Fenneropenaeus merguiensis). HPV is thought to slow down growth and cause mortality in the juvenile stages of banana shrimp. Genetic improvement through selection of shrimp resistant to viral diseases is one approach to address this issue. This is the first detailed report on an aquaculture species that investigates whether viral titre varies among families and is heritable, and thus whether viral titre per se is a possible candidate trait for selection to produce resistant stock.In shrimp farming, major production losses are caused by viruses. Hepatopancreatic parvovirus (HPV) is one of the viral pathogens that infect banana shrimp (2 = 0.41). Genetic correlations of HPV with body traits  ranged from −0.13 to −0.38. HPV titre was negatively correlated with raw and cooked body colour .HPV titre was measured by quantitative polymerase chain reaction of DNA extracted from 1137 offspring (from 48 full-sib families). Estimated heritability of HPV titre, based on the linear animal mixed model, was moderate (hThis is the first study based on a large dataset that provides evidence that viral titre may have a genetic component in penaeid shrimp or even in any aquaculture species. The moderate heritability estimated for this trait suggests that resistance to HPV may be achieved by selecting for low HPV titre. With moderate and negative correlations, selection for resistance to HPV should gradually improve body traits and colour of banana shrimp. Hepatopancreatic parvovirus (HPV) is a viral pathogen that infects penaeid shrimp and also frequently co-infects shrimp with other viruses –3. HPV wVarious biosecurity practices in hatchery and farming systems, e.g., pathogen screening in broodstock, water filtration and disinfection before stocking, have been advocated to control the spread and impact of shrimp viruses . On the In addition to the biosecurity practices, genetic improvement is another option to control the impact of viral diseases by selecting for disease resistance . The maiLitopenaeus vannamei), i.e. against taura syndrome virus (TSV) [2 = 0.22 to 0.30) and white spot syndrome virus (WSSV) [2 = 0.01 to 0.07).Heritabilities of resistance against viral diseases have been reported in several aquaculture species. In farmed fish, estimated heritabilities of resistance to viral infection are generally moderate to high and can range from 0.24 to 0.79 . In marius (TSV) , 14 withs (WSSV) , 16 withTo date, heritabilities of viral resistance in aquaculture species have been estimated by using survival data after challenge testing. Survival data was considered as a binary variable  that was recorded at the end of the experiment, usually at the time when 50 % of the tested animals are dead or mortality rates flatten out , 11, 15.Although many comprehensive models have been developed to increase the accuracy of genetic estimates from survival data, setting an experimental infection to obtain survival information still requires a dedicated testing design that considers inoculation approaches, challenge dosage, stock density, testing capacity and operational costs . All intAn alternative to measuring the phenotypic impact on the host of the virus is to measure the viral titre itself, so that viral titre may be the selection criterion, provided it is heritable. Indeed, Van Koevering et al.  and BosiOne may postulate that using viral titre as a selection criterion irrespective of its heritability cannot yield improvement in survival, due to poor correlations between survival and viral titre and thus that classical challenge testing cannot be replaced by screening for virus titre. However, from a logical point of view, the idea that selection of resistant individuals will reduce or eliminate virus titre and thus, reduce or eliminate the negative impacts of a given virus on a given aquaculture species, is appealing.Overall, in view of all these potentially desirable aspects of using viral titre as a selection criterion, and notwithstanding the efficacy of viral titre versus survival as a selection criterion, it is remarkable that, until this report and that of Knibb et al. , there hHepatopancreas samples of 1137 banana shrimp were collected at harvest time after 140 days of grow-out; shrimp had an average body weight of 16.5 g. These 1137 individuals were the offspring of 48 full-sib families with family size ranging from 9 to 30 offspring. They were cultured within a single grow-out pond in a commercial banana shrimp farm at Cardwell, Australia. Details on rearing conditions, breeding practices, genotyping and pedigree construction are in , 28. The2, 250 nM primers and 4 μL (i.e. 160 ng) normalised DNA. Cycling conditions were as follows: a first step at 95 °C for 10 min, 35 cycles at 95, 60 and 72 °C each for 25 s. The HPV titres were expressed as copy number per μg DNA based on the standard curve.Essentially standard curves were produced using known DNA concentrations, and the DNA amounts in the new samples, which were estimated with reference to the standard curve. The genomic DNA of 1137 banana shrimp was extracted using a DNeasy Blood and Tissue Kit (QIAGEN) following the manufacturer’s instructions. DNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and normalised to a concentration of 800 ng/μL. HPV titres were quantified based on an absolute quantitative PCR method using the SensiMix HRM kit (Bioline) and validated HPV primers, HPV140F (5′-CTACTCCAATGGAAACTTCTGAGC-3′), HPV140R (5′-GTGGCGTTGGAAGGCACTTC-3′) . QuantitThe hepatopancreas samples were separated into two main groups. The first group consisted of 897 samples for which HPV titres had not been quantified. The second group included 240 samples for which HPV titres had been determined in our previous study . In thisIn our previous study , each saThe shrimp HPV titres that were quantified by qPCR were transformed to the base 10 logarithm termed log(HPV). All preliminary statistics were analysed using the IBM SPSS Statistics 22 software . The logP < 0.05) and was included in the model to estimate heritability of log(HPV). The proportion of log(HPV) variation explained by the qPCR batch in the statistical model was only 3.8 %.We used histograms to graphically represent the distribution of the HPV titre data and assess its normality. The empirical frequencies of log(HPV) followed the theoretical curve drawn from a normal distribution, which indicated that the HPV titre data was approximately normally-distributed. ANOVA analysis was conducted to examine statistical differences of log(HPV) means between the 48 families studied. The general linear model was used to investigate the relationships between fixed factors and log(HPV). The fixed factors included sampling locations around the pond, sampling time, the person who sampled the hepatopancreas, sex, DNA quality, qPCR batch and qPCR run date. Among these factors, only the effect of qPCR batch was statistically significant (mn = log(HPV), μ = estimated population mean, Qm = qPCR batch; m = 1–25, an = additive genetic effect of individual shrimp in the pedigree; n = 1 to 1137, emn = residual.The heritability of log(HPV) was estimated using a linear animal mixed model in ASReml 4.1 . The modijklmn = traits under study, μ = estimated population mean, Si = sampling batch within a single grow-out pond; i = 1–8, Tj = sampling time; j = AM and PM, Ok = operator; k = 1 and 2, S1 = Sex; l = M and F, Qm = qPCR batch; m = 1–25, an = additive genetic effect of individual shrimp in the pedigree; n = 1 to 1137, eijklmn = residual.The phenotypic and genotypic correlations of log(HPV) with commercial traits such as body weight, body length, head length, body width, abdominal tail weight, meat yield, raw and cooked colour, and ‘flesh streaks’, were estimated using a series of two-trait animal mixed models in ASReml 4.1 . ASReml i and xj are the estimated heritabilities based on Eqs. i and σj are their respective standard errors. The observed z-score was tested against a large-sample normal distribution (α = 0.05).In this study, heritability of log(HPV) was estimated by using the model that included the significant effect i.e. qPCR batch Eq. . In addimentclass2pt{minim47,1089 = 7.334, P < 0.001; Fig. P < 0.001). The log(HPV) of the family with the highest HPV titre was more than 2.5 fold higher than that of the family with the lowest HPV titre. There were also large differences, up to sixfold, in log(HPV) among individual shrimp within families. An example of log(HPV) variation within the family with the lowest (i.e. highest HPV resistance) and that with the highest HPV titre is in Fig. The log(HPV) values averaged for each of the 48 families ranged from 3.52 ± 0.16 to 6.29 ± 0.20, and showed significant differences across families (ANOVA FP < 0.001) with a correlation coefficient (r) close to 1, r = 0.98,  and genotypic (rg) correlations of log(HPV) with body and carcass traits are in Table The heritability of log(HPV) was moderate, i.e. 0.41 ± 0.08 (Table P > 0.05).The additional analysis that included other systematic effects in the model Eq.  yielded Overall, we found that qPCR for HPV detection in banana shrimp was highly reproducible when the same sample was tested even months apart. Such high reproducibility of qPCR has been reported elsewhere , 34 and The current work was completed over several months and faster turnaround is probably needed for applied selection programs. However, recent technologies and qPCR machines that are capable of analysing 96 samples, even up to hundreds of samples at a time, have become available, at relatively low costs; our calculations are that the same work can now be completed in weeks instead of months. DNA assays such as immunofluorescence titration and virus particle counts are fast and inexpensive methods to measure viral titres, although perhaps at the cost of accuracy , 25. Morprima facie evidence of a moderate heritability indicates that this uncontrolled variation was not sufficient to obscure genetic effects. Another advantage of using viral titre from qPCR analysis to assess resistance to virus is that shrimp samples can be directly collected at harvest times under farm condition, and transferred to the laboratory for viral quantification. This procedure of testing the animals from commercial conditions does not require challenge facilities, thus investments related to challenge testing  are reduced or even completely avoided. This approach also guaranties that the assessed parameters are relevant to commercial operations, whereas there is always concern about whether dedicated challenge testing is fully relevant to commercial production. The moderate heritability of viral titre suggests that selective breeding for low viral titre is possible, and may result in shrimp that are resistant to the virus.This is the first report, apart from our preliminary study , which pNot only was there substantial HPV titre variation among families, there was also substantial variation among individuals within each family, which suggests that selection should consider both within- and between-family variation.rg ~ 0) were reported between body weight and resistance to viruses and bacteria, e.g., viral nervous necrosis (VNN), vibriosis and furunculosis in various cultured marine fishes [C. rogercresseyi [Oncorhynchus mykiss) [per se.The genetic correlation between HPV titre and body weight was negative and the magnitude of the correlation was moderate (−0.33), which suggest that selection to increase HPV resistance (i.e. by reducing HPV titre) would improve growth performances (e.g. increase body weight) in banana shrimp and vice versa. Our results contrast with those of a previous study on pacific white shrimp for which highly negative genetic correlations between body weight and WSSV resistance were found for two breeding lines (−0.55 and −0.64) . In marie fishes –38. Howecresseyi , while a mykiss) . To someImproving HPV resistance in shrimp is also expected to increase some carcass characteristics  as indicated by the negative (favourable) genetic correlations between the two traits. Besides growth performances and carcass characteristics, raw and cooked colours of banana shrimp are expected to be improved (more pronounced dark or red colour) by selecting for HPV resistance. Another finding of our study was the negative genetic correlation between HPV titre and condition of the hepatopancreas (yellow hepatopancreas). This could be a desirable trait from a commercial perspective because selection for reduction in HPV titre (i.e. increase in HPV resistance) will likely decrease the incidence of shrimp with yellow (presumably undesirable) hepatopancreas. Unfortunately, to our knowledge, genetic correlations between viral resistance and condition of the hepatopancreas have never been reported in any other aquaculture species for comparison with our findings.To date, the nature of the defence mechanisms against viruses in shrimp is still unclear. Various studies –3, 41 haThe favourable heritability of HPV titre and genetic correlations that we obtained in this study indicate promising opportunities to improve HPV resistance in banana shrimp without compromising other important traits.In this study, we do not report any hard data that specifically relate HPV titre to survival. What data that are available are only circumstantial. For example, as reported above, we observed negative genetic and phenotypic correlations of HPV titre with measures of growth; this suggests that HPV titre has an impact on production traits but may not particularly relate to survival. There are data on counts of offspring per family but they likely reflect problems of differential fertility and other genetic or stochastic events in the hatchery and prior. Indeed these counts were not significantly correlated with family means of log(HPV).per se, is that HPV is often detected with other viruses [One unresolved matter is the degree to which HPV titre and survival are correlated, or not, and whether such a correlation is evident only at a certain threshold titre, i.e. perhaps below this threshold there is no correlation; shrimp may have HPV but are not “sick” or “diseased” or do not show any specific clinical signs. Indeed, various studies , 4, 5 ha viruses , 41; morTo what degree many of these uncertainties and unknowns can be best resolved by returning to more classical challenge testing is debatable. Perhaps we could test predefined HPV titres against ensuing survival; this approach presumably would identify any effects of HPV titre and relate the effects specifically to HPV rather than to other viral infections. However, we would then be returning to the swath of issues and problems with challenge testing referred to in the discussion such as costs and potential inadequacy with industry practices, etc.ipso facto, is that if we can eliminate the virus by selection, as suggested by this study, then concordantly, we will eliminate the negative effects of the virus.In conclusion of this section, it is possible that selection for viral titre at the levels recorded here may not achieve desirable outcomes for situations of extreme viral titres and catastrophic collapse, i.e. correlation between HPV titres reported here and survival to HPV is poor as the pond evolves towards collapse. Notwithstanding these uncertainties, what we can say Penaeus monodon) after feeding animals with infected black tiger shrimp postlarvae, while Sivakumar et al. [There are two major types of transmission, horizontal and vertical, although the two are not mutually exclusive. Horizontal transmission refers to all possible situations in rearing environments that diseases can be transmitted from infected animals to healthy ones in the same facility. There is circumstantial evidence for horizontal transmission of HPV: Catap et al.  detectedr et al.  reportedr et al. , 45. AllIn our study, shrimp were cultured communally in the same grow-out pond, thus presumably, they had equal chances to be exposed to diseases excepting prior infections due to vertical transmission. Variation in this type of  transmission in the pond is more like an environmental factor, and by itself should not contribute to the additive genetic variation of HPV titre and the repeatable differences among families.There is a possibility that vertical transmission, from parents to offspring, may have contributed to the differences of HPV titres among families. Several previous studies investigated the potential for vertical transmission, from infected parents to offspring, of different farmed shrimp viral diseases, e.g., MBV and IHHNV. All these studies confirmed the existence of a parental contribution to viral infection in the offspring, however the proportion of virus transmitted to the offspring depended on the severity of the infection in the parents –48.From a scientific perspective, vertical transmission if not swamped by horizontal transmission, could lead to inflated or completely artefactual genetic parameter estimates. Unfortunately, estimation of the maternal effect was not possible with our data structure and we cannot discount that the maternal effect from vertical transmission may have merged with, and inflated, the additive genetic estimates. In other words, there is considerable risk that the heritability is biased but that our design cannot avoid this pitfall. The only publications so far for farmed fish that are communally reared at early stages show that maternal effects are negligible, at least for traits such as growth and condition index , 50. SucWhile we have no direct evidence for vertical transmission of HPV in shrimp, if it occurs and contributes to the differences in HPV titres among families, then on-farm selection for families with low HPV titres will still result in lower HPV titres in the next generation through a reduction in the viral titre of the parents.The additive genetic variation of HPV titre suggests that resistance to HPV in banana shrimp can be improved by selective breeding. Selection for HPV titre may also improve other traits of commercial importance such as body weight, meat yield, condition of the hepatopancreas, and shrimp colour. This is the first major report on viral resistance as a quantitative trait and the genetic parameters of HPV titre, for an aquaculture species.


Lactobacillusdominant) and CST IV  were found in 41% and 59% of samples, respectively. We did not find association of any CST to postpartum period (six or twelve months), HPV infection or cytology . However, five bacterial genera were associated with cervical lesions , with significant odds ratio (OR) of 40 (2.28–706) for the presence of Moryella and 3.5 (1.36–8.9) for Schlegelella. Longitudinal analysis of samples at postpartum that regressed , progressed  and maintained the cytology  evidenced Gardnerella with a significantly higher abundance in regressing lesions. In the current study, we report the first data on the cervical microbiota of HIV-positive women in the postpartum period. Consistent with previous studies of HIV-negative cohorts, HIV-positive women present a stable cervical microbiota of high-diversity in the postpartum period. Our results highlight that specific microbiota species may serve as sensors for changes in the cervical microenvironment associated with cervical lesions.The cervical microbiota composition and diversity of HIV-positive women in the postpartum period is unknown. Using a high-throughput bacterial 16S rRNA gene sequencing, we identified four community state types (CSTs). CST III ( The primary factor associated with the development of cervical cancer is the persistent infection by the human papillomavirus (HPV). Additional factors correlated with HPV persistence include immunodeficiency caused by HIV, smoking, use of oral contraceptives and, more recently reported, cervicovaginal dysbiosis2.Cervical cancer is the fourth most common cancer among women worldwide, and over 500,000 new cases are diagnosed each year, leading to more than 200,000 deathsLactobacillus sp. . However, in a state of dysbiosis, there is a marked reduction of Lactobacillus and a high diversity of bacteria, with increased abundance of anaerobic bacterial species5. Cervicovaginal dysbiosis is asymptomatic in some cases, turning the definition of normal/dysbiotic microbiota a point of current discussion. However, many women with cervicovaginal dysbiosis develop symptomatic bacterial vaginosis (BV), characterized by vaginal discomfort and homogeneous malodorous vaginal discharge7. Both symptomatic and asymptomatic dysbiosis have been associated with the risk to preterm birth, acquisition of sexually transmitted infections (STIs) and increased risk of pelvic inflammatory disease9. In general, previous studies on cervical microbiome have described five community state types (CSTs), I to V3. Whereas CSTs I, II, III and V are dominated by Lactobacillus species , CST IV is characterized by a high diversity and predominance of anaerobic bacteria as Gardnerella vaginalis, Atopobium vaginae, Prevotella sp. and other anaerobic bacterial species3.The classically-defined normal cervicovaginal microbiota is dominated by one or more Lactobacillus sp., combined with increased diversity of cervicovaginal microbiota, are risk factors for HPV acquisition, persistence, development of cervical intraepithelial neoplasia (CIN) and cervical cancer2. A study conducted with Korean women with and without CIN showed that those carrying Atopobium vaginae, Garderella vaginalis and L. iners in the absence of L. crispatus had an almost 6x higher risk for CIN. That study also showed the synergistic effect of this microbial pattern and oncogenic HPV infection on a very high risk (odds ratio of 34.1) of CIN10. In a longitudinal study with samples collected over a 16-week period, women with highly diverse or L. iners-dominated cervicovaginal microbiota were more likely to be HPV-positive, while a microbiota dominated by L. gasseri was associated with more rapid clearance of HPV infection11.It has been shown that reduction of 12. It is known that HIV-positive women more often present with high-risk HPV infection and development of cervical disease13. However, little is known about the influence of the cervicovaginal dysbiosis in HIV/HPV coinfected women. Comparison of HIV-infected and HIV-uninfected women showed that microbiota dominated by L. crispatus are associated with a lower prevalence of HIV/STIs14 and with decreased prevalence of HPV in HIV-infected woman15. In contrast increased microbiome diversity has been demonstrated in high frequency among HIV-positive woman14. The cervicovaginal dysbiosis may also generally affect maternal health, leading to postpartum complications17. A longitudinal study of woman followed-up during pregnancy and postpartum showed that the cervicovaginal microbiota composition is modified at six weeks postpartum, with increased bacterial diversity and decrease in L. crispatus moieties18.The high diversity of cervicovaginal microbiota is strongly correlated with local inflammation and it is also thought to increase the risk for HIV acquisitionBased on this information, we hypothesize that postpartum HIV-positive women have a highly diverse cervicovaginal microbiota, which could be associated with an increased risk for developing persistent HPV infection and consequently CIN. Therefore, our study aimed to evaluate the postpartum cervical microbiota profiles of HIV-positive women displaying diverse cervical intraepithelial neoplasia.+ T-cell count and HIV viral load values of the studied subjects were of 579 cells/μl and 344 copies/ml at 6 months after labor, and of 548 cells/μl and 409 copies/ml at 12 months after labor, respectively, and those differences were not significant. The frequency of HPV-negative women was significantly higher at 12 months than at 6 months postpartum (8% versus 37%). However, the frequency of HPV-positive women with lesions (LSIL/HSIL) was higher at 12 months compared to six months postpartum  and another dominated by Prevotella (CST IV-B.2)  Fig. .Lactobacillus-dominated CSTs (except for L. iners), generally seen in other studies. Additionally, we performed bacterial profile analysis by reconstruction of the 16S gene to confirm the results obtained in the read analysis with QIIME, and the two methods showed a high degree of similarity, ranging from 61 to 86%  Fig. . The PhyWe next analyzed the distribution of the samples classified as CSTs III and IV  with respect to the postpartum period, HPV status and cytology . However, no significant differences were observed in any of the comparisons . However, due to the fact that the LEfSe algorithm still suggests significant associations , we decided to keep all five bacterial taxa in further analysis for confirming their association with cervical lesions.We carried out analyses to identify differences in the microbiota composition, independent of the CST classification, through the linear discriminant analysis (LDA) effect size (LEfSe) algorithm as a way to discover potential biomarkers linked to a lesion status. The LEfSe analysis comparing the microbiota between normal and lesion samples showed significant differences in the abundance of ons Fig. . When weons Fig. , only MoSchlegelella thermodepolymerans and Moryella presented ORs of 3.5 (1.4–8.9) and 40.1 (2.28–706), respectively, in lesions compared to normal samples  to estimate the risk of carrying the bacterial taxa found in the previous analysis in cytological lesions. Gardnerella vaginalis presented significant differences in its relative abundance in samples that regressed the cytological status, showing a higher abundance in lesions compared to normal cervices of the same subjects  for their association with the lesion status. A total of 25 women had longitudinal samples available to analyze; 16 maintained status , 5 had lesions that regressed , and 4 had lesions that progressed . Only cts Fig. .Figure 421. In the present study, we showed that HIV-positive women with HPV infection at 12 months postpartum presented a higher frequency of cervical premalignant lesions than at 6 months postpartum. The persistence of HPV infection is an essential but not sufficient factor for the development of CIN and cervical cancer, and recently the cervical microbiota dysbiosis has been proposed as a key player in the development of cervical diseases10. Several studies showed an association of high-diversity cervical microbiota with HPV infection, CIN, cervical cancer and HIV acquisition23. However, the profile of such microbiota in HIV-infected women in the postpartum period remained largely unknown.HIV-infected women present a higher risk of HPV infection, persistence and development of cervical cancer compared to HIV-negative counterpartsL. iners, while CSTs IV (A and B) are high-diversity communities dominated by anaerobic bacteria. We did not find CSTs I, II and V described in previous studies which are dominated by L. crispatus, L. gasseri and L. jensenii, respectively3. L. crispatus (CST I) is present in high frequency in the vaginal microbiota of healthy white (European) and Asian women24. The absence of this CST among our samples is congruent with the observation that L. crispatus is associated with reduction in HIV acquisition and is generally absent in HIV-infected women22. Furthermore, the composition of CSTs is likely different in distinct ethnic groups. Studies showed that women of African ancestry have a higher prevalence of CST IV when compared to those of Asian and European ancestries24. The Brazilian population is highly mixed due to its colonization history, with large admixing of European and African descendants26. Therefore, the ethnic background of the participants in this study may also explain the high proportion of CST IV (A and B) found in the samples.In this study, we identified four CSTs in postpartum HIV-positive women, which for the sake of definition in previous reports have been named CSTs III, IV-A, IV-B.1 and IV-B.2. CST III is dominated by Lactobacillus sp., while CST IV-B shows a specific high proportion of Prevotella, Gardnerella, Atopobium, Mobiluncus and other bacterial taxa20. Herein, we describe a further stratification of CST IV-B into IV-B.1 (Gardnerella-dominant) and IV-B.2 (Prevotella-dominant). These two CSTs formed two distinct clusters, but no significant differences were observed in their diversity by Shannon index and PD whole tree analysis. Additionally, CST IV has been associated with HPV infection, CIN and HIV acquisition23. In this study, we showed that CST IV comprised a high proportion (59%) of the samples, but distinct CSTs were not associated with HPV status, the presence of CIN or the postpartum period (6 versus 12 months). A previous study reported a high frequency of CST IV among HIV- and HPV-positive women27. Additionally, HIV-positive women do not show significant variation in their microbiota irrespective of their HPV status27. Our data are in agreement with previous reports27 and suggest that HIV-positive women present a stable community of high-diversity bacteria in the postpartum period that is not altered by CIN status, HPV infection or time postpartum. Addionally, our results are consistent with a study that analyzed the microbiota from an HIV-negative women cohort with normal cytology, which showed a persistent and highly diverse microbiota in the postpartum period of samples collected for up to one year after delivery28. On the other hand, another study of vaginal microbiome among European HIV-negative women showed an increased prevalence of CST IV with increasing severity of cervicovaginal lesions, irrespective of HPV status29. Our findings prompt further investigation on the stability of microbiome in HIV-negative women with cervical lesions at different time points postpartum compared to HIV-positive women.CSTs IV-A and B are characterized by a high diversity of bacteria and modest to absent proportion of Bifidobacterium, Moryella, Schlegella and Aerococcus) and one in high abundance (Gardnerella) associated with cervical lesions in initial analyses. The Moryella and Aerococcus genera belong to the Firmicutes phylum. Recent studies associated this phylum with colorectal cancer31, but the role of these bacteria in cervical cancer remains unknown. The Aerococcus genus modulates the immune response by the production of proinflammatory cytokines12. Moryella was first identified in abscesses of clinical samples32, suggesting it may also play a role in immune modulation. Additionally, Gardnerella vaginalis is associated with high-diversity microbiome and is also able to modulate the production of proinflammatory cytokines in the cervical region22. Whereas inflammatory responses are required to eliminate sexually transmitted infections effectively, a hyperactivated chronic inflammation in the cervix has been correlated with increased risk of CIN and HIV acquisition33.Since the CSTs found herein were stable over time, we performed an association analysis of individual bacteria with cervical lesions. We found four bacterial genera in low abundance  and 3.5x (1.36–8.9) respectively, whereas the three other genera were not significant. These results and the Chi-square statistics reinforce the strength of the association of these two bacteria with cervical lesions.The five genera originally associated with cervical lesions in the analysis depicted above had their OR estimated for their presence in comparison with normal cytology. The OR of the Gardnerella vaginalis significantly changed its relative abundance in women in which the lesion regressed. However, among women who changed from normal cytology to cervical lesions, G. vaginalis levels remained statistically unchanged. These data may suggest that G.vaginalis may either induce the developmnent of cervical lesions or may colonize the cervix after lesions develop, and our data cannot discriminate between these two possibilities. The lack of additional associations may be due, at least in part, to the small number of paired samples analyzed in each category.The availability of longitudinal samples from many study participants (at 6 and 12 months postpartum) allowed us to evaluate the robustness of particular bacteria as biomarkers for cervical lesions. Upon longitudinal analysis of the taxa that were significantly associated with lesions in the former tests, none of them varied significantly among women that remained with the same status  over the two time points analyzed Fig. . On the L. crispatus dominant types. Moreover, we found three bacterial genera associated with cervical lesions  through distinct analyses. Little is known about the function of these bacteria on cervical microbiota homeostasis, and it is not clear how these bacteria modulate the development of cervical cancer, whether they arise before or after cervical lesions appear and whether they are a necessary component for the development of malignancy. We propose that distinct microbiota species may serve as sensors for changes in the cervical microenvironment, being able to modulating it or being modified by it towards health or disease.The cervical microbiota displays a complex interaction with the local environment and with the immune system, playing a central role in the cervical homeostasis. Here we demonstrated that the cervical microbiota communities of HIV-positive women at postpartum are stable, generally presenting a high diversity of bacteria and a lack of Eighty women enrolled at the Program for HIV-infected Pregnant Women at the Federal University of Rio Janeiro (UFRJ), Brazil, participated in this study. HIV-positive serological status was confirmed by an HIV rapid or ELISA test and subsequent Western blot following recommendations for HIV diagnosis by the Brazilian Ministry of Health. All participants were under antiretroviral treatment at the time of sample collection. Cervical smear collection was carried out from 2009 to 2010, and two time points were collected for each patient, at six and 12 months postpartum, respectively. However, both time points have been retrieved only for 25 subjects, totaling 50 (25 × 2) paired samples, that were used in the longitudinal analyses. As selection criteria for the longitudinal analyses, we selected samples of subjects with cytology information in the two time points collected. Individuals with ASCUS cytology at any one of the time points were excluded from the analyses. For 80 subjects, unpaired (single timepoint) samples were used in the analysis; the 6-month postpartum time point was available for 26 women, and 12-month timepoint was available for 54 women. Overall, a total of 105 individual samples have been analyzed.41. Clinical, obstetrical and sociodemographic variables were also retrieved from the medical records and from a questionnaire answered by the participants. All participants of this study were mixed-race people of European and African ancestries. The study was approved by the Ethical Committees of UFRJ  and of the Brazilian National Cancer Institute (INCA) (protocol #142/10). All participants signed an informed consent before enrollment in the study. All experimental procedures involving sample collection were performed in accordance with the Brazilian National Ethics Committee guidelines and regulations.Cytological Pap smear results were obtained from patients’ medical charts and were classified according to the 2001 Bethesda reporting guidelines42. All PCR products were analyzed by electrophoresis on 1.5% agarose gels, and positive samples were purified with the Illustra GFX PCR DNA and Gel Band Purification Kit  and sequenced by Sanger. The sequences obtained were analyzed through BLAST search, and HPV types were assigned based on the HPV type that best matched the query samples.Sample collection was performed with two endocervical cytobrushes; one was placed in 100% ethanol for Pap smear diagnosis, while the other was placed into 1 ml of phosphate-buffered saline and stored at −80 °C for DNA extraction. Total genomic DNA was extracted from cervical samples with the RBC Genomic DNA Extraction Kit , according to the manufacture’s protocol, and was stored at −20 °C until use. HPV DNA was detected by PCR amplification of an L1 gene fragment using consensus primers MY09/MY11 and GP5/GP6, and reaction conditions previously described43. PCR reactions were carried out using 5 μl Taq 10X buffer, 1.5 μl 50 mM MgCl2, 0.4 μl 25 mM dNTP mix, 25 pmol/μl each primer and 0.3 μl Taq Platinum polymerase (5U/μl). The final volume was adjusted to 50 μl with RNase/DNase free water . An initial denaturation step of 95 °C for 5 min was carried out, followed by 35 cycles of denaturation , annealing  and extension , and a final elongation step of 6 min at 72 °C. PCR products were visualized by electrophoresis in 1.5% agarose gels and bands of the expected size of 700–1,000 bp were purified with the Illustra GFX PCR DNA and Gel Band Purification Kit. Purified products were quantified in a NanoDrop apparatus , and 0.4 ng of DNA was used to prepare each library with the Nextera XT DNA Sample Preparation kit . Library concentrations were assessed by qPCR with the KAPA Library Quantification kit . Libraries were diluted to a final concentration of 10 pM, loaded onto the flow cell and clonal clustering with cBot (Illumina Inc.) was carried out. Sequencing by synthesis was performed on an Illumina HiSeq. 2500 system (Illumina Inc.).The V3-V6 region of the bacterial 16S rRNA gene was PCR-amplified using the primers 338F and 1061R, which have been reported to amplify over 90% of the bacterial sequences present in the Greengenes database44. Operational taxonomic units (OTUs) were assigned to the reads using QIIME’s pick_closed_reference_otus.py script against the Greengenes Database, and the Uclust algorithm was used to group and assign taxa45. Sequences were clustered into OTUs using a 97% similarity threshold. OTUs with a number of sequences lower than 0.005% of the total number of sequences generated were discarded and those remaining were summarized at the species level. A rarefaction curve was constructed for each sample  was used for classification. The taxa presented and their relative abundances as estimated in EMIRGE were reported and compared to the results obtained in QIIME for a subset of the samples to investigate possible divergence in taxonomic assignment between the two methods.The BCL2FastQ2 Conversion Software  was used to demultiplex data and convert BCL files to FASTQ file formats. Formatted reads were subjected to the FastQC  for quality filtering using the split_libraries_fastq.py script of QIIME20. The alpha diversity of each defined CST was estimated using the Shannon index and the Phylogenetic Diversity (PD) whole tree methods. Clusterization, diversity analyses and plots were carried out using the R environment.Unsupervised hierarchical clustering based on Bray-Curtis dissimilarity and average linkage was applied to define clusters according to abundance and taxa diversity of each sample. The clusters found were associated with vaginal microbiome CSTs classified in previous studies48, which uses Kruskal-Wallis and estimates the effect size of the comparisons, was used for evidencing potential biomarkers that distinguish normal from lesion samples. A 99% confidence interval has been used in this analysis. We corrected the p-value obtained with LEfEe for multiple testing using the Bonferroni’s correction in the R package. The odds ratio (OR) and 95% confidence interval values for the presence of specific taxa associated with risk of lesions were estimated using Microsoft Excel for Windows, and p-values were calculated according to Sheskin49. We calculated the unadjusted OR using the Haldane’s modification, which adds 0.5 to all cells containing zero counts to allow statistical calculations. The OR was adjusted to confounder variables  using logistic regression. The relative abundance of specific bacterial taxa was compared in groups of paired samples that maintained the lesion status  or changed the status between samples  with the Wilcoxon test in SPSS.Patients’ sociodemographic and clinical data have been summarized with frequency and medians. Frequencies of cervical cytology results and HPV status were compared between the two timepoints studied (6 and 12 months postpartum) using the Pearson’s chi-square test in the SPSS environment . The same test was used to compare the distribution of CSTs between different cervical cytology results , between different HPV statuses (HPV+ and HPV−) and between the two time points postpartum (6 and 12 months). Shannon indexes of the different CSTs were compared using the Mann-Whitney test in SPSS. The Linear Discriminative Analysis (LDA) Effect Size (LEfSe) program of the Galaxy environmentAll raw data files are available on Sequence Read Archive (SRA) upon accession number PRJNA392046.Supplementary information


Cervical cancer is one of the most common and lethal cancers in West Africa. Even though vaccines that protect against the most common Human papillomavirus (HPV) strains, 16 and 18, are currently in use in developed countries, the implementation of these vaccines in developing countries has been painfully slow, considering the pre-eminence of HPV-associated cervical cancer among women in those countries.We performed serological and PCR-based assessment of blood and tissue specimens obtained from women undergoing cervical cancer-related surgery at a major urban hospital in Bamako. Since several therapeutic HPV vaccines are currently in clinical trials, we also assessed willingness to participate in HPV cancer vaccine trials.Blood and biopsy samples of 240 women were evaluated for HPV types 16 and 18 by serology and PCR. Knowledge regarding the HPV vaccine and autonomy to decide to vaccinate their own child was assessed with a standardized questionnaire.HPV 16 and 18 were identified in 137/166 (82.5%) cervical cancer biopsy samples by PCR. Co-infection with both HPV 16 and 18 was significantly more frequent in women over 50 years of age than in younger women (63.0% vs. 37.0%). 44% of study participants said they would be willing to vaccinate their child with HPV vaccine. Only 39% of women participating in this study reported that they would be able to make an autonomous decision to receive HPV vaccination. Permission from a male spouse or head of household was identified as important for participation by 59% of the women.This study provides strong support for the introduction of currently available HPV vaccines in Mali, and also provides key information about conditions for obtaining informed consent for HPV vaccine trials and HPV vaccination in Mali. Nearly 85% of the half a million cervical cancer (CC) cases diagnosed each year occur in developing world countries. There are 27,300 new cases of CC identified in West Africa every year, and 16,500 deaths per year are attributed to CC ) and thuThe main risk factor of CC is infection with high-risk types of human papillomavirus (HPV) . As defiThe most recent HPV typing data trace back to 1995 and 2002 which showed that high-risk HPV types 16 and 18 could be identified in 54% to 62% of CC cases in Mali . To addrHôpital Gabriel Touré has an annual admissions rate of about 16,500 patients and serves two million patients in the direct community surrounding the hospital (Bamako proper) and 12 million in total across the wider region. It is the primary referral hospital for cervical cancer in Mali and 95% of cervical cancer patients diagnosed in Bamako are referred to this hospital.During the study period more than 3,344 patients were seen in the department of gynecology, of which 10% were diagnosed with cervical cancer (roughly 334). This study was performed over 20 months during that period and captured nearly 88% of the patients who had cervical cancer confirmed by biopsy during that period.From June 2011 to January 2013, all patients with histologically confirmed CC were approached by clinicians at the Gabriel Touré Hospital and asked whether they would be willing to participate in a study of HPV infection and in a survey. 240 women agreed to participate; informed consent was obtained for their participation. No compensation was offered in exchange for participation in the study. 240 women consented to the use of their cervical biopsy specimen and provided blood for serology; 235 women answered the survey questions (five declined to participate in the survey portion of the study). Cervical biopsy specimens and serum samples were processed at the Laboratory of Applied Molecular Biology at the University of Bamako.A standardized questionnaire was read to the participants in order to collect general and medical information and willingness to participate (WTP) in an HPV vaccination program and vaccination decision-making capacity. The questionnaire used in this study was adapted from a pilot study published by our group in 2013 . The strWhole blood samples (4 mL) were collected and centrifuged at 4000 rpm for 20 minutes; serum samples were transferred in cryovials, stored at -80°C and sent to an immunology laboratory  for serological marker detection of HPV subtypes 16 and 18 (see below). Cervical biopsies were collected in 400 μL of 10X Dulbecco's Phosphate-Buffered Saline  and stored at -80°C for further examination by polymerase chain reaction (PCR) assay for detection of HPV 16 and 18 DNA.Total DNA was extracted from biopsies using the QIAamp DNA Blood Mini Kit  following the manufacturer's instructions: 15–25 mg tissue sections were applied to a clean single-edge blade using strict aseptic technique to prevent contamination or cross-contamination of samples. The samples were digested overnight at 55°C in the lysis Buffer AL containing protease K , followed by precipitation of DNA with absolute ethanol in a spin column. The DNA was eluted with Buffer AE . DNA purity and concentration were quantified using a BioPhotometer . Two sets of primers specific to the E6 oncogene were used to detect HPV types 16 and 18 DNA , reverse and forward primers for HPV 16 or HPV 18 ; PCR-quality water was used to reach a final volume of 25 μL. To avoid false positive and/or negative results, a negative control (no template DNA) and an HPV-positive DNA sample were included in each assay. PCR amplification was performed using a DNA Thermocycler , as follows: 5 min at 94°C, 40 cycles  and a final 7 min extension period at 72°C.The PCR assays were performed as described by others ,22 with As a control of the PCR product size, 12 μl of PCR product were loaded on a 2% 0.5X TBE buffer agarose gel, stained with 0.3 mg/mL ethidium bromide, and photographed by UV transillumination with a KODAK EDAS 290. The molecular weight of the PCR products was determined by comparison to DNA molecular weight marker XIV .7 microspheres/mL in the dark at 4°C. A total of 5,000 of each VLP16- and VLP18-microspheres were combined with 50 μl of 1:4 diluted blood samples in a dark 96-well plate. To each well, 25 μl of HPV type-specific mAbs conjugated to PE  were added, followed by an overnight incubation at room temperature. Samples were transferred to a filter plate  and washed in PBS; the VLP-microspheres were re-suspended in 200 μl of PBS containing 1% BSA for analysis on a BioPlex Suspension Array System .The presence of antibodies to HPV types 16 and 18 was detected by a VLP-based competitive Luminex immunoassay (cLIA) using a multiplexed Laboratory Multi-Analyte Profiling (LabMAP3) assay system  as previously described ,24. BrieThe reference standard used for analysis was a serial two-fold dilution of a pool of serum samples from African green monkeys hyperimmunized with monovalent HPV L1 VLP types 16 and 18. Dilution-corrected serum values (in mMU/mL) were computed based on a four-parameter logistics fit of the standard curve on each assay plate. Samples that exceeded the limits of quantitation for the standard curve were retested at higher dilutions. Samples were considered Ab seropositive for the respective HPV subtype if the cLIA signal was ≥11 mMU for HPV 16 and ≥10 mMU for HPV 18. All standards, controls, and samples were tested in duplicate.http://www.graphpad.com) was used to calculate 95% confidence intervals. The Fisher test was applied to determine significant differences between groups. p<0.01 was considered significant.GraphPad  in Bamako, Mali. The informed consent document was translated from French to Bambara. The consent form was read aloud to individuals who were illiterate. All participants provided a signature or fingerprint indicating their informed consent. All participants were over 18 years old.Between June 2011 and January 2013, all of the patients undergoing biopsy for cervical cancer at Hôpital Touré, the primary referral hospital for cervical cancer in the region of Bamako, were approached to participate in this study. 88% (240) of the more than 270 women who had biopsies during that period agreed to have their serum obtained and their biopsy specimen evaluated for HPV testing. Of these 240 women, 235 agreed to participate in the structured interview.Characteristics of the study participants are summarized in The mean age of CC diagnosis was 49.0 years (SD: 13.6): 142 participants (60.4%) were diagnosed between the ages of 20–50 years, and 92 (39.1%) were diagnosed between 50–90 years. 134 participants (57.0%) reported having previous sexually transmitted infections (STIs).Cervical biopsy samples (N = 240) were tested by PCR using human β-globin pair primers as a control for sample integrity; 166 (69.2%) were PCR-positive for human β-globin. Of these 166 samples, 31 specimens were not positive for either genotype  . For theIn samples where HPV 16 and/or 18 was detected (n = 135), mono-infection with HPV 16 or HPV 18 was found in 65  and 17  cervical biopsy samples, respectively . Co-infeThe age distributions of mono- and co-infected women were different . SignifiAnother means to detect HPV infection (or the efficacy of HPV vaccination) is to measure the presence of anti-VLP-16 and -18 antibodies in the serum. Blood was obtained from 160 of the 240 patients; serum antibodies were assessed using a cLIA assay to measure the presence of anti-VLP 16 and 18 antibodies in the serum. HPV 16 antibody-positive blood samples were more frequent  than HPV 18  , which w235 women participated in the oral survey. Two hundred and four participants (86.8%) were able to recall that they had previously received any vaccine, and 228 (97.0%) said they would like the HPV vaccine to be available to young adolescent girls in Mali . Nearly This study reveals important information on HPV prevalence and subtype distribution among women diagnosed with CC in Mali, and to our knowledge, it is the first such study conducted in Mali since 2002 . This stet al. [et al) and with co-infection (31.9% of the 166 participants in our study vs. only 11.1%). These differences may be due sample size or to differences in the methods used; in the Bayo et al study, the authors used the PCR enzyme immunoassay method [In our study, HPV 16 and/or 18 was detected by PCR for 81.5% of CC patients. This prevalence of the infection is consistent with the last study published in 2002 by Bayo et al. , where 9y method  while wey method . Studiesy method . We founy method . The samThe mortality rate of cervical cancer in Mali is very high (69%). Vaccines that protect against HPV infection are in use in the developed world, and therapeutic vaccines are in clinical trials . Our resCo-infection has been shown to be associated with persistent HPV infection and therefore has also been associated with development of CC . In a prAs has been reported previously, serology  was less sensitive than the PCR assay to detect type-specific HPV infection in cervical cancer patients. This assay is primarily used for measuring effective sero-conversion soon after HPV vaccination. Regardless, the prevalence of HPV 16 and 18 by these two methods (one or both subtypes found in 81.5% of women by PCR and 43.5% of women by the less-sensitive antibody test) indicate that these genotypes are associated with CC among Malian women.HPV prophylactic vaccines were being introduced in Mali at the time of this study and therapeutic HPV cancer vaccines were under development. Thus, we also assessed the willingness of these women to participate in vaccine trials, and perhaps more important, their ability to autonomously give consent for participation in a clinical study. Among these participants who already were affected by cervical cancer, 93.6% were willing to participate in a CC vaccine trial. In addition, 97% of the participants agreed that the HPV vaccine should be made available to adolescent girls. This data and similar data published in a companion study  provide The ability of the female participants in this study to make autonomous decisions reflects local customs. Most of the participants identified men (husbands and fathers) as the primary decision-makers regarding their participation in HPV vaccination trials. In our parallel study, we also assessed autonomy regarding vaccination  among coSeveral studies have shown that women’s autonomy is positively related to children’s health outcomes . Since tWe also note that the women in this study reported that their age of sexual debut was 16, consistent with published studies for this region of the world . ProphylEven though cervical cancer screening is available in certain health clinics in Bamako, lack of public awareness about HPV and CC is one of the most important barriers to effective CC prevention. Additional studies by our group  have demIn conclusion, this study provides important information on HPV prevalence and subtype distribution among women diagnosed with CC in Mali, and to our knowledge, it is the first such study conducted in Mali since 2002 . Our stuS1 FileThis questionnaire was adapted from a pilot study published by our group in 2013 .(DOCX)Click here for additional data file.


To investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.Defined solution samples of TB and a mixture with lutein/zeaxanthin were exposed to blue light. Thermal degradation processes were ruled out using controls not subjected to irradiation. All samples were analyzed using optical microscopy, UV/Vis spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Degradation kinetics were determined based on changes in absorbance; intermediates were identified by analyzing mass differences of characteristic fragment ion peaks within the fragmentation patterns, and assignments were verified by NMR.TB demonstrated a photochemical degradation, which can be triggered by lutein/zeaxanthin. Intermediates vary depending on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of TB occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, within the presence of lutein/zeaxanthin the decomposition of TB indicates the formation of methoxyamine and sulfonyl arin. Thermal degradation processes were not observed.TB demonstrated a photodegradation that may be triggered by lutein/zeaxanthin and results in the formation of cytotoxic decomposition products. Our findings contribute to understand degradation mechanisms of TB and may elucidate previous clinical and experimental observations of cellular toxicity after TB application. Trypan blue is a water-soluble diazo dye that was introduced in 1904 by Paul Ehrlich for the treatment of African trypanosomiasis (sleeping sickness). Since thIn this account, an interaction of trypan blue with photodegradable antioxidants, such as lutein and zeaxanthin seems obvious. These compounds are highly concentrated within the retina, but almost absent within tissues of the anterior segment. The aim ®; Alchimia, Padova, Italy) and a solution mixture of 0.4 mg/ml trypan blue with 10.0 mg/ml lutein/zeaxanthin  were used. While lutein and zeaxanthin are poorly soluble in water, the purchased substances contain specially treated “water-soluble lutein”, as disclosed by Sousa-Martins. as an internal reference at 298 K. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, and m = multiplet. Preparation of the samples included removal of all volatile materials under vacuum (0.001 mbar at 25 °C), filtration, and re-dissolution in DMSO-d6. Several other deuterated solvents had been tested for their ability to completely dissolve the samples, among them CDCl3, CD2Cl2, C6D6, acetone-d6, and MeCN-d3, but all proved to be inferior to DMSO-d6.NMR spectra were recorded on a Bruker AVANCE III 700 MHz instrument  using 32 scans for Normally distributed variables with equal variances were compared using the Student t-test. Variables with unequal variances were compared using the Welch-test for independent samples. Pearson’s correlation coefficient was applied for normally distributed variables; otherwise Spearman’s correlation coefficient was used. A p value less than 0.05 was considered statistically significant.UV/Vis spectroscopy within a bandwidth of 220–750 nm revealed substance-specific absorption spectra of trypan blue and lutein/zeaxanthin, as shown in TB580 = 2.274–0.003 t (t in hours) and concentration by cTB580 = 100–0.125 t (cTB in percent). Likewise, the degradation of trypan blue in the presence of lutein/zeaxanthin resulted in a drop of the 580 nm absorption by ETB580 = 1.744–0.008 t and concentration by cTB580 = 100–0.455 t (cTB580 in percent). The absorbance at 379 nm of lutein/zeaxanthin declined by EL/Z379 = 4.473–0.016 t  and the concentration by cL/Z 379 = 100–0.358 t (cL/Z in percent). The extinction of lutein/zeaxanthin diacetate at 460 nm and 505 nm declined similar . However, due to the spectroscopic overlap of lutein/zeaxanthin and trypan blue the exact estimation of degradation kinetics is limited. Changes of compound-specific absorbance during the process of irradiation are depicted in Thermal degradation processes were ruled out by comparing absorption spectra of control samples not subjected to irradiation, which were stored at 4°C or room temperature (22–24°C) for 120 hours, respectively. No significant shift of absorption maxima was observed for substance-specific bands at 325, 379, 460, 505 and 580 nm (p > 0.05). With respect to potential photodynamic processes of trypan blue, a linear decline of absorbance was noted during the period of blue-light irradiation. As shown in Dye solutions were examined by high-resolution optical microscopy. While pure trypan blue dissolved homogenously in phosphate-buffered saline and did not present any precipitates before and after irradiation; the mixture of trypan blue and lutein/zeaxanthin revealed microscopically fine homogenous yellow granules of lutein/zeaxanthin, which changed during the irradiation process into heterogeneous brown clots as shown in +) and dimethyl sulfate (m/z = 745.6 [M– 127.2]+) are split off. Significant correlations between the amount of intermediates and the duration of light exposure were found for methoxyamine  or phenol (m/z = 651.4 [M– 127.2–94.2]+), respectively. Correlations between the amount of intermediates and irradiation time were found for sulfonyl arin  δ = 8.08 , 7.69 , 7.68 , 7.32 , 7.06 , 6.89  ppm. Signals from -OH, -SO3H and -NH2 functionalities were not detected due to interactions with residual water; signals from -CH3 groups were obscured by DMSO signals. Lutein/zeaxanthin signals are shown in 1H NMR  δ = 6.71 , 6.69–6.60 , 6.39 , 6.33 , 6.22 , 6.19–6.09  ppm; signals from -CH3, -CH2-, and -CHOH were obscured or unassignable. Methoxyamine is a colorless volatile liquid in polar solvents, which quickly decomposes into methane and nitroxyl and could not be detected by NMR. Yet, dimethyl sulfate was detected by its signal at δ = 4.00 ppm after trypan blue irradiation,[Nuclear magnetic resonance spectrometry was used for verification. Trypan blue signals were detected as shown in Figs adiation, as shownSo far, the intraocular application of trypan blue has been considered relatively safe within the anterior segment, while clIn the following, we will focus on reliably evidenced degradation metabolites and mechanism to reduce the room for speculations. While the degradation is quite complex for the involved solutions, formulations and processes, we like to emphasize that the photochemical degradation is also accompanied by the formation of volatile substances and reactive oxygen species, which are not explicitly analyzed hereinafter.Our results demonstrate a slight but continuous self-degradation of trypan blue under blue-light irradiation. Decomposition products were concluded from mass spectrometric analyses, which indicate the formation of methoxyamine (methylhydroxylamine), dimethyl sulfate, phenol and sulfonyl arin while the preferred degradation pathway depends on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of trypan blue occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, results of trypan blue decomposition in the presence of lutein/zeaxanthin indicate the formation of methoxyamine and sulfonyl arin. In this context, the self-sensitized photodegradation of lutein/zeaxanthin is well understood, and does not generate methoxyamine and sulfonyl arin by itself. PhotodegThe mechanism of lutein-triggered photochemical degradation of trypan blue is of clinical relevance, primarily because of its widespread use as intraocular surgical adjuvant. Corresponding to our findings, the blue-light degradation of the widespread commercial dye crystal violet in the presence of the antioxidant riboflavin has also been recently revealed under the formation of reactive oxygen species. While thIn 2008 Engel et al. were able to disclose mechanisms of indocyanine green self-decomposition by singlet oxygen under light exposure. Their fiAs previously discussed, trypan blue-associated toxicity might be reduced by lowering the intraocular dosage and the duration of tissue-exposure. However, potential risks are not limited to the time of surgery, but may also emerge postoperative from residues of trypan blue on ocular tissues. Trypan blue may last intraocular for hours to days, and can diffuse form the anterior to the posterior segment. Even thoIn conclusion, our findings contribute to the understanding of trypan blue photodegradation, which can be triggered by adjuvant substances, such as lutein or zeaxanthin, and results in the generation of cytotoxic products. For this reason, trypan blue should be considered with care and intraocular residues need to be avoided to keep application safety high.S1 Fig(PDF)Click here for additional data file.S2 FigThe absorbance decreases as follows: lutein/zeaxanthin , trypan blue  and lutein/zeaxanthin diacetate , which indicates that all compounds are decompesed/consumed during the photochemical reaction.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 File(SAV)Click here for additional data file.S2 File(SAV)Click here for additional data file.


This theme is controversial because no major medical organization recommends universal neonatal circumcision and no major medical organization calls for banning it either. The argument that this procedure must be kept within the purview of medical professionals is found across all major medical organizations. In addition, the organizations advise medical professionals to yield to some degree to parents’ preferences, commonly based in cultural or religious views, in the decision to agree to circumcise . CircumcThe practice of neonatal circumcision exerts a protective factor avoiding the genesis of penile cancer. While the presence of phimosis is a strong risk factor for penile cancer, neonatal circumcision appears to be a protective factor , 9. The Infection by high-risk HPV group is probably the major cause of anogenital cancers. High transmission potential with a low impact on herd immunity means extensive vaccination would be required to substantially reduce the incidence of cancer of the cervix and penis caused by high-risk HPV types. Further, vaccination of males against HPV appears to represent an expensive measure for prevention of penile cancer, particularly when one considers that high-risk HPV is present in only half of penile cancers. HPV vaccination of males should nevertheless help reduce cervical, anal and perhaps oropharyngeal cancers.On the other hand, lack of circumcision is a risk factor for phimosis and balanitis which themselves are risk factors for penile cancer. This would explain why invasive penile cancer is rare in circumcised menCircumcised men are consistently less likely than uncircumcised men to have HPV infection at the glans/corona and urethra. Several studies showed that male circumcision is associated with an overall reduction in the prevalence of genital HPV infection in men -16. ThesIn recent study, our group found HPV in 46.66% of our patients with phimosis, of whom 50% had high risk HPV genotypes. Of asymptomatic cases 16.36% were HPV positive but only 1 sample showed high risk HPV. We detected a significantly high rate of HPV genital infection in patients presenting with phimosis compared with asymptomatic men (p=0.00167). The prevalence of high risk HPV genotypes in patients with phimosis was also statistically significant (p=0.0004). We found a robust association between phimosis and the genital HPV prevalence in men and a significant frequency of high risk HPV. However, more studies are needed to adequately assess the effect of male circumcision on the acquisition and clearance of HPV infections. The focus of the treatment with vaccination or circumcision should be men in early age range . Taking


Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, predominantly represented by cervical cancer and oropharyngeal squamous cell carcinoma. Because of the prevalence of the virus, persistence of infection, and long latency period, novel and low-cost methods are needed for effective population level screening and monitoring. We review established methods for screening of cervical and oral cancer as well as commercially-available techniques for detection of HPV DNA. We then describe the ongoing development of microfluidic nucleic acid-based biosensors to evaluate circulating host microRNAs that are produced in response to an oncogenic HPV infection. The goal is to develop an ideal screening platform that is low-cost, portable, and easy to use, with appropriate signal stability, sensitivity and specificity. Advances in technologies for sample lysis, pre-treatment and concentration, and multiplexed nucleic acid detection are provided. Continued development of these devices provides opportunities for cancer screening in low resource settings, for point-of-care diagnostics and self-screening, and for monitoring response to vaccination or surgical treatment. Globally, cervical cancer is the third leading cause of cancer death among women, with >550,000 new cases diagnosed annually, resulting in >288,000 deaths per year . CervicaIn addition to the genital tract, HPV infects other anatomic sites, including the oropharynx (tonsillar area). U.S. incidence of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is predicted to surpass that of cervical cancer by 2020 . OverallHPVs are small, 50–55 nm in diameter, non-enveloped double-stranded DNA viruses that infect the skin and mucous membranes, causing both benign and malignant conditions. Worldwide, ≈5% of the total global cancer burden is attributable to HPV . Many HPE1–E7) that support viral DNA replication and two late genes (L1 and L2) that encode capsid proteins necessary for viral assembly and release. The compact viral genome necessitates viral dependence on host factors to carry out the life cycle, thus viral replication is linked to that of host cells [E6 and E7 cooperate to induce cellular immortalization and genomic instability, affecting multiple factors involved in cell cycle progression, cell survival, differentiation, and inhibition of apoptosis [HPVs encode only eight genes, which enable host infection and viral replication. HPV infection initiates in proliferating basally-localized mucosal keratinocytes, often at sites of injury . The genst cells ,28. Virast cells . In partpoptosis . Corruptpoptosis .E6 and E7 in host cells mediates degradation of the tumor suppressors p53 and retinoblastoma (Rb) and alters the activity of proteins that regulate cell cycle, leading to cellular transformation  characterized by cytological abnormalities . Persistent HPV infection results in progression to high-grade lesions . In productively infected cells, transcription of viral oncogenes ormation . The majE6 and E7, the molecular-level response of the ‘host’ to an oncogenic HPV infection is an area of active investigation. MicroRNAs (miRs) can function both as oncogenes and tumor suppressors. Both upregulation of oncogenic miRs and loss of tumor suppressive miRs in host cells have been reported in HPV-associated malignancies [In addition to research on HPV-encoded oncogenes such as gnancies ,32. Exprgnancies , suggestMultiple miRs have been reported as dysregulated in HPV-associated cervical and oropharyngeal cancer, summarized in recent reviews ,34. MechIt should be noted that a given miR is not specific for a single malignancy, highlighting the need for multiplexed analysis of a panel of miRs to potentially enhance the specificity of disease detection. Although the majority of miRs are intracellular, some miRs are also present in the circulation where they may circulate bound to specific proteins or as components of exosomes ,40. DeteHPV+ OPSCC are associated with distinct survival strata, thus early identification of this patient cohort is needed to tailor care to the unique biology of HPV+ tumors . CurrentE6,E7) or host miR response to an oncogenic HPV infection  in biological fluids, such as a cervical swab or oral rinse, would facilitate early discovery of malignant transformation and thus improve patient outcomes. In the case of cervical cancer, it is relevant to note that the vast majority (>85%) of cervical cancer deaths are in women in developing countries that also lack access to routine cervical cancer screening or vaccination against HPV infection [Low cost: A sensor that is developed using simple chip fabrication steps can be mass-produced, thus making it amenable for low-resource settings.Portability: The platform should be portable and easy to operate with the possibility for battery operation and minimal need of infrastructure.Simplicity of assay protocol: An easy-to-use device that is portable will eliminate the need for a sophisticated clinical laboratory facility or highly skilled, trained personnel. The final integrated platform needs to be an automated sample-to-answer device.Signal stability: A sensing signal that is stable, reproducible and insensitive to surrounding noise.Sensitivity and selectivity: A sensor should be sensitive enough for the target of interest and have the requisite specificity comparable to the gold standard.Rapid and robust: The device should generate results rapidly, allowing medical personnel to diagnose disease and start immediate treatment (screen-and-treat). Additionally, the platform should be robust and not require any refrigeration or other external equipment to perform the test.Multiplexing: The platform should be flexible enough to detect more than one target biomarker.Due to the long clinical latency between infection and oncogenesis (often tens of years) and the lack of clinical treatment for persistent HPV infections that have not yet induced abnormal cellular changes, the availability of technologies for monitoring of viral oncogene expression  or the oral cavity, light-based detection systems for oral cancer screening, or HPV-associated serologic markers (HPV biomarkers). These have served extensively for screening of cervical and oropharyngeal cancers and remain in use today. ® Imaging System (TIS)  showed a significant improvement in specificity for detection of high grade lesions, however, concerns remain regarding the overall cost of assay per test and its utility in low-resource settings [® system for detecting high grade lesions was ≈76% compared to ≈68% detection sensitivity when conventional Pap smear test was utilized. Additionally, ThinPrep had greater specificity (86%) compared to the conventional method (79%) [The Pap test  is the oldest known test that has served as the standard for cervical cancer screening. Since its introduction into standard clinical use in the 1950s, this method has reduced death from cervical cancer by 60% in women under 55 . The Papsettings . Sensitiod (79%) .®, MonoPrep® and SurePath®) and studies have shown that the sensitivity is improved by 1.29× for the monolayer technology compared to the conventional cytology method for high grade squamous disease [Monolayer cytology has been in use since the 1990s. The main difference from the conventional Pap method is the liquid storage of the cervical specimen in a solution that preserves cellular morphology while lysing other components in the sample including blood cells and microbes. Many cross-sectional studies have been performed related to conventional and monolayer cervical smear ,60. The  disease ,62.VIA is predominantly used in low-resource settings. It is a quick and simple visual inspection of the cervix using acetic acid  to highlight precancerous lesions. The procedure eliminates the need for laboratories and transport of specimens, requires very little equipment and provides women with immediate test results (one minute after the application of 4% acetic acid) . As a scE6 and E7 oncogenes associated with high-risk HPV [E6 and E7-specific serology for the diagnosis of this disease is still questionable [Current efforts aimed at appropriate targeting for immune therapy for cervical cancer are associated with HPV. Serologic markers could be tumor-specific antigens associated with expression of the risk HPV . While ctionable . Anothertionable . Immunoltionable . Also imtionable . Since itionable .Routine oral cancer screening is important and has been shown to significantly reduce mortality . ScreeniWhile the traditional tools for cervical and oral cancer screening continue to be widely used, recent developments for several lab-based non-invasive methods have been made to uncover differential expression levels of miRs that may more accurately report on physiological status or disease progression. Some of the commonly available techniques include (i) microarray; (ii) quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and (iii) next-generation sequencing. Microarray is one of the most powerful high-throughput techniques capable of monitoring the expression of thousands of miRs in a single experiment ,88,89,90Although qRT-PCR requires extreme care to avoid human sample handling error and contamination, it can be considered as a gold standard due to its superior performance in terms of sensitivity, specificity and quantification ability relative to other available miR profiling methods. In qRT-PCR, the small miR is reverse transcribed to cDNA, followed by conventional qPCR. The amplification is achieved by using a miR-specific probe and a stem-loop/poly(A) primer. Either the SYBR Green or TaqMan technique is employed to measure the fluorescence signal of the amplicon. This technique is moderately expensive, relatively sensitive, specific and capable to quantify compared to microarray . Two of E6/E7 oncogenes. Qiagen’s Digene Hybrid Capture® 2 HPV DNA test  [® HPV HR  [® HPV 16/18, Cobas® HPV Test by Roche  [® by GenProbe  [® HPV Test by Roche, Clart human papillomavirus 2 by Genomica  [® HPV [E6/E7 oncogenes is PreTect® Proofer by Norchip  [® HPV system weighs over 150 kg and is 166 cm wide while the Hologic Panther system weighs an incredible ≈363 kg and is over 175 cm in height [PCR-based detection of miR is technically challenging and costly and has not been used to screen for OPSCC. Salivary diagnostics based on PCR DNA detection  are available to detect the presence of the virus . A disadCA, USA) , HologicMA, USA) , CervistIN, USA)  and AptiMA, USA)  are the , Spain) , INNO-Li, Spain) , Genomic) [® HPV  and Abbo) [® HPV . One oth Norway) . Howevern height ,118. Then height .Despite the expense, high-throughput next-generation sequencing is becoming the dominant technology in the discovery and experimental validation of miR biomarkers, as it can distinguish between closely related miR sequences. The process starts with the preparation of a cDNA library from the RNA sample, followed by the sequencing of millions of individual cDNA molecules from the library. The sequence reads are then aligned against a miR sequence database after extensive bioinformatics processing to identify the known and unknown miRs present in the sample. The number of sequence reads thus make the technique very quantitative. This technique is suitable for both new miR discovery and study of known miRs. However, one main disadvantage of next-generation sequencing is that it shows the same biases in quantification analysis due to involvement of enzyme processing of the miRs, similar to qRT-PCR and microarrays, which can result in misrepresentation of the miR complement . Some ofR2 = 0.685) compared to moderate (R2 = 0.806) and highly (R2 = 0.806) expressed miRs [r = 0.974) of microarray platform for duplicate miR samples, but observed considerable variation (r = 0.443) in the results associated with two assay platforms when analyzing 84 miR samples [D and the PCR efficiency are highly temperature-sensitive and length-dependent. Hence, as the two techniques involve different reacting species and kinetic mechanisms at different reaction times and temperatures, variability in the hybridized and amplified yield is not surprising. While alternative detection strategies that address these issues are needed, screening via Pap smear, VIA and qPCR are the current options for large-scale screening in the developed and developing world.Many challenges still remain to be addressed if the recent discoveries in miR biomarker research are to be successfully advanced, particularly related to the detection accuracy and reproducibility of the quantified results. Although it is better to profile miR expression by microarray followed by qRT-PCR, it is difficult to validate and correlate miR expression data obtained using different profiling methods ,132. Higsed miRs . Further samples ,135,136. samples . The hybThe molecular diagnostic research community has been actively engaged for years in the development of a highly sensitive biosensing platform for cancer biomarker detection. These biosensing assay platforms use a probe-functionalized sensing element where the target capturing event is measured most commonly by means of an optical or electrical signal. Among the reported optical based quantification technologies, most rely on fluorescence labeling such as a molecular beacon , FörsterCapacitance, conductance and field-effect transistor (FET) electrode sensors are typically sensitive to sample ionic strength, as the thickness of the screening electrical Debye layer is comparable in dimension to the target nucleic acid molecules ,148. TheE6/E7 genes or miR biomarkers, there is a significant need for integrated systems that provide rapid results in a sample-to-answer format. Microfluidic systems currently under development afford the advantage of forming such an integrated system due to flexibility in design and the necessity for small volumes. Additional advantages are realized in low-resource settings including portability, possibility of automation to reduce the need for personnel, decrease in power consumption and easy disposability. Modular multi-stage microfluidic platforms with sensors can also offer standardized pretreatment units for different samples thereby eliminating the need for steps like centrifugation that are not suitable for field applications and even PCR amplification which introduces normalization issues. While offering these advantages, microfluidic systems do not compromise on the sensitivity and selectivity of the assay [While there are several nucleic acid-based screening platforms available for detection of HPV DNA, he assay . HoweverNormalization and variability issues can be minimized with sample selection and improved/standardized sample processing techniques and, if possible, elimination of PCR amplification. miRs are commonly found in body fluids, such as blood, saliva, spinal fluid and urine. The composition of blood is very complex as compared to other biofluids and hence miR isolation processes cannot be transposed from one fluid type to another. For profiling studies, both plasma and serum contain miR biomarkers ,157,158.® MTBDRplus assay was validated for detection of tuberculosis with high sensitivity [Recent work has focused on development of microfluidic systems capable of analyzing bio-specimens including blood ,168,169,sitivity . One of sitivity . In addisitivity .While these microfluidic systems bring the field a needed step closer to cancer screening, several of these platforms involve extensive integration of multiple systems and require peripheral detection systems. The use of complex fabrication systems and numerous assembled modules make it challenging to transition the device to high scale manufacturing. Their complexity also introduces robustness issues due to contamination and target loss that are particularly acute for quantification of low-copy-number miRs. Although some commercially available medical diagnostic products are FDA-approved, like the Focus Dx by Quest Diagnostics , Handy Lab by Becton-Dickinson , Abbott’s i-Stat Corp , IQuum by Roche, and Cepheid’s GeneXpert , they are still not suitable for low-resource settings ,193,194 In contrast, Slouka et al. and Senapati et al. developed a simple, single-channel integrated diagnostic platform as shown in The working principle of the pretreatment chip developed by Slouka et al. was based on electrophoretic extraction (by application of direct current voltage) of negatively-charged nucleic acid molecules from the sample reservoir through a thin agarose gel layer into a microfluidic channel as shown in Downstream from a pretreatment unit was a pre-concentration unit consisting of a cationic exchange membrane. The goal of the pre-concentration unit was to form an ionic plug using electric field and block the passage of the extracted nucleic acids. This resulted in the accumulation of the extracted nucleic acids in a specific location thus improving the sensor sensitivity. The location of the concentrated molecules depended on the flow rate of target molecules in one direction opposed by the ionic plug force in the other direction .As for the biosensing unit for this particular integrated platform, an ion-selective nanomembrane was used for molecular detection of nucleic acid molecules. In prior studies, the group showed the application of anion-exchange membranes for detection of negatively charged molecules using the charge inversion phenomenon ,199,200.The integrated platform was constructed by combining these three components using low-cost polymeric polycarbonate materials, thus reducing the overall cost of the assay. The external circuitry eliminates any on-chip micro-electrode fabrication and hence reduces manufacturing challenges and cost. Another advantage of the platform is its modular design which allows for rapid prototyping of the different components. Oral cancer cell line samples were used to successfully verify the platform’s performance for the detection of target miR in under 45 min. Current research is focused on extension of this microfluidic platform to a panel of miR biomarkers, with insertion of multiple sensors (≈10 targets) into the membrane sensor module and by using a reasonable sample volume. Advantages of this multi-target platform are reduction of miR degradation and loss, removal of inhibitors, and incorporation of a low-cost but sensitive membrane sensor. The key remaining issue is whether the current platform will offer sufficient sensitivity and specificity to quantify low-copy number miRs. If PCR amplification is necessary, a new module must be designed for low-copy number miRs or to quantify under-expressed cohorts. Such modules could be digital PCR or solid-state nanopore sensors, with single molecule resolutions for the lower-copy-number targets. It is hence quite possible that a second microchannel needs to be inserted to divert the sample to a second module. This more complex integration may lead to increased target loss and robust designs must be carefully implemented. The design strategy would again be to minimize the pretreatment and isolation steps, perhaps by dilution to reduce the concentration of the inhibitors. High-throughput digital PCR will allow such dilution and would be an attractive low-copy-number complement to the current high-copy-number microfluidic platform. Nanopore sensors may require preconcentration if the sample is diluted. Extension to multi-targets without PCR amplification would be a significant advance of the current nucleic acid quantification technology and should be the next technical development.Significant challenges remain for development of robust biosensors used for disease detection in the developing world. For example, while cervical cancer is consistently decreasing in the U.S., it remains a significant public health challenge in developing nations. There is an unmet medical need for a low cost, easy to use, point-of-care diagnostic that can be used for self-sampling or clinic-based sampling of cervical cells. This would facilitate screen-and-treat in the same clinic visit, providing a major advantage to patients that lack access to transportation. Such a device would also simplify analysis of patient response to surgical procedures such as loop electrosurgical excision procedure (LEEP) and for more routine monitoring of vaccine efficacy.Any diagnostic platform is only as good as the validity of the biomarkers it detects. Hence, an important future direction is the discovery and approval of more specific miR biomarkers for cervical/oropharyngeal cancer. Redundancy with multiple targets would minimize false positives and negatives. Over-expressed miRs are easier to quantify, provided the specificity is not compromised by other interfering miRs with similar sequences that could compete for the oligo probes. Optimal fragmentation of long HPV DNA will also enhance the sensitivity and selectivity. This will require proper selection of target sequences and fragmentation enzymes. Other than target discovery and selection, more advanced pretreatment units that can remove interfering debris and lipids of certain samples will also advance the platform. Portable instrumentation, large-scale manufacturing designs and field validation would then be the final steps. Like any other new platform, the road to commercial and clinical realization is long and tortuous. However, the unique advantages of emerging nucleic acid biosensor technologies may fundamentally transform health care in low-resource developing countries.In addition to the examples highlighted, a robust nucleic acid-based biosensor would have utility in a large variety of applications. Development of low cost miR-based screening for recalcitrant cancers with low survival rates such as pancreatic cancer or ovarian cancer may enable population-based screening to improve the incidence of early detection and thereby improve overall survival from these diseases. Portable units would also enable use in environmental applications such as monitoring for invasive aquatic species or for rapid point-of-care detection of emerging pathogens.