of TTN  under the human alpha skeletal muscle actin 1 (ACTA1) promoter, enabled us to delete Ttn exons 4–6 in adult skeletal myocytes upon doxycycline (dox) administration  on day 21 of dox-treatment, and 17 ± 1.9%  lower weight on day 30 ). On day 31 of treatment, the hang time was reduced to only ~20% of the initial value  Fig. . MUT miclue Fig. .Histological analysis by hematoxylin-and-eosin staining of skeletal muscle revealed reduced myofiber diameters and some centralized nuclei in MUT Fig. , indicatImmunostaining against different TTN epitopes Fig.  on parafElectron micrographs of fast-type M. gastrocnemius revealed severe ultrastructural changes in TTN-depleted myocytes after ~30 days of dox-treatment, while yet-unaffected MUT cells still showed the regular, WT-like sarcomere pattern Fig. . In affeImmunogold-EM demonstrated that Z-disc-like rods/aggregates in MUT cells were ACTN2-positive Fig. . Antibod2+-ATP but excluding calcium). Force-indentation curves recorded on myofibers of pectoralis muscle showed significant softening in MUT. The Young’s modulus  decreased from 7.89 ± 4.65 kPa in WT to 3.92 ± 3.06 kPa in MUT, whereas the indentation depth at 3 nN indentation force increased from 268.5 ± 160.2 nm in WT to 461.4 ± 267.5 nm in MUT   Fig. . Tensile19) Fig. . Specifi70% Fig. . Thus, t29, demonstrated downregulation in MUT by 34 ± 12%  coding for MyHC-IIb and alpha-1 skeletal-muscle actin (Acta1) were not significantly altered in MUT vs. WT, although there was a trend toward reduction in MUT  relative to actin protein Fig. . No cons12% Fig. . At the MUT Fig. . On immuMUT Fig. . Cronos Myh4 mRNA, whereas actin transcripts (Acta1) were unaltered  revealed increased signals for remaining N2A (~3.6 MDa) and Novex-3 (~0.7 MDa) in dox-treated MUT  and M-band-associated (TTN-binding) Trim63 (MuRF1)32. However, none of these molecules was significantly altered at transcript level in dox-treated MUT vs. WT M. gastrocnemius 90 was significantly increased in MUT  or the opposite regulation of two isoforms (FHL1)  and ankyrin-repeat-domain-2 (Ankrd2) was highly increased in dox-treated MUT vs. WT muscles, at both transcript  muscles from CIM patients , intermediate , and low expression ratios   Fig. . The act 4) Fig. .Fig. 8PrInterestingly, we found strongly reduced TTN:actin ratios in muscles with intermediate- or low-MyHC content, compared to those with high-MyHC content  vs. 0.45 ± 0.15 (intermediate My:Ac) vs. 0.39 ± 0.21 (low My:Ac)) Fig. . The difBoth the essential and the regulatory myosin light chains were consistently reduced or missing in low-My:Ac CIM muscles  carrying TTN-truncating mutations, which suggested that contiguous TTN is critical for the initial stages of sarcomere assembly43. Further analysis of hiPSC-CMs with an A-band-TTN truncation implicated that intact TTN forms an essential mechanical link between sarcomeric myosin and the protocostamere-actinin (Z-body) complex as a prerequisite for sarcomere assembly13. Our constitutive TTN-KO model supports the view that full-length TTN provides a mechanical connection between the Z-bodies and the myosin filaments required for cardiac sarcomerogenesis. The A-band-bound Cronos isoform, which we recently found to support formation of sarcomeres in hiPSC-CMs31, does not rescue cardiac sarcomerogenesis in vivo in the absence of Z-disc-anchored titin.Interestingly, our proof-of-concept homozygous TTN-KO mice failed to develop aligned cardiac sarcomeres. This finding is consistent with previous reports of genetically modified miceAlthough TTN is widely thought to stabilize adult sarcomeres, direct proof has been missing. Our inducible, skeletal muscle-specific TTN-KO mouse now unequivocally demonstrates that Z-disc-anchored TTN maintains the sarcomeric integrity in mature muscle cells. While this function is provided most likely by the half-sarcomere-spanning titin isoforms (such as N2A), it remains to be seen whether Novex-3, which was also deleted in our model, has an additional role in sarcomeric maintenance.5 after depletion of Z-disc-anchored TTN. However, disintegration of the sarcomeres occurred independent of whether Cronos was present (M. gastrocnemius) or not (diaphragm), which excludes a role of Cronos in maintaining the structure of adult sarcomeres. Remarkably, there was a tendency for extended lifespan of remnant A-bands in muscles expressing Cronos. Overall, however, the protective effect of Cronos on the A-bands, if any, appeared to be rare and transient, and it did not prevent rapid detachment of Z-discs from A-bands upon TTN-KO. Generally, the changes in sarcomeric protein levels and ultrastructure with TTN-loss were similar in all muscle types studied. Thus, Cronos may play a role in stabilizing the A-band of healthy fast-type muscle sarcomeres, but it is unable to substitute for the function of full-length TTN in sarcomere proteostasis.We found differential expression of Cronos in fast-type vs. fatigue-resistant murine skeletal muscles and exploited this fact to address the potential role of Cronos in rescuing the sarcomere structure46. Mechanosensitive regions in titin’s Z-, I- and M-band segments associate with molecules involved in hypertrophic/atrophic signaling, which are activated by stress or strain47. In the Z-disc, the TTN N-terminus interacts with the T-CAP/CSRP3 complex, a postulated key component of Z-disc-based mechanosignaling37. We found that TTN-loss in adult muscle profoundly increased T-CAP and CSRP3 expression. Previously, transgenic mice overexpressing T-CAP were reported to develop less apoptosis than WT animals under elevated cardiac afterload, suggesting a role for the protein in regulated cell death48. CSRP3 is involved in myocyte autophagy, next to its roles in myocyte cytoarchitecture and myotube differentiation49. Considering these functions, the upregulation of T-CAP and CSRP3 in our MUT mouse may represent an adaptive mechanism to prevent muscle wasting—which however fails, as TTN becomes increasingly unable to provide mechanical support.The role of TTN as a contributor to myocyte mechanical properties was confirmed here, as both passive stiffness  and active developed tension were reduced in dox-treated MUT vs. WT myofibers. An additional property of TTN is its mechanosensor function40. Members of this protein family can modulate transcription factors and are deregulated under various muscle stresses50. The increase of Ankrd1 and Ankrd2 in our TTN-KO mice suggests deregulation of hypertrophy/atrophy signaling. Furthermore, the N2A-element and many Ig-domains in the TTN spring interact under stress with small HSPs, such as CRYAB51. Chaperones are important regulators of myocyte protein turnover and provide a first-line protective response to many types of stress52. At least part of the increase in CRYAB expression observed upon TTN-inactivation may be related to disrupted sHSP-titin interactions. The N2A-element of TTN also associates with HSP90 in complex with co-chaperone Smyd2; together, they protect the Z/I-band structure of the sarcomere54. HSP90 and another Smyd protein, Smyd1, also have crucial roles in chaperoning the myosin filament during sarcomere development and maintenance55. The abnormal expression of these (co)chaperones in our MUT muscles may indicate the (failed) attempt to protect sarcomeric proteins from pathological aggregation and excessive degradation.Other mechanical signaling molecules in myocytes binding to the N2A-region of I-band TTN are the MARPs57, were both elevated. Their increase suggests a role in sarcomeric protein degradation at early stages of myofibril breakdown58 in our MUT model. Furthermore, we observed substantially higher ubiquitination of TTN and other muscle proteins in dox-treated MUT vs. WT muscles as a sign of increased activity of the ubiquitin-proteasome system and alternative pathways for targeted degradation52. This finding is consistent with previous studies in mouse models of muscle atrophy demonstrating that TTN becomes ubiquitinated at lysine residues mainly in the A-/M-band60. Interestingly, the muscle E3 ubiquitin ligases, Fbxo32 (atrogin-1) and Trim63 (MuRF1), were not significantly altered in our TTN-KO mice, although they are usually induced during muscle atrophy32. While TRIM63 binds directly to TTN at the M-band61, the E3 ligase has never been shown to ubiquitinate TTN; however, it does ubiquitinate many other sarcomere proteins, preferentially in thick filaments62. We speculate that other, yet-to-be-identified, E3 ligases may be activated by reduced supply of TTN, causing increased ubiquitination of remaining TTN protein. Moreover, two markers of autophagic-lysosomal activity, SQSTM1/p62 and LAMP264, were upregulated in MUT muscles. SQSTM1 binds via neighbor of BRCA1 gene-1 protein to the titin-kinase domain in M-band TTN36, thus linking stress-induced autophagic signaling to the sarcomere47. Although it is still unknown whether/how much TTN protein is degraded via autophagic pathways, the increase in autophagy markers upon TTN-KO suggests involvement of autophagic processes in the sarcomeric disintegration. Collectively, these results demonstrate that the TTN-loss increases the activity of all components of the PQC machinery, which jointly mediate the accelerated degradation of the myocyte proteins.Various other components of the cellular PQC machinery became activated as a consequence of the TTN-loss. The TTN-binding calpains, CAPN1 and CAPN365. Trim63 activation was speculated to cause the preferential myosin-loss in CIM patients26. Other proteins found to be elevated in CIM patient muscles are inflammatory molecules, transcriptional repressors, and mechanosensitive myocyte proteins65. We are not aware of any genetic animal model resembling the hallmarks of human CIM, i.e., the preferential loss of thick filaments and the pathophenotypic appearance of the myocyte/sarcomere ultrastructure, as closely as induced depletion of TTN.An intriguing finding of this study is the remarkable similarity in the pattern of sarcomere disintegration and myocyte protein changes between our TTN-KO mice and CIM patients presenting with peripheral muscle wasting. In TA muscles of CIM patients, progressive reduction in thick-filament proteins, such as MyHC, MyBPC and myomesin-1, was accompanied by a significant loss of TTN protein already at early stages of the muscle atrophy when the MyHC:actin expression ratio was only modestly reduced. Nebulin, actin, and α-actinin levels showed little changes, whereas TTN-binding mechanosensitive proteins, chaperone HSP90, and the levels of ubiquitinated TTN increased progressively. A large body of evidence has demonstrated that CIM patient muscles upregulate, among others, components of the PQC machinery, including chaperones, proteases, and E3-ubiquitin ligases67. Importantly, passive mechanical loading attenuated muscle wasting and myosin loss in this model68. Thus, mechanosensitive factors within the muscles are involved in the pathomechanisms of CIM. Our findings suggest the participation of TTN-based mechanosensing via TTN-binding signaling molecules. Mechanical silencing of TTN due to muscle unloading may cause the loss of TTN in CIM, because mechanosensitive pathways normally triggered by TTN strain fail to be activated and regulate sarcomere protein turnover. Despite upregulation of TTN-associated factors, muscle wasting proceeds, and the pathologically lowered TTN levels cause sarcomeric disintegration. Presumably, loss of TTN precedes loss of myosin not only in our TTN-depletion mouse model but also in CIM. If this holds true, the TTN-KO mouse provides a new tool for studying the pathogenesis of CIM and may also help in the search for potential therapeutic approaches.In mechanically ventilated, deeply sedated and/or pharmacologically paralyzed ICU patients, there is a complete loss of mechanical stimuli of the skeletal muscles, including external strain (no weight bearing!) and internal strain (no activation of the contractile apparatus!), which presumably is an important trigger of this special myopathy. A sophisticated rat model has been developed to assist in defining the contributions of mechanical ventilation and muscle unloading to CIMAll animal procedures were performed according to national guidelines (TierSchG and TierSchVersV) and were approved by local government authorities .Ttn gene targeting construct are available in the GenBank® database (accession no. JN959336.1). Validation of inserted cassette integrity and correct targeting are described in supplementary methods  and to Flipase (Flp)-expressing transgenic mice (Flp-Deleter)69 to generate the lacZ-neo cassette-deleted, heterozygous, conditional Ttn KO mice (Ttntm1c/+).The titin knockout-first mice were generated by injecting EPD0145_2_G01 ES cell clones from the EUCOMM  Consortium into BAlb/C blastocysts. The specifics and the complete DNA sequence of the tm1c/+ or Ttntm1c/tm1c) to the B6;C3-Tg102Monk/J mouse line, purchased from the Jackson Laboratory , which expresses Cre-recombinase under the control of the tetracycline-responsive regulatory element (tetO/TRE) and a reverse tetracycline-controlled transactivator (rtTA) under the control of the human actin, alpha 1 skeletal muscle (ACTA1) promoter28.The inducible skeletal muscle-specific Ttn KO mice were generated by crossing the conditional Ttn KO mice 26Sortm1Sor/J strain. The latter is the R26R Gtrosa26 targeted mutant mouse70 carrying lacZ flanked by loxP sites  hybrid line, generated by mating the B6;C3-TgPrimers used for genotyping are shown in Supplementary Table tm1c/tm1c;ACTA1-rtTApos;tetO-crepos and gender-matched WT littermates, Cre-mediated recombination was achieved by administering the tetracycline analog doxycycline. Doxycycline hydrochloride  was dissolved in drinking water at a concentration of 2 mg/mL supplemented with 5% sucrose and was provided ad libitum in light-proofed water bottles for 27–38 days. Treatment was terminated when mice had reduced their body weight by 10–20%. Tissues were then collected from mutant and WT mice for further experiments.In 8–30 week-old TtnMice were sacrificed by cervical dislocation and embryos, adult hearts and skeletal muscles were dissected. Body weight was recorded and muscle weights measured relative to tibia length. Tissues were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for paraffin- or cryo-embedding, or with Zamboni’s fixative for electron microscopy, and placed on a shaking table at 4 °C overnight. Cryosections of M. triceps brachii were stained with hematoxylin or hematoxylin/eosin to observe differences between dox-treated WT and MUT. Digital images of hematoxylin/eosin-stained cryosections of triceps brachii were processed and analyzed using ImageJ software , to measure cell number, fiber cross-sectional area, and number of nuclei. The percentage of centrally nucleated fibers was determined for at least 225 (up to 488) fibers per muscle, using photomicrographs taken at two different positions along the muscle. The cross-sectional area was determined on at least 160 fibers per animal at different positions along the entire muscle.71 was used to test muscle strength of dox-treated Ttntm1c/tm1c;ACTA1-rtTApos;tetO-crepos and WT mice. Briefly, mice were placed on a wire mesh consisting of 6 mm squares of 1 mm diameter wire, surrounded by 5 cm edging to prevent climbing on the other side. Immediately after placing the mouse in the center, the screen was turned upside down, fixed in this position 40–50 cm above a soft-bedded cage, and the hang time was measured. The test ended when a hang time of 15 min was reached; if not, the test ended after two sessions. The maximum hang time was recorded.The 4LHT or inverted screen testTtn exon 20_24 probe was derived from Ttn N2A sequence positions 3372–3483  according to the manufacturer’s protocol. RNA quality and quantity were estimated by comparing the fluorescence signals of the 28 S and 18 S rRNA bands on agarose gels. Reverse transcription was carried out using Oligo(dT) and Random primers, and RNA polymerase M-MLV RT  according to the manufacturer’s instructions. Transcript-specific primers are reported in Supplementary Table Total RNA was prepared from tissues using Trizol−∆∆CT). The hypoxanthine-guanine phosphoribosyl transferase gene  served as the reference gene, unless otherwise stated. The qPCR was performed on the StepOne™ Real-Time PCR System (Applied Biosystems) using the following 2-step program: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation for 15 s at 95 °C and annealing/extension for 1 min at 60 °C, terminated by a final denaturation step at 95 °C for 15 s. For qRT-PCR reactions, the GoTaq qPCR Master Mix reagent was used. Amplicon specificity was confirmed by examination of the dissociation curves. A distinct single peak indicated that a specific DNA sequence had been amplified.Quantitative real-time PCR (qPCR) analysis was performed on samples obtained from at least 3 animals per genotype and used for quantification of relative gene-expression data using the RQ  by cutting them in very small pieces and incubating them for 20 min on ice. After boiling the probes for 3 min at 96 °C, the debris was spun down for 5 min at 20,000 × 34. Proteins up to 250 kDa were resolved by standard SDS-PAGE . For simultaneous separation of TTN and proteins up to 250 kDa, we combined both methods, using for the stacking gel an agarose-stabilized 1.8–2.5% gel and for the running gel the standard SDS-PAGE recipe . Electrophoresis was performed at 2 mA/plate at room temperature overnight. After separation, proteins were transferred onto PVDF membranes (Millipore) by using the Trans-Blot® Turbo™ transfer system (Bio-Rad). After transfer, total protein blot stain (TPBS) on the membranes was performed with Coomassie brilliant blue . Myosin or actin bands were used as an additional loading control.The concentration of total protein in each sample was assessed using a Bradford reagent (Sigma). For the separation of giant myofibrillar proteins, protein lysates (7–30 μg) were resolved overnight in agarose-stabilized 1.8–3% slab gelsDetailed information on the primary and secondary antibodies is provided in Supplementary Table Muscle tissues were prepared for immunohistochemistry using standard protocols. Briefly, samples were fixed with 4% formaldehyde solution in PBS and embedded in paraffin; 10-µm-thick sections were prepared. After deparaffinization and rehydration, antigen unmasking was achieved by shortly boiling the slides, submersed in 1 mM EDTA (pH 8), using a microwave oven. After incubation for an additional 20 min at a sub-boiling temperature, slides were washed in distilled water and incubated in blocking buffer (0.1% Triton X-100 and 10% goat serum in PBS) for 1 h. Antibodies were diluted in staining buffer . The primary antibodies were applied overnight at 4 °C, the secondary antibodies for 2 h at room temperature, with three washes in between incubations. Detailed information on the primary and secondary antibodies is given in Supplementary Table n = 3 gastrocnemius muscles per group) after dox-treatment for 21 or ~30 days. Images were recorded using a Nikon A1 confocal microscope. The striation pattern of at least 90 muscle fibers per animal was evaluated.Immunofluorescence staining on paraffin sections was performed using anti-titin Z-disc TTN Z/I-2080) and anti-α-actinin antibodies  at a thickness of 50 μm, blocked in 20% normal goat serum  in PBS and were incubated with primary antibodies diluted in PBS containing 5% NGS overnight at 4 °C. Antibody references and corresponding dilutions are provided in Supplementary Table N = 2 animals, 250 Z-discs/group), we calculated the Z-disc circumference by using ImageSP software . Z-disc boundaries were clearly identified by their electron densities and perimeters were obtained via ImageSP polygon measurement. Full image information was transferred to Microsoft Excel for statistical analysis.On electron micrographs of WT and KO (10–20% BWL) ultrathin sections of gastrocnemius muscle tissue  supplemented with 0.5% Triton X-100. Under a binocular , single myofibers were manually dissected and placed in relaxing buffer  at room temperature. The nanoindentation technique was applied as follows: dissected myofibers in relaxing buffer were placed in a fluid chamber, in which the cover glass had been coated with the glue Cell-Tak ; fibers thus adhered tightly to the cover glass as a prerequisite for reliable force-indentation measurements. A pre-calibrated cantilever  with a mounted spherical polystyrene bead  periodically indented the myofiber up to a pre-set indentation force of 3 nN. During the measurements, the AFM tip was moved vertically toward the sample surface at constant velocity of 2 μm s−1. Upon indentation of the myofiber, the AFM cantilever became deflected. The relative position of a laser beam, focused on the cantilever tip and reflected back to a photodiode detector, was used to quantify cantilever deflection and generate force-vs.-indentation curves. From the force acting on the cantilever, the piezo displacement, and the deflection sensitivity, the indentation depth was calculated. By subtracting the deflection of the cantilever from the piezo movement after contacting the myofiber, the force-distance curves were converted to force-indentation curves. The slope of the force-indentation curve indicated the lateral stiffness of the myofiber. The Young’s modulus was obtained by fitting the force-indentation curve from the contact point to the end (maximum indentation) using the Hertz model corrected for a spherical indenter , according to the equation:F is the indentation force, E is the Young’s modulus, R is the radius of the sphere directly contacting the sample surface (R = 5 μm in the current experiments), v is the Poisson’s ratio of the sample , and δ is the measured indentation depth.To determine the mechanical stiffness of WT and MUT skeletal muscle cells, single isolated fibers were probed by nanoindentation measurements using an atomic force microscope . Deep-frozen tissue was defrosted and skinned overnight in ice-cold low ionic-strength buffer  supplemented with 0.5% Triton X-100 and protease inhibitor cocktail . After washing three times with relaxing buffer, myofiber isolation was carefully done on ice, avoiding excessive stretching. Single myofibers were attached between a motor and a force transducer  and mechanical experiments run at room temperature. Fiber cross-sectional area was estimated from the fiber diameter (assuming a circular shape) measured at slack length. For passive tension measurements, fibers were stretched from slack length in six equidistant steps . After each step, force relaxation was allowed for during a 1-min hold period. The peak force value immediately after each stretch was converted to stress . For active tension measurements, fibers were pre-stretched  in relaxing buffer for 2 min and then transferred to activating solution , while force was recorded. Maximum active force was normalized to fiber cross-sectional area (Ca2+-activated tension). All solutions used for mechanical experiments contained protease inhibitor cocktail .Dissected vastus lateralis muscle from ~30-day dox-treated WT and MUT mice was skinned overnight in ice-cold relaxing buffer  and 5 control immobilized ICU patients with lesions in the central or peripheral nervous system . All ICU patients with CIM had been mechanically ventilated for longer than two weeks at the time of muscle biopsy. The biopsies are from patients recruited in a study aiming at improving the diagnostic precision of CIM, i.e., comparing results from percutaneous muscle biopsies with results from biopsies obtained with a disposable microbiopsy instrument. Thus, ICU patients were included who had developed a general paralysis in response to long-term mechanical ventilation and were referred to the clinic for diagnostic purposes. Control samples displaying high myosin:actin ratios were obtained on the first day on the ventilator prior to any myosin loss. In these patients, a myosin loss was evident first after more than one week of immobilization and mechanical ventilation. There is no indication that the inclusion of these patients may have introduced any bias which may have impacted on the results. Each biopsy was frozen in liquid propane chilled by liquid nitrogen and stored at −140 °C or −160 °C until analyses. Written informed consent was obtained from patients’ close relatives prior to the study. The study was approved by the Ethical Committee on Human Research at Karolinska Institutet, Stockholm and Uppsala University Hospital, Sweden . Informed consent was obtained from all patients or close relatives .Percutaneous conchotome muscle biopsies were taken from the t-test or Mann–Whitney test and differences with p-values lower than 0.05 were considered significant. Variance analyses of repeated measurements were performed by repeated measures two-way ANOVA, followed by Sidak’s test for multiple comparisons; here, differences with p values lower than 0.01 were considered significant. Sample size was estimated based on sample availability, previous experimental studies performed in our laboratory, and the literature. Experiments were not randomized or blinded.Data organization, scientific graphing and statistical analyses were performed using Microsoft Excel (2013) and GraphPad Prism (version 7). Results are presented as the mean ± SD, mean ± SEM, or as the difference between means ± SEM, as indicated. Normality was assessed using the Shapiro–Wilk test. Significant differences between two groups were determined by two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


A follow-up triage test was completed for 84.2%, 85.8%, and 50.1% of HPV-positive women in Guatemala, Nicaragua, and Honduras, respectively. Of those with a positive triage test, 84.7%, 67.1%, and 58.8% were treated in Guatemala, Nicaragua, and Honduras, respectively. First-time screening was highest in Nicaragua (55.8%) where self-sampling was also widely used (97.1%). The Scale-Up project demonstrated that large-scale cervical cancer screening and treatment intervention in a high-burden, low-resource setting can be achieved. Self-sampling and ablative treatment were key to the project's achievements. Data monitoring, loss to follow-up, and triage methods of screen- positive women remain critical to full success.The Scale-Up project introduced vaginal self-sampling and low-cost human papillomavirus (HPV) testing as the primary approach for cervical cancer screening in selected public health centers in Guatemala, Honduras, and Nicaragua. We evaluate the country-specific accomplishments in screening: target-coverage, triage, and treatment. Between 2015 and 2018, cervical cancer screening was offered to women at least 30 years of age. Triage of HPV-positive women was based on visual inspection with acetic acid or Pap. Aggregated data included total women screened, use of self-sampling, age, time elapsed since last screening, HPV results, triage tests, triage results, and treatment. A total of 231,741 women were screened for HPV, representing 85.8% of the target populations within the project. HPV positivity was lower in Guatemala (12.4%) compared to Honduras and Nicaragua (14.5% and 14.2%, respectively,  •Central America successfully implemented HPV testing in public sector clinics.•HPV testing using self-sampling holds great promise to increase coverage of cervical cancer screening.•Follow-up of screen positives is a critical component step for an effective screening program.•Thermal ablation of precancerous lesions may be critical in increasing treatment coverage.•Basic health information systems are required for monitoring and mitigating loss to follow-up. These fcareHPV, a low-cost test specifically designed for use in low-resource settings using either cervical or vaginal samples  project showed that screening with cervical or vaginal HPV testing performed better than visual inspection with acetic acid (VIA) or Pap testing in resource-limited settings . START-U samples .Building upon this evidence, PATH worked with Central American ministries of health (MOHs) to introduce a new model of cervical cancer screening under the Scale-Up project. Scale-Up was implemented in selected areas in Guatemala, Honduras, and Nicaragua from 2015 to 2018. Essential elements of the project's implementation were: a) raising awareness at the community level, b) facilitating adoption and encouraging procurement of HPV testing, c) using vaginal self-sampling for test specimens to facilitate rapid screening uptake and d) developing indicators to be monitored throughout the activity. El Salvador followed a different timeline for screening and implemented a different treatment algorithm; thus, results from this country are reported elsewhere .Here we report on the country-specific accomplishments in screening target coverage, triage, and treatment indicators in Guatemala, Honduras, and Nicaragua. Collectively, these results can inform decision-making in other low-resource settings, as countries consider more efficient and effective models for secondary prevention of cervical cancer.22.1The MOHs of Guatemala, Honduras, and Nicaragua defined geographic target areas  and screThe Scale-Up project was planned in three phases: preparation, pilot testing, and expansion; project details have been described previously . Each co2.2careHPV , a signal-amplification batch diagnostic test for high-risk HPV DNA detection. The test allows qualitative detection of 14 high-risk types of HPV DNA in cervical and vaginal specimens. QIAGEN and PATH trained local laboratory microbiologists, cytologists, and technicians to run the test. PATH also developed quality assurance guidelines  or community-based locations ; less commonly, providers collected specimens during a pelvic exam at clinic sites. In Honduras, self-sampling was promoted in one province only, the metropolitan region of Tegucigalpa, from 2017 onward. In all three countries, HPV samples were sent to referral laboratories within approximately one week of collection, and women could collect the test result in one month from the clinic. Women who provided a sample outside of the clinic also had an option to receive their HPV test results from a community health worker. A triage appointment at the primary health care center was scheduled for HPV-positive women. Triage results and treatment eligibility for the HPV-positive women were evaluated, using primarily VIA in Guatemala and Honduras, and cervical cytology in Nicaragua. For women outside of the country's target age range for HPV testing, or if HPV tests were temporarily not available at the primary health care center, screening by VIA or Pap was offered.Guatemala and Nicaragua selected self-sampling using Women with triage-positive lesions eligible for ablative treatment were treated with cryotherapy, although thermal ablation was introduced and subsequently used in Guatemala in 2017 and in Honduras in 2018. If not eligible for ablation because the transformation zone was not fully visible or the lesion occupied >75% of the transformation zone, women were referred to a higher level of care.2.3Indicators were collected and compiled in an aggregated and anonymous form that was sent at least quarterly to PATH. In addition to the number of tests provided per country, the information collected included (1) women screened with HPV testing, (2) women providing self-collected samples, (3) women in the target age range, (4) time elapsed since last cervical cancer screening, (5) HPV-positive women, (6) women receiving a triage test, (7) women positive in triage, and (8) women treated for precancer.Each country had a pre-established number of tests to be used in the project. Target-coverage was estimated as the number of women screened divided by the number of tests allocated to a country during the project The estimates cannot correct for repeated tests during the period.2.4P-values <.05 values were considered statistically significant.Data were analyzed using Excel and Stata/SE 15.0 Whenever appropriate, estimates of different indicators and their respective 95% confidence intervals (95% CI) are presented. 2.5The funder had no role in manuscript development, data collection or analysis. The corresponding author had full access to the data presented and had final responsibility for the decision to submit for publication.2.6All the data analyzed were provided to PATH aggregated with no individual identifiers. No informed consent was requested as women were managed according to country based national guidelines.33.1N = 85,226), 76.3% in Honduras , and 52.3% in Nicaragua . Nearly all women for whom age data were available fell within the country-specific target age range established for screening (97.7%)  in the project target areas, HPV testing was the most common method used (59.7%), with 52.0% of screenings done with HPV testing in Guatemala ( (97.7%) . Among w (97.7%) .Table 2CHPV screening through self-collected samples was high in Nicaragua (97.1%) and Guatemala (90.2%) and reached 74.0% in the metropolitan region of Tegucigalpa, Honduras, starting in 2017, considerably increasing the volume of tests performed in the area .Fig. 3Cu3.2As shown in 3.3Guatemala achieved a significantly higher proportion of HPV-positive, triage-positive women who completed treatment with 2676 (84.7%), followed by Nicaragua with 1657 (67.1%), and Honduras with 1230 (58.8%) . HonduracareHPV test-failure rate was approximately 10%. Some plates had to be repeated due to suspected well-to-well contamination or due to unspecific test running failure  with an overtreatment rate of 13% . In thatAny gap in time between diagnosis and treatment creates opportunity for loss to follow-up whereas women with a positive HPV test through self-sampling had to be recalled to a clinic for triage. In El Salvador, women under a screen-and-treat protocol had approximately twice the treatment completion rate compared to women referred to colposcopy . In ScalSimilarly, the introduction of thermal ablation for same-day treatment contributes substantially to higher treatment percentages . We commAny cervical-cancer screening program should aim to reduce cervical cancer mortality and thus requires monitoring measures of effectiveness. An estimated 85% of treatment-eligible women must be treated for precancerous lesions to show an impact at the population level . EthicalThere are myriad challenges to delivering HPV screening results to women and engaging HPV-positive women to complete triage and treatment if needed; these include limited longitudinal health information systems, requiring women to appear for multiple appointments and travel to specialized facilities, lack of trained providers able to provide follow-up, and lack of treatment availability. Under the Scale-Up project, providers from primary health care centers involved in the project recommended that women return to the clinic for results approximately one month after screening, but there was no call-and-recall system established to reinforce follow up which can easily derive in losing effectiveness of the screening activities.Although mobile phones are widely available, no specific communication was undertaken at the primary health care level to recall women. Many settings are now exploring how best use the mobile technology through automatic or directed SMS as a way for women to keep in regular contact with medical services . InclusiThe Scale-Up project was not designed to report on coverage or population-level impact, but we could estimate the capacity of the system by reporting the target coverage performance which was high. Individual-level data were not available to the Scale-Up project and duplicate testing or shorter intervals between screening tests were not measured; a limited set of individual data from the MOH of Honduras and Nicaragua were retrieved for additional quality control checks. Further, access to pathology records was not available for Scale-Up and no population-based cancer registry exist to measure of impact. Honduras is now in the early stages of developing a population-based registry that will reduce this limitation in the near future.5The Scale-Up project successfully launched and implemented a new cervical cancer screening strategy in a high-burden region. The project worked with three Central American MOHs to update national guidelines, train local staff, and engage local champions to introduce key innovations in screening  and improve knowledge of and capacity for clinical and laboratory management of HPV tests. Efforts to gather specific indicators were important for project evaluation and accountability to stakeholders. At the time of completing this manuscript, the MOHs of Guatemala and Honduras are planning to continue with HPV testing, while the MOH of Nicaragua intends to re-initiate and expand HPV testing at a future date. The limited quality of available triage approaches remains a challenge, but new approaches with the potential to improve visual evaluation of the cervix for both screening and triage purposes will likely be available soon .The Scale-Up project provides evidence that moving toward the elimination of cervical cancer with high-precision screening is possible in low-resource settings. Yet this will only be attainable if, in addition to primary prevention through HPV vaccines, well-organized efforts for follow up are used at each screening step. Scale-Up countries have generated encouraging evidence that HPV testing, self-sampling, and thermal ablation can overcome many previous obstacles to screening and timely treatment, resulting in high volume of women screened and treated for cervical precancer and cancer lesions in LMICs.Francesca Holme:Conceptualization, Visualization, Formal analysis, Data curation, Writing - original draft, Writing - review & editing.Jose Jeronimo:Conceptualization, Data curation, Writing - review & editing.Francisco Maldonado:Conceptualization, Visualization, Data curation, Writing - review & editing.Claudia Camel:Conceptualization, Visualization, Data curation, Writing - review & editing.Manuel Sandoval:Conceptualization, Visualization, Data curation, Writing - review & editing.Benito Martinez-Granera:Conceptualization, Data curation, Writing - review & editing.Mirna Montenegro:Conceptualization, Data curation, Writing - review & editing.Jacqueline Figueroa:Conceptualization, Data curation, Writing - review & editing.Rose Slavkovsky:Visualization, Data curation, Writing - review & editing.Kerry A. Thomson:Writing - original draft, Writing - review & editing.Silvia de Sanjose:Visualization, Formal analysis, Data curation, Writing - original draft, Writing - review & editing.Francesca Holme, Francisco Maldonado; Claudia Camel; Manuel Sandoval; Benito Martinez-Granera, Mirna Montenegro, Jacqueline Figueroa, Rose Slavkovsky, Kerry A. Thomson and Silvia de Sanjose declare no competing interests.Jose Jeronimo was the co-owner and Deputy Manager of Onco Prev International, a Peruvian company, from 2012 through March 2017. Onco Prev offers cervical cancer screening services and in 2016 also began positioning for distribution of medical devices including colposcopes and the Liger thermo-coagulator. Onco Prev International did not commercialize any medical instrument during the time JJ was part of that company. JJ declares that he has never received an honorarium, travel money, or compensation from any company manufacturing thermal ablation equipment.


Human papillomavirus (HPV) infections are found in children, but transmission modes and outcomes are incompletely understood. We evaluated oral samples from 331 children in Finland who participated in the Finnish Family HPV Study from birth during 9 follow-up visits (mean time 51.9 months). We tested samples for 24 HPV genotypes. Oral HPV prevalence for children varied from 8.7% (at a 36-month visit) to 22.8% (at birth), and 18 HPV genotypes were identified. HPV16 was the most prevalent type to persist, followed by HPV18, HPV33, and HPV6. Persistent, oral, high-risk HPV infection for children was associated with oral HPV carriage of the mother at birth and seroconversion of the mother to high-risk HPV during follow-up . Children acquire their first oral HPV infection at an early age. The HPV status of the mother has a major impact on the outcome of oral HPV persistence for her offspring. Cutaneous warts are common in children and are acquired mostly through horizontal transmission, but also through vertical transmission; lesions can persist asymptomatically for years . Vertical transmission can be further categorized as periconceptual (time around fertilization), prenatal (during pregnancy), and perinatal (during birth or immediately thereafter)  as described  and covering the entire oral mucosa as described , which detected 24 low-risk, putative high-risk, and high-risk HPV genotypes as follows: 6 low-risk genotypes ; 3 putative high-risk genotypes ; and 15 high-risk genotypes  and Stata version 15 (https://www.stata.com) software packages.Predictors of incident HPV infection and genotype-specific HPV clearance or persistence were analyzed by using the most prevalent high-risk HPV types . To model incident infections and genotype-specific HPV clearance, Poisson regression analysis was used. For persistence, a generalized estimating equation (GEE) modeling was used. In the univariate GEE model, all covariates recorded at baseline and previously implicated as potential risk factors for HPV infections were tested  children . ParticiWe collected general background information for the general health of the children recorded at the 36-month visit as given by their parents . Hand wa>2 genotypes) were recorded, HPV16 was present in 56% (14/25) of these samples. When analyzed by sex, we found differences in HPV prevalence at 1-, 2-, 12-, and 36-month visits, but none at the 6-year visit. HPV positivity at birth or later was unrelated to the mode of delivery. Overall, 41.4% (135/329) of the children remained negative for all oral samples collected during the follow-up.We also provide an overview of oral HPV infections in children who had HPV genotypes and their point prevalence during the follow-up period . The preIncident HPV infection (baseline negative) was determined for 107 (32.8%) of 326 children: 107 cases/5,754 person-months at risk (PMR), which resulted in an incidence rate of 18.6 cases/1,000 PMR. Ten children (9.4%) had multiple-type infections among this group of incident infections. Kaplan-Meier analysis showed that there were no significant differences in the acquisition of oral HPV between the different species  Figure. A total of 99 children cleared their oral HPV infection during the follow-up, resulting in a clearance rate of 19.1 cases/1,000 PMR . The mean clearance times for clades 10 (HPV6/11 and their closest relative), 9 (HPV16 group), and 7 (HPV18) genotypes were not significantly different: 28.6, 34.2, and 30 months, respectively  Figure.A total of 14.9% (48/323) of the children had persistent oral HPV infection. The mean time of persistence was 20.6 months (range 0.1–92.2 months). We provide type-specific HPV persistence times . The mosWe summarized the predictors of incident, cleared, and persistent oral high-risk HPV infections in these children . All estHPV infections in the oral cavity have been detected in young children, but the outcome of these infections has remained unknown. We found that the prevalence of HPV and multiple-type infections was highest and the spectrum of HPV genotypes was widest at birth. The mode of delivery had no association with oral HPV carriage, and some sex differences were found in oral HPV prevalence during the early months, but not at the end of the follow-up period. Results indicate that none of the HPV genotypes present at birth would promote acquiring another specific HPV genotype, not even an HPV from the same clade. Although most of the oral HPV infections were cleared during the 6-year follow-up period, persistent oral HPV infection was found in 14.9% of these children. The 6 children who had multiple-type HPV infections at birth still harbored those infections at the most recent visit. Thus, clade α9 resulted most frequently in the full-time persistence of oral HPV infection during the early childhood, followed by α7 as the second most frequent clade.HPV acquisition at birth has been regarded to be caused by vertical transmission, although controversial opinions have been reported , followed by subsequent hybridization with specific probes for HPV6, 11, 16, 18, 31, 33, and 39  has been controlled by models for panel data (GEE and panel Poisson). Trottier et al. published their first results from the HERITAGE study on perinatal transmission and risk for HPV persistence in children . We do not know yet whether these children will continue to remain HPV negative later in life. We suspect that these children might be less prone to HPV infections in general, and would be interesting to evaluate again later in life.In our study, 41% of children remained constantly HPV negative during the follow-up period  might also benefit from vaccination, as has been the case with hepatitis B virus–infected newborns or children (Additional information on oral human papillomavirus infection in children during the first 6 years of life.


Evaluate the significance of BMRT HPV assay viral load and its performance for secondary screening.BMRT-HPV reports type-specific viral loads/10,000 cells. We tested 1,495 physician collected, stored specimens from Chinese Multiple-center Screening Trial (CHIMUST), that were positive by Cobas, SeqHPV, and/or Cytology (≥LSIL); and 2,990 age matched, negatives in a nested case control study. We explored the relationship between BMRT HR-HPV viral load and cervical lesions, determined alternative CIN2+ cut-points by ROC curve, and evaluated BMRT HR-HPV for primary / secondary cervical cancer screening.P<0.05, among which HPV16, 33 and 58 showed the strongest relationship (P<0.01). The viral load of HR-HPV also increased with the grade of cervical lesions (P<0.05). The sensitivity for CIN2+ and CIN3+ of BMRT was comparable to Cobas , specificity was higher than Cobas . When using HPV16/18 viral load(log cut-point ≥3.2929), plus the viral-load of 12 other subtypes (log cut-point ≥3.9625) as secondary triage, compared with Cobas HPV16/18+ plus cytology ≥ASC-US as triage, the sensitivities for CIN2+ and CIN3+ were similar (P>0.05). However, the BMRT HR-HPV viral load combined with subtypes did not require cytology.The viral loads of HPV16/18, 12 other subtypes HR-HPV and 14 HR-HPV were statistically different in all grades of cervical lesions (BMRT is as sensitive as Cobas4800 for primary cervical cancer screening. BMRT HR-HPV viral load combined with subtypes can be used as a secondary strategy for cervical cancer screening, especially for areas with insufficient cytological resources. Persistent high-risk human papillomavirus(HR-HPV) is a necessary cause of cervical cancer, HPV-based cervical screening can identify >95% of pre-cancerous cervical lesions  , so highThe relationship between HR-HPV viral load and the progression of cervical precancerous lesions remains controversial. Several studies from our team have shown that the higher the HR-HPV viral load, the higher the risk and severity of cervical lesions ,7. In paBMRT  is a PCR based assay developed in 2015. It measures the number of cervical cells by quantitatively detecting the number of single-copy genes (TOP3 genes) in cervical exfoliated cells, and then reports HPV viral load  for standardized quantitation. This assay can detect 14 high-risk HPV subtypes and 7 Medium and low risk subtypes; and it provides viral load per unit cell of each subtype.In this study, we selected physician-collected specimens from the Chinese Multi-Center Screening Trial (CHIMUST) for BMRT testing. Using this assay, we investigated the relationship between viral load and cervical lesions and assessed the effectiveness of a triage strategy using HPV viral load combined with HPV subtypes.This study of BMRT-HPV was a nested case–control study within the Chinese Multi-center Cervical Cancer Screening Trial (CHIMUST) (Registration number: ChiCTR-EOC-16008456). The CHIMUST protocol was approved by the ethics committee of Peking University Shenzhen Hospital(IRB:PUSH2016001) and Cleveland Clinic Institutional Review Board (IRB:15–1549). From August 2016 to January 2018, the trial provided screening for 10885 women who had not been screened in the past 3 years. The women needed to be non-pregnant sexually active women, ages 30 to 59 years with no prior pelvic radiation, and with an intact uterus. They needed to agree to return for further testing if their screening was positive. Women were recruited from Beijing and the following five provinces: 1. Jiangxi 2. Hebei 3. Hubei 4. Guangdong 5. Inner Mongolia Autonomous Region. ALL participants signed an informed consent document before enrollment.All participants provided a self-collected vaginal sample and a physician-collected endocervical sample. The physician-sampling was performed following self-sampling. The physician placed a vaginal speculum to expose the cervix, then obtained a cervical exfoliated cell sample at the squamocolumnar junction of the cervix with a sampling brush and then placed the brush into a 20mL PreservCyt® solution  for testing. All samples were tested with the PCR-based high-risk HPV assays: Cobas and SeqHPV . The physician-collected samples were also tested by Cytology using the Hologic I2 imager system (computer assisted cytology). Women positive by either Cobas or SeqHPV (self or direct) were referred for colposcopy.In this study, using BMRT-HPV which reports type-specific viral loads/10,000 cells in the specimens, we tested only the physician-collected samples. There were 1,495 cases with adequate remaining sample from CHIMUST who were positive by Cobas, SeqHPV, and/or Cytology (≥LSIL) and 2,990 age matched, negatives in a nested case control study. see HPV L1 gene, including 14 HR-HPV genotype , and 7 MR and LR-HPV genotypes . For this study, a 14 type high-risk BMRT assay was used. All types and viral loads specifically refer to high-risk HPV. To control DNA quality and determine the relative viral copy numbers in the samples, a single-copy gene encoding DNA topoisomerase III (human TOP3) was amplified in the reaction. The experimental procedure was conducted according to the kit manufacturer’s instructions. PCR was performed on an ABI Prism 7500 Fast Dx System . Five-point standard curves for HPV and the cellular log-phase were established for absolute quantification. The standard curves for HPV and TOP3 were Y = –3.85633(log10X) + 36.93833 and Y = 3.34656(log10X) + 38.51644, respectively. The number of viral copies was normalized according to the cellular input and log10-transformed Therefore, the normalization of HPV type-specific viral loads was performed as follows: viral load = log10 copies/10,000 cells, where Cn HPV is the quantity of HPV DNA and Cn TOP3 is the number of human cells. Perfectus,v1.0 was used for genotyping and quantitative analysis of HPV nucleic acids (Bioperfectus Ltd). 189 women did not have colposcopy with Cobas and/ or BMRT HR-HPV positive. 2 cases of Cobas and 1 case of cytology were technical failures. The final complete dataset of 4293 cases was analyzed, with an average age of 45.4±7.2 years. This included 748 cases of BMRT HR-HPV(+) and 810 cases of Cobas(+). The pathological diagnosis showed: normal cases (989), CIN1 (150), CIN2 (74), CIN3 (43) and cervical cancer (5). The histological results were grouped into 1139 cases of ≤CIN1 , 122 CIN2+ , and 48 CIN3+ (CIN3 and carcinoma). See P<0.05). This was especially true for HPV16, 33 and 58 (P<0.01). The viral loads of HPV16/18, 12 other subtypes HR-HPV, 14 HR-HPV, HPV16, HPV33 and HPV58 increased linearly as the cervical lesions changed from ≤CIN1 to CIN3+ .Whereas the viral load of HPV18, 31, 35, 39, 45, 51, 52, 56, 59, 66 and 68 were not predictors in all grades of cervical lesions (P>0.05).Tables P<0.001) and an optimal cut-point of 3.2968 copies/10,000 cells . HPV33 had an AUC of 0.862  and an optimal cut-point of 3.5533 copies/10,000 cells . HPV58 had an AUC of 0.710  and an optimal cut-point of 4.4323 copies/10,000 cells .We created ROC curves from the type-specific HR-HPV viral load data to determine the most useful cut-point for the identification of CIN2+ . AccordiP<0.05), among which HPV16, 33 and 58 showed the strongest relationship. (P<0.01). The viral loads of HR-HPV were positively correlated with cervical lesions (P<0.05). It could predict that sensitivity and specificity value for CIN2+ infected by HPV16/18, 12 other subtypes HR-HPV and 14 HR-HPV were 92.6%, 76.3%, 77.0% and 48.0%, 51.2%, 48.1% when cut off values of the log viral load achieved 3.2929, 3.9625 and 3.9625, respectively. This suggests that HPV viral load is a type-specific biological indicator of cervical cancer [At present, the relationship between viral load and cervical lesions is still controversial. One of the important reasons for inconsistent results is that there is no standard unified high-risk HPV quantitative detection method. One of the key challenges of viral load is that sampling differences can result in varying numbers of cells in a sample. This lack of a standard has created a formidable obstacle to using viral load. BMRT can simultaneously identify the HPV type(s) and quantify the sample by testing housekeeping genes and the cells. This avoids the influence purely by the number of cells in the sample and can more accurately represents the viral load. So far there are few studies on simultaneous genotyping and viral load analysis for high-risk HPV. In a prior study we used micro-cutting technology to accurately obtain the viral load of specific subtypes of HPV in cells from cervical lesions. This work showed that HPV16/52/58 viral load was closely related with the grade of cervical lesions . Dong all cancer –15, so wLi  confirmeOur study shows that BMRT-HPV, as the primary screening algorithm for cervical cancer, has similar sensitivity to Cobas, which is similar to the result of the Chen’s study  This sugThe ideal secondary cervical cancer screening strategy should have the same characteristics as the primary screening strategy. It should be sensitive and specific for CIN 3 or cancer, reproducible, inexpensive, and not require an extensive heath care infrastructure. Admittedly, a highly specific secondary screening algorithm will, in most circumstances, compromise the overall sensitivity. In this study, the secondary screening algorithms #4,#5,#6 after BMRT-HPV primary screening have the same sensitivity for CIN3+ as Cobas primary screening, and the specificities are increased by 8%-10%, while colposcopy referral rate is also decreased significantly. This indicates that these three above secondary screening algorithms are feasible. The 2014 ASCCP transitional guidelines for cervical cancer screening. recommended HPV-16/18 typing combined with cytology as a secondary screening strategy for HPV primary screening [In this study, the BMRT test was performed on the remaining samples taken by the physician using a nested case-control design. Eight patients were BMRT HR-HPV(+) but Cobas, SeqHPV and cytology were negative, and therefore colposcopy was not performed. Considering that only 8 women who were only positive for BMRT HR-HPV were not recalled, it is believed that there is little influence on the overall data presented.In conclusion, type-specific HR-HPV viral load is closely related to the severity of cervical lesions. BMRT HR-HPV viral load combined with subtypes can be used as a secondary strategy for HPV positive women in primary screening. This may have important application to low resource regions and we plan to test this in the future.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Sexually transmitted infections (STIs) cause a major public health problem that affect both men and women in developing and developed countries. The aim of the study was to estimate the prevalence of 11 STIs among women who voluntarily participated in the study, while seeking gynecological checkup. The existence of an association between the presence of pathogens and symptoms and various sociodemographic risk factors was assessed.hlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis, Candida albicans and Human Papillomavirus (HPV). Positive HPV samples underwent genotyping using a microarray system.A total of 505 vaginal and cervical specimens were collected from women above 18 years of age, with or without symptoms related to gynecological infections. Nucleic acid was extracted and samples were tested by real-time PCR for the following pathogens: CGardnerella vaginalis, 35% for Ureaplasma parvum, 8% for Candida albicans, 6.7% for HPV, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis, 2% for Trichomonas vaginalis, 0.8% for Chlamydia trachomatis, 0.4% for Mycoplasma girerdii, 0.2% for Mycoplasma genitalium and 0.2% for Neisseria gonorrhoeae. Lack of symptoms was reported in 187 women (37%), among whom 61% were infected. Thirty-four samples were HPV positive, with 17 high risk HPV genotypes (HR-HPV); the highest rates being recorded for types 16 (38%), 18 (21%) and 51 (18%). Out of the 34 HPV positives, 29 participants had HR-HPV. Association with various risk factors were reported.Of the 505 samples, 312 (62%) were screened positive for at least one pathogen. Of these, 36% were positive for This is the first study that presents data about the presence of STIs among women in Lebanon and the MENA region by simultaneous detection of 11 pathogens. In the absence of systematic STI surveillance in Lebanon, concurrent screening for HPV and PAP smear is warranted. Sexually transmitted infections (STIs) cause a major public health problem; they affect both men and women in developing and developed countries. There are more than 30 types of STIs caused by different pathogens including bacteria, viruses and parasites that may differ in their clinical manifestations . STIs arThe development of new technologies that include nucleic acid amplification platforms, such as multiplex PCR assays, have improved the diagnosis and reporting of various infections , 8. The There are limited STI surveillance reports published in the MENA region. In Lebanon, routine screening for all potential STIs is cost-prohibitive, limiting the knowledge about transient carriers and transfer of pathogens. Premarital sexual intercourse is becoming more common in Lebanon , 11. ThiThe aim of the study was to estimate the prevalence of eleven STIs among women seeking gynecological checkups, evaluate the occurrence of concomitant STIs, and assess the existence of an association between the presence of pathogens and symptoms and various socio-demographic risk factors. Determining the presence of STIs is essential to improve the reporting system in the country and to develop appropriate health care strategies that focus on preventive and protective measures.A cross-sectional study was conducted in Beirut, Lebanon between November 2016 and December 2017. An estimated number of 377 subjects was determined using the Raosoft online sample calculator with alpha 0.05 and a confidence interval of 95% . ParticiWomen aged 18 years and above, presenting with or without symptoms of possible reproductive tract infections were included. Whereas, women who underwent hysterectomy or claimed not to have any kind of previous sexual intercourse were excluded. Since specimen collection may include a mild invasive procedure, pregnant women were excluded from this study for safety reasons. On the other hand, all nonpregnant women who were approached, voluntarily participated in the study, knowing that the screening was free of charge.This study was approved by the institutional review board of Saint George Hospital and Makassed General Hospital. A written informed consent was obtained from all eligible women before entering the study. Women were informed about the study, their participation was voluntary and they had the right to withdraw at any moment. The risks and benefits were clearly explained. Results were communicated to the gynecologists who reported them to patients.N = 61) of the questionnaires. For women who had a positive HPV results in our selected sample, follow-up with their healthcare providers showed that only 17 of them underwent a Papanicolaou (PAP) smear test simultaneously. Data were reported in our results to determine if there are discrepancies between presence of the HPV infection and PAP smear pathological results.The healthcare practitioners, including physicians, midwives or nurses, were responsible for collecting the samples and filling the questionnaires with the patients. Data collection included information about the socio-demographic profile of patients, their lifestyle, habits as well as their medical and sexual history. The questionnaires were completed having less than 10% missing data on some questions except for the item on job occupancy which had 15% missing data. Clinical symptoms such as painful urination, abdominal pain, discharge, itching, painful intercourse, bleeding in urine and bleeding post sexual intercourse were recorded. Clinical symptoms were not filled in 12%  were collected from each woman and placed in a single tube containing universal transport media  and stored prior to testing at − 80 °C.DNA from 200 μl of each sample was extracted from the collected tubes using a QIAamp DNA extraction kit  according to the manufacturer’s protocol. An artificial single stranded molecule was used as an internal control  for all Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Ureaplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis and Candida albicans.Four multiplex PCR assays that include two quadriplex and two duplex assays were used to test for the presence of 10 pathogens: Primers-probes sets were purchased from Metabion  and Thermofisher  Table S. PositivA real time PCR monoplex reaction that target the L1 open reading frame of HPV was performed using the GP5+/6+ consensus primers pairs (GP5+ primer: 5′-TTTGTTACTGTGGTAGATACTAC-3′ and GP6+ primer 5′-GAAAAATAAACTGTAAATCATATTC-3′) . A univePositive HPV samples were screened for HPV genotypes using EUROArray HPV kit . The kit includes amplification and hybridization steps that identify 30 different HR and LR HPV types. The kit is based on the amplification of E6 and E7 viral oncogenes fragments and their detection via hybridization with immobilized DNA probes , 17.The assay was performed according to the manufacturer’s instructions. The first reaction was a Pre-PCR step that included 10 μl of PCR Mix A and 10 μl of PCR Mix B and 5 μl of the extracted sample DNA. The PCR reactions were performed on the CFX96 real-time PCR machine . Each PCR product was mixed with 65 μl of Hybridization buffer and incubated at 45 °C for 60 min on a microarray slide. Slides were washed with buffer, dried and read on the Microarray Scanner and EUROArray Scan software that detects signal strength in terms of immunofluorescence units.Data analysis was performed using SPSS (version 20.0). Descriptive analysis including frequencies, percentages and averages were used when appropriate to describe the population under study. Prevalence was calculated to indicate the number of subjects carrying STIs among the recruited sample. A chi-square test was used to assess the association between exposures and outcomes expressed as categorical variables. The threshold for significance was set at alpha 0.05. Univariate and multivariate logistic regression were used to calculate odds of infection relative to variables that showed significance at the bivariate level. Odds ratios were considered significant if the 95% confidence interval excluded 1.N = 312) of the participants. Bacteria were identified in 58% (N = 295) of the patients; 8.1% were positive for C.albicans (N = 41), 6.7% for HPV (N = 34) and 2% for T.vaginalis (N = 10). The pathogens causing bacterial infections were 36% for G.vaginalis (N = 184), 35% for U.parvum (N = 175), 4.6% for U.urealyticum (N = 23), 3.6% for M.hominis (N = 19), 0.8% for C.trachomatis (N = 4), 0.4% for M.girerdii (N = 2), 0.2% for M.genitalium (N = 1), and 0.2% for N.gonorrhoeae (N = 1) , three , four  or six coinfections  N = 257) reported having genital clinical symptoms, of whom 64% (N = 165) were positive for the tested pathogens. A high rate of U.parvum and G.vaginalis coinfection was observed (N = 103), among which only 49% were seen as symptomatic. No significant association was however observed between the presence of studied pathogens (when detected as single or multiple infections) and the presence of any symptoms (p = 0.330).Among the 505 women, 50% (N = 187) of the women, of whom 61% (N = 114) were carrying the pathogens  had varied HPV genotype combinations of HR-HPV and LR-HPV (Table S) with different prevalence rates and percentages. Figure N = 13) and 18 . A lower prevalence of LR-HPV was observed where 11 LR-HPV genotypes were identified, with the highest number of patients being recorded for types 54  and 42 . HPV genotypes were grouped on the basis of their presence as single, double, triple or more genotypes (Table SN = 15). HPV genotype 16 was found as a single genotype in five women (33%). As the number of coinfecting HPV genotypes increases, the percentage of women positive for more than one genotype decreases from 24% (N = 8) for 2 types, to 12% (N = 4) for 3 types, reaching 3% (N = 1) for 8 simultaneous genotypes (Table SThe HPV positive samples (V Table S with dif) and 18 1%, N = 7) and 18 1%, N = 7) and 18 1%, N = 7Further investigations were conducted for the 17 HPV positive patients who had PAP smear.N = 12, 71%). Five patients had normal cytological results in the presence of HR-HPV  had taken HPV vaccine. Of the vaccinated women, 7 (35%) were positive for HPV. By observing the genotype combination present in the 7 women, two had one of the HR-HPV genotypes present in all HPV vaccines; one female was positive for HR-HPV type 16 and another for HR-HPV type 18 along with other HPV genotypes. Three women were infected with LR-HPV genotypes.A total of 505 women aged between 18 and 69 years, with a mean age of 38 years (SD = 10.8), were enrolled in this study; a third of the women who participated were between the ages of 30 to 39 years old 32.8%). The majority were Lebanese (63.6%) and housewives (63.6%)  and the presence of infection. Eighty-six percent (86%) of single women had an infection compared to 61% of married women and 40% of divorced/widowed women. No association was observed between nationality and the presence of infection.p = 0.000).A significant association was observed between the presence of infection and age groups, with a higher proportion among younger women aged between 18 and 29 (73.9%) compared to older women (50 to 69) (43.8%) . Coinfections were mostly observed in women aged between 18 and 29 years, while women aged 50 and above were either not colonized (56%) by the studied pathogens or colonized by a single pathogen (25%) . Moreover, it was noticed that as age increases the presence of HPV decreases: 11 women ranged between 30 and 39 years, 5 women between 40 and 49 years and 4 women between 50 and 59 years.Other findings revealed that a combination of ≥3 HPV genotypes were most likely found in age groups 20–29 and 30–39 while older age groups, 40–49 and 50–59, had either one or two genotypes (Table Sp = 0.015).Women who reported to smoke and drink alcohol were 32 and 16% respectively. The only significant association between behavioral risk factors and the presence of infection was the consumption of alcohol with 73% being infected among consumers compared to 59% among non-consumers . The presence of infection was significantly associated with the type of contraceptive (P = 0.036), with a higher proportion among IUD users.Only 162 women (32%) were using contraceptives, with intrauterine device being the most frequently used  and N.gonorrhoeae (0.2%) including one asymptomatic coinfection. Our results were comparable to other studies in the Middle East and North Africa region (MENA) and the Eastern Mediterranean Region (EMR) which reported a prevalence ranging from 2.5 to 4.2% for C.trachomatis and 0.1 to 0.6% for N.gonorrhoeae [This study presented a low prevalence of both orrhoeae –22. The orrhoeae , 23–27. U.parvum was the most frequently detected microorganism with an overall prevalence of 34.9%, and the most isolated single pathogen. U.parvum was frequently associated with increased positivity for G.vaginalis, however, no significant association was found between the detection of both pathogens and symptoms. Neither U.parvum, U.urealyticum, nor G.vaginalis seem to be significantly associated with symptoms in females when isolated as a single pathogen; their role in the female urogenital tract therefore remains unknown, although reported cases have shown that these infections can cause cervicitis and bacterial vaginosis [Our study has found that among the mollicutes, aginosis –30.C.albicans, whose overall prevalence was lower than G.vaginalis, was not associated with symptoms and was mostly observed among women aged between 30 and 39 years (50%). Candida species may persist for months, years and live in symbiosis with vaginal microbiota without causing any symptoms [andida or alteration in the protective defense mechanisms [symptoms , 32. Howchanisms , 34.T.vaginalis especially in asymptomatic individuals [T.vaginalis infection in our study was higher (2%) than the one found in a previous study conducted in Lebanon (1.2%) [T. vaginalis was also reported to be low in other industrialized countries [New diagnostic assays such as nucleic acid testing improved the detection of ividuals . This man (1.2%) . T. vagiountries , 37.HPV genotypes were investigated to provide an insight into the need for HPV vaccination at sexual debut or early adolescence age. The prevalence of HPV in our study sample was 6.7% which is in concordance with the results of a meta-analysis that was conducted in 2012 on HPV in the MENA region . The ageData on the prevalence and genotype distribution of HPV infection in Lebanon are limited. The most frequently detected HPV genotypes among women were the HR-HPVs. Our study found that HPV16 (38%), HPV 18 (21%) HPV51 (18%), HPV 26 (15%), HPV39 (15%), and HPV 73 (12%) were the six predominant genotypes in the sample. If the immune system fails to clear the HR-HPV types, infections will be associated with high risk for cervical disease progression leading to cervical cancer .WHO has issued a document on controlling cancer in the Eastern Mediterranean region where only 35% of cervical cancer were reported at early stages while the rest were discovered at later stages; the latter are usually associated with bad prognosis and minimal recovery rate . AnotherIn any country, it is necessary to know the HPV genotype distribution to assess the effectiveness of vaccines recommended by the gynecologists. The predominance of HR-HPV in our study favors the recommendation of HPV vaccination. Currently available HPV vaccines are Gardasil, Gardasil 9, and Cervarix , 47; allHPV vaccines are still expensive in Lebanon, not yet recommended by all pediatricians and not offered by the Lebanese government as part of the national immunization program. The ministry of health in Lebanon should implement a systemic approach for HPV vaccination similar to that initiated by an international organization, GAVI, who helped 30 countries to introduce HPV vaccines into their national immunization programs with the aim to protect around 40 million girls from cervical cancer by 2020 , 50.In this study, STIs were common in single (86%) and married women (61%) indicating that sexual behavior could be associated with infection . Women mThe use of IUD in women was high reaching 54%; 72% of those were positive for the infection. Previous studies showed an association between IUD and pelvic inflammatory diseases among women with sexually transmitted infection . The risThis study has also shown an association between STIs and alcohol consumptions (73%). The latter could have an impact on the sexual behavior of women and their sexual arousal , 53–56.In Lebanon and the MENA region, this is the first study that presents data about the prevalence of STIs among women by simultaneous detection of 11 pathogens and describes the distribution of circulating HR-HPV and LR-HPV genotypes. It also identifies various risk factors associated with STIs.Hemophilus ducreyi, Treponema pallidum and blood born viruses  were not investigated. The sample chosen is convenient and limited in its majority to married women, it also excluded non pregnant women. Future research directions should include STI screening for men and women of different age groups at the national level.However, the prevalence of other STIs such as HSV, The results of this study should trigger the healthcare professionals to implement reproductive healthcare services that are accessible and affordable for all. Awareness campaigns are also necessary for the population to adopt a healthy sexual behavior.Additional file 1: Table S1. Nucleotide sequences of primers and probesAdditional file 2: Table S2. HPV genotypes distribution patterns detected in 34 samplesAdditional file 3: Table S3. Comparison of Pap smear results with HPV genotyping findings of 17 HPV positive patients