Significantly increased levels of proteins in the tumor cells were confirmed using the IHC method for all studied proteins. The expression of βIII-tubulin, CA IX, and survivin increased significantly after treatment with both cytostatics (PTX and DOX). Depending on the type of tumor, a significant increase in all proteins was observed in IC samples after PTX treatment, and CA IX expression after DOX treatment. In CIS samples, a significant increase of βIII-tubulin and survivin expression was observed after a DOX treatment. The results suggest that βIII-tubulin, survivin, and CA IX may be significant drug resistance markers and the clinical regulation of their activity may be an effective means of reversing this resistance.Breast cancer is the most common cancer in females. The aim of this study was to determine the effect of paclitaxel (PTX) and doxorubicin (DOX) therapy on the βIII-tubulin, carbonic anhydrase IX (CA IX), and survivin expression in chemically-induced rat mammary tumors. Animals with induced mammary carcinogenesis were randomly divided into treatment groups and an untreated group. The total proportion of tumors, the proportion of carcinoma in situ (CIS), and invasive carcinoma (IC) were evaluated. Protein expression in tumor tissue was determined using IHC. Statistical analysis of the data, evaluated by Fisher-exact test and unpaired  Breast cancer is the most common cancer in women today. It is the fifth leading cause of death (6.6%) and also causes the highest number of deaths (approximately 15%) related to women’s cancer . PaclitaMany studies have examined the prognostic association of the expression of various proteins in tumor tissues with the response to treatment. The development of tolerance to taxanes is largely attributed to microtubule changes and, in particular, the overexpression of specific ß-tubulin isotypes .p < 0.05). Their results suggest that high expression of both proteins may be responsible for docetaxel drug resistance in patients with advanced breast cancer.ß-tubulin exists in the form of several isotypes that are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. In particular, the βIII isotype is overexpressed in many aggressive and metastatic tumors and may serve as a prognostic marker in certain types of cancer . HoweverSurvivin, like βIII-tubulin, is considered a potential target for the treatment of cancer, mainly due to its dual role. It acts as a suppressor of apoptosis and also plays an important role in cell proliferation. Survivin dysfunction leads to cell cycle defects, including disruption of cytokinesis, centrosome dysregulation, and multinucleated cell formation . In addiThe expression of CA IX proteins is induced by hypoxia in various tumors. Through their ability to regulate pH and create an acidic environment they increase the chance of tumor cell survival in hypoxia conditions and provide an increased ability to migrate . Under pIn the present study, we investigated whether levels of βIII-tubulin, CA IX, and survivin could change in induced breast cancer in rats as a result of treatment with PTX and DOX.A1.Non-treated group: n = 13 tumors.Treated group: n = 49 tumorsTreatment with paclitaxel: B1.Carcinoma in situ (CIS): n = 32 tumors.Invasive carcinoma (IC): n = 30 tumors.Stage of tumor: A2.Non-treated group: n = 29 tumors.Treated group: n = 34 tumorsTreatment with doxorubicin: B2.Carcinoma in situ (CIS): n = 43 tumors.Invasive carcinoma (IC): n = 20 tumors.Stage of tumor: To detect the tumor stage, the slides were stained by hematoxylin-eosin . We divißIII-tubulin showed cytoplasmic positivity A,B. In tIn the group of non-treated animals, 16 IC tumors were found. From the paclitaxel group, four of them were ßIII-tubulin negative (80%) and one sample was ßIII-tubulin positive (20%). Five tumors from doxorubicin-treated rats were ßIII-tubulin negative (45.5%), while six tumors were ßIII-tubulin positive (54.5%). Samples of CIS showed two (25%) negative tumors in the paclitaxel group and 10 (55.5%) negative tumors in the doxorubicin group . A comprp < 0.05). However, when CIS treated tumors were compared with CIS non-treated tumors the results did not show statistical significance (p > 0.05). In the doxorubicin group, the IC treated tumors did not show statistical significance compared to untreated IC tumors but statistical significance was confirmed (p > 0.05) in the comparison of CIS tumors. The evaluation of staining in untreated compared to treated tumors revealed that IHC expression of βIII-tubulin increased significantly (p > 0.05) in breast tumors after treatment with both paclitaxel and doxorubicin  and seven (38.9%) negative samples in the paclitaxel and doxorubicin group, respectively, while the IC subgroup had three (60%) negative samples in the paclitaxel group and seven (63.6%) negative samples in the doxorubicin group . All in p < 0.05). The difference in CA IX expression between CIS treated vs. CIS non-treated tumors was statistically non-significant (p > 0.05) in both groups. Similar to the ßIII-tubulin, expression of CA IX in the paclitaxel and doxorubicin treated groups, staining significantly increased in tumor samples compared with non-treated animals  while IC tumors treated with doxorubicin were not statistically significantly different from non-treated tumors. The difference in survivin expression between CIS paclitaxel treated vs. CIS non-treated tumors was statistically non-significant (p > 0.05). On the other hand, statistical comparison of CIS doxorubicin-treated and untreated tumors showed statistical significance (p < 0.05). The overall expression of survivin in the group of non-treated compared with treated animals significantly increased staining in tumor samples (p < 0.05) after both paclitaxel and doxorubicin treatment  and, in comparison to CIS tumors from the doxorubicin group, we also found that statistical significance was confirmed (p > 0.05). These results indicate that βIII-tubulin expression may be an early event in the malignant process after doxorubicin therapy. In addition to cytoplasmic activity, the stromal activity of the protein was detected. According to Person et al. [p > 0.05) and 94.1% of tumors were positive after doxorubicin treatment (p > 0.05). These results are not consistent with the study of Yuan et al. [Several studies have debated the possible correlation between taxane resistance and high ßIII-tubulin expression. Expression of the neuron-specific βIII-tubulin isotype correlated with taxane resistance in vitro and in vivo and has been extensively studied as a clinical biomarker of taxane efficacy . Our stun et al. , βIII-tun et al. , the expn et al.  who obsen et al.  found 60n et al.  determinn et al.  analyzedAs previously mentioned, the results of this work show that βIII-tubulin is overexpressed in tumor tissue after cytostatic treatment. The results justify further studies considering the validation of this marker for its potential role as a predictive biomarker in certain types of breast cancer. Several studies have clearly indicated that the expression of βIII-tubulin can be induced in normal as well as neoplastic cells exposed to a toxic microenvironment characterized by hypoxia and nutrient deficiency .p < 0.05). The difference in CA IX expression between CIS treated vs. CIS non-treated tumors was statistically non-significant (p > 0.05) in both groups. These results suggest that CA IX expression may be a significant predictor of tumor invasiveness. In the paclitaxel group, 77.6% of tumors were positive and 79.4% of tumors were positive in the doxorubicin group. Similar to the ßIII-tubulin expression of CA IX in the paclitaxel and doxorubicin non-treated groups, staining significantly increased in tumor samples after treatment compared with treated animals. The overexpression of CA IX in mammary tumors that was demonstrated in this study after treatment with cytostatics is likely to indicate an increase in drug-induced resistance.Hypoxic cancer cells overexpress CA IX, which provides a tumor-favorable intracellular pH, contributing to stromal acidosis and facilitating tumor invasion and metastasis . In recep < 0.05) while IC tumors were not statistically significantly different. On the other hand, statistical comparison of CIS doxorubicin-treated and untreated tumors showed statistical significance (p < 0.05). Only a small number of studies have looked at the development of breast cancer cell resistance to paclitaxel. Kreger et al. [p < 0.05). From these results, it can be deduced that increased survivin expression is a response to PTX treatment. Our conclusion is consistent with the results of He et al. [p < 0.05). These results suggest that survivin may be a significant marker of established drug resistance and clinical regulation of its activity may be an effective means of reversing this resistance. Several types of survivin inhibitors are already known but in preclinical studies only. Some inhibitors have also been administered at the clinical level but the second phase of the clinical trial has not been surpassed. The possibility of targeted therapy using survivin inhibitors is a great challenge for the future as the regulation of aberrant survivin activity represents great potential for improving cancer therapy. In relation to our previous results [Survivin, also known as BIRC5, is essential for cell division and can inhibit apoptosis and increase angiogenesis. It is normally only expressed in actively propagating cells, but its increased expression has been shown in almost all types of malignancies. In addition to tumor cells, survivin has also been shown in autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, where its cytokine-dependent expression correlates with decreased apoptosis and inflammation. For oncologists looking for a new targeted treatment, the proteins needed for cell proliferation and the proteins related to apoptosis are at the top of the most-wanted list. Therefore, as a protein essential for mitosis and capable of inhibiting apoptosis, survivin is a promising protein for targeted therapy . We deter et al. , based or et al. . Patiente et al.  who exame et al.  examinede et al.  observede et al.  demonstr results  and recead libitum. Three to six animals were placed in one cage. During the experiment, the rats were palpated and weighed weekly. After 4–6 weeks, the first tumors appeared in all rats. The number of tumors in each animal differed from only one up to twelve tumors, averaging 3 to 6 tumors per animal. Finally, we found 125 separated tumors in 44 rats. The experiment was conducted in two stages with different animals. In the first stage, the animals were treated with paclitaxel, and doxorubicin was administered in the second stage. Rats were randomly divided into a control  and a paclitaxel or doxorubicin group. Rats in the paclitaxel group (n = 20) were treated with 6 doses of intraperitoneal paclitaxel (5 mg/kg). The doxorubicin group (n = 10) of female rats with tumors was treated with 6 doses of intraperitoneal doxorubicin (5 mg/kg). These rats received the first dose on the day the first tumor appeared. The next dose was given every 3–4 days. Altogether they were given 6 doses. Untreated groups (n = 07 in both cases) served as a control group that received the same amount of physiological saline instead of treatment. Four days after the last dose the animals were euthanized by an anesthetic overdose . Mammary tumors were immediately excised and samples were taken for histopathological and IHC examination.Sprague-Dawley (SD) rats are DMBA-sensitive . The susceptibility peak is at 55–60 days of age. After metabolic activation in the mammary gland, the carcinogen metabolites interact with rapidly proliferating cells in the terminal end buds, forming subsequent mutations that, in turn, play a role in their transformation into malignant cells . Breast Formalin-fixed, paraffin-embedded tissue sections from 44 female rat mammary tumors were IHC analyzed for expression of ßIII-tubulin, CA IX, and survivin proteins .2O2 for 10 minutes, the specimens were incubated with primary antibodies. Antibody ßIII-tubulin was concentrated in a ratio of 1:200 and 1:350 for CA IX, leaving them for 30 minutes at room temperature. Sections with survivin at a 1:50 dilution were incubated for 60 minutes at room temperature.Deparaffinization, rehydration, and antigen retrieval were performed on PT Link (Dako PT100) using the EnVision™ FLEX Target Retrieval Solution, at low pH 6.0  and high pH 9.0 (CA IX). Following incubation in 3% HTM FLEX Wash Buffer 20×). Each IHC run contained a positive control and a negative antibody control .Specimens were then incubated with the EnVision™ FLEX+ Rabbit /Mouse (survivin) LINKER for 15 min at room temperature followed by incubation with a Universal LSAB2 KIT/HRP visualization reagent for 20 min at room temperature. The enzymatic conversion of the subsequently added EnVision Flex 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 5 min at room temperature resulted in the precipitation of a visible reaction product at the site of antigen. The specimens were then counterstained with hematoxylin and embedded in Pertex. All the described steps were followed by buffer washes for 5 min  = 0% of positive cells (no positivity).1+ = 1–10% of positive cells (weak positivity).2+ = 11–90% of positive cells (intermediate positivity).3+ = 91–100% of positive cells (strong positivity).The semiquantitative immunohistochemical method was evaluated according to the following scale:Samples marked as − (minus) and 1+ were considered as negative, samples marked as 2+ and 3+ were considered as positive.2) test to calculate statistical significance. A significance limit of 5% (p = 0.05) was set for the p-value and the result was considered statistically significant if the p-value was less than 0.05.We used the “Pearson’s chi-square” (χ


C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories.C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital.In this study, we validated C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement.The analytical sensitivity of C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively.When testing rapidly processed clinical samples, all three C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%.At the POC, the C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.This study describes reliable POC options for the detection of the equine pathogens: The online version contains supplementary material available at 10.1186/s12917-021-02986-8. C. psittaci (C. psittaci) has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry [C. psittaci from equine placental membranes to humans causing severe respiratory illness [C. psittaci, EHV-1 does not pose a risk to humans [As for other mammalian hosts, infectious diseases are important causes of morbidity and mortality in horses, incurring significant economic losses for stakeholders. Endemic and emerging equine pathogens pose significant risks not only to equine health, but in some cases to humans , 2. Horsindustry –5. Furth illness , 7. Equio humans . Nonetheo humans –10.Methods for nucleic acid detection, such as quantitative real-time PCR (qPCR), remain a ‘gold-standard’ in veterinary diagnostics; however, this testing is usually performed in well-equipped veterinary diagnostic laboratories with molecular capabilities and trained personnel , 11–13. C. psittaci [To date, numerous isothermal amplification and end-point detection methods have been established , 13. Perpsittaci  and EHV-psittaci , 19, 20,psittaci , 20. AnoC. psittaci [The aim of this study was to compare the previously described psittaci  and newlC. psittaci, we employed the primer set from our previously established LAMP assay for all testing [For  testing , whereas testing . Our newC. psittaci or EHV-1 gDNA as template  was prepared separately, with each replicate tested in a separate run.The detection limits of the three LAMP assays  were evaluated using ten-fold serial-diluted, purified and quantified C. psittaci and EHV-1 rtLAMP assays . The detection limit for both ays Fig. .C. psittaci and EHV-1 cLAMP assays   assays using the thermal block for amplification. The analytical sensitivity of both ays Fig. . InteresC. psittaci primer set and EHV-1 primer set designed in this study, we tested DNA extracted from a range of bacterial and viral species using rtLAMP assays  over a six-week period , and more accurately evaluate reagent stability (C. psittaci DNA with lower genome copy number). Amplification was continuously achieved weekly for all samples up to the endpoint of six weeks independent of LAMP assay types  of 100% and specificity of 97.5%, with overall agreement of 98.86%  accredited Veterinary Diagnostic laboratories were compared to the For EHV-1, 35 DNA samples, also extracted from swabs from equine abortion cases, were tested by both rtLAMP and the EMAI Veterinary diagnostic laboratory reference qPCR assays, whereas another eight DNA samples were tested by both rtLAMP and the IDEXX Veterinary diagnostic laboratory reference qPCR assays Table . CompareC. psittaci and three for EHV-1), rtLAMP testing was repeated to confirm results, whereas all results from the diagnostic laboratories testing were deemed valid and from conforming runs.For all discrepant samples (one for Two penile samples were excluded from cLAMP testing as immediate discoloration of the mix (to yellow and/or orange) was observed upon addition of the swab suspension indicating that template from these types of samples were of low pH. However, samples yielding an orange colour post amplification were deemed “ambiguous” and reported as negative , respectively  training. Training included: sample processing, primer and reaction preparation, use of Genie Fluorometer for rtLAMP, and interpretation of results. On both occasions, we noted time from rapid sample processing to interpretation of results when assays were performed by technicians with and with no prior molecular diagnostic experience. On average, a technician with prior experience took 1 h to rapidly process and test up to eight samples using the rt- and/or cLAMP assays, while for newly trained technicians took 1.5 h. The assays were demonstrated to be robust, and easy to perform at our POC setting.In order to assess the LAMP technology for POC deployment, we performed pilot testing of clinical samples using the C. psittaci rtLAMP  foetal tissue and placental swab samples from equine abortion events were opportunistically tested for C. psittaci and EHV-1. Furthermore, our testing of ‘real-world’ clinical samples  in equine clinical setting represent a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.Although attractive to use and available for many veterinary and human pathogens, LAMP technology is still in the early stages of POC and/or field use , 22–24. C. psittaci rtLAMP assays in our previous study [During the initial LAMP assay optimisation, previously published , 19 and us study , there aus study . In prevus study , 19..To the best of our knowledge, both DNA targets were evaluated here for the first time with commercial colorimetric and SYBR Green dyes as end-point assays. The colorimetric LAMP assays, with 10 copies/μL determined as 100% detection limit, were slightly less sensitive than the rtLAMPs. Interestingly, the commercial colorimetric mix also gave false positive results for EHV-1 when using the primer set described by Nemoto et al. , whereasC. psittaci, and a total of 43 EHV-1 DNA samples . C. psittaci rtLAMP was in 98.86% agreement with the reference qPCR assay, confirming its sensitivity and specificity of the assay and its suitability for POC diagnostic use. Although preliminary validation of 43 samples indicated 91.43 and 100% agreement for EHV-1 rtLAMP respectively, with the reference qPCR assays run at two different laboratories, additional comparison testing with a larger sample set is required.In order to validate the rtLAMP assays from this study, two accredited Veterinary Diagnostic laboratories performed comparative reference qPCRs on 88 C. pecorum, a livestock and koala pathogen [As a proof of concept for POC use, we previously evaluated a rapid swab processing technique employing a simple vortexing and heat lysis which allowed the application of crude DNA extracts in isothermal detection of pathogen . As demopathogen . As LAMPNo such trend was observed for discrepant results from the colorimetric LAMP assays . These discrepant colorimetric results could be due to several reasons, such as pH change of the WarmStart Mix, DNA contamination from opening the tubes to add SYBR Green DNA, or non-specific amplification and/or primer dimerization , 17. NevC. psittaci isothermal assays  for the first time in an equine clinical setting in Australia. Rapid diagnosis of infectious causes of equine pregnancy loss is important for equine veterinarians and stud owners to determine appropriate disease treatment and management. In the case of C. psittaci, the information is also critical to alert attending staff to the zoonotic risk [In this study, to the best of our knowledge, we applied and evaluated the use of tic risk , 4, 14. tic risk .C. psittaci rtLAMP assay, performed in Genie III, was demonstrated to be robust and easy to use. In 2019, we performed a much smaller pilot trial using cLAMP, performed on heating block with result denoted as a color change, on only 11 samples. The feedback about the POC use of these assays was that after training they were easy to use and interpret. On both occasions, we achieved 90–100% congruence between the LAMP assays and the reference qPCR, however we must note that we have tested C. psittaci-suspected abortion material that typically contains high pathogen loads and the colorimetric LAMP assay was only evaluated on 11 samples. A previous study showed that the C. psittaci infectious load (ranging from 1 × 102 – 2 × 106 genome copies/μl) is high in DNA extracted from placental and foetal tissues [C. psittaci and EHV-1 isothermal POC testing.On the 2017 occasion where we tested 34 swab samples, the If the LAMP assays are demonstrated to be reliable for a specific pathogen detection using ‘real world’ clinical animal samples then translation of ‘from lab to the stable’ diagnostic testing would be feasible , 22, 29.C. psittaci, we applied our previously described isothermal assay and primers targeting a 263 bp of the C. psittaci-specific ORF_607 gene [https://blast.ncbi.nlm.nih.gov/Blast.cgi). In this study, we utilized both LAMP Designer 1.15 software which yields a single set of six LAMP primers  and open-source Primer Explorer v5 software which yields five sets of four LAMP primers  to evaluate primer design for EHV-1 gene target, and to compare them to the previously published primers [https://blast.ncbi.nlm.nih.gov/Blast.cgi) and primer characteristics using online tools OligoEvaluator  and OligoAnalyzer . Following sequence analyses and preliminary isothermal testing with the sets of newly designed primers , we selected the most optimal primer set for EHV-1, designed by Primer Explorer. As the Primer Explorer yields set of four LAMP primers , we additionally designed LF and LB primers  to 10− 1 copies/μl (tested as five replicates)  was prepared separately for each replicate, from the same quantified DNA . Dilutions were tested in separate runs by three operators.The newly designed and previous primer sets for EHV-1 were tested and compared in rtLAMP assay using quantified DNA, extracted from viral culture. Similarly, the C. psittaci, and newly developed EHV-1 rtLAMP assays was evaluated against a range of previously screened and confirmed DNA extracts from other viruses, cultured bacteria and horse tissues  to 10− 1 copies/μl (tested in five replicates)  assay using 2X WarmStart® Colorimetric LAMP Master Mix  was performed in 25 μL reaction volumes, consisting of 12.5 μL 2X WarmStart® Colorimetric LAMP Master Mix, 2.5 μL primers mix  and 1 μL (for sensitivity assays) or 5 μL  template, run at 65 °C for 30 min on a thermal block. Using the colorimetric method, reactions must be bright pink in colour prior to amplification, indicating that the reaction has a high pH, which is essential for the pH-dependent colorimetric LAMP reaction. Furthermore, the primers and DNA template should be used as an aqueous solution  so as not to interfere with the required pH for the colorimetric LAMP assay. Post amplification, the tubes were cooled to room temperature and examined by eye. Positive (target species DNA) and negative (MilliQ water and/or LAMP mix only) controls were included in each assay. As per manufacturers chart, positive reactions turned yellow, while negative reactions remained pink  to 10− 1 copies/μl (tested in five replicates)  was diluted to 1000x in PCR-grade water in a total of volume of 1 ml. The sgLAMP assays were performed in 25 μL reaction volumes, consisting of 15 μL No Dye Isothermal Master Mix ISO001 , 5 μL primers mix  and 1 μL (for sensitivity assays) or 5 μL template (for testing samples), run at 65 °C for 30 min on a thermal block. After 30 min incubation, 2 μl of 1000x SYBR Green dye was added to the tubes. A visible colour change from orange  to bright yellowish green  was used for results interpretation  was tested weekly for a period of six weeks. The isothermal master mixes were assayed as outlined above every week using 1 μl of the serial-diluted C. psittaci DNA  LAMP isothermal master mixes and primers were kept refrigerated (4 °C) for up to six weeks. To test the stability of the reagents, serially diluted, purified and quantified C. psittaci, and the newly developed EHV-1 rtLAMP assays were evaluated against DNA samples, extracted from swabs from equine abortion cases, that had been previously tested with: a) the reference C. psittaci [n = 88 samples) and EHV-1 [n = 35 samples) diagnostic qPCR assay at Elizabeth Macarthur Agricultural Institute (EMAI) Veterinary Diagnostic Laboratory, Menangle, NSW, Australia; or b) reference singleplex EHV-1 RealPCR™ (n = 8 samples) diagnostic qPCR assay at IDEXX Veterinary Diagnostic Laboratory, Brisbane, QLD, Australia  swabs for use with the LAMP assays in order to reduce the time involved in sample processing and DNA extraction prior to the diagnostic assay. All the swabs were collected by qualified veterinarians as a part of routine health and diagnostic investigations. The testing and use of these swabs were approved by University of the Sunshine Coast Animal Ethics approval exemption .C. psittaci and EHV-1 if amplification time was > 23.50 min as this was considered below detection level (BDL). In the c- and sgLAMPs a sample was considered negative if there was no clear colour change (i.e. colour change was ambiguous). Inhibition of amplification by swab suspensions was also assessed with ‘spiking’ experiments, where we added 10 μl of C. psittaci and/or EHV-1 DNA to five 25 μl aliquots of each swab suspension that tested negative. In order to perform comparison to the reference in-house qPCR assays, the remaining volume of the swab suspension was used for DNA extraction using QiaAMP DNA mini kit, as per manufacturer instructions  , and the remaining 12 were individual Transystem plastic shaft Amies gel swabs . The rapid swab processing was performed in a Biosafety Cabinet. A 300 μl aliquot of sterile MilliQ water was added to sterile 1.5 ml Eppendorf tubes. The dry and/or Amies gel swabs were placed in tubes and the shaft was cut off so that the swab fit within the tube. For Amies gel swabs, if present, any excess gel media was carefully removed. The mucosal swabs were vortexed for 1 min and heat lysed at 95 °C for up to 10 min. The swab suspension was briefly centrifuged, cooled to room temperature and a 5 μl aliquot was then added to the LAMP reactions as outlined above  > 35 and below the signature melt threshold were considered negative  Laboratory, NSW, Australia, on two separate occasions. In August 2017, 34 rapidly processed horse mucosal and tissue swabs were tested with the C. psittaci qPCR assay  and rtLAMP assay (samples from 2019 visit)  [We compared the results for: a) the same DNA samples previously tested with reference assays run in the veterinary diagnostic laboratories, with the rtLAMP assays; b) rapidly processed swabs suspensions tested with the real-time and colorimetric LAMP assays; c) rapidly processed swabs tested with LAMP and with DNA extracs of the same samples tested with in-house qPCR assays; and, d) samples tested at the POC using rapid processing and LAMP and DNA extracts from the same samples with in-house qPCR assays. The performance of the tests evaluated on the same samples was done using EpiTools online epidemiological calculators using diagnostic test evaluation modules (wotests) . For DNAAdditional file 1. Newly designed EHV-1 LAMP primers sequences utilized in this study. Primer sequences and their lengths are outlined in the table.Additional file 2. EHV-1 LAMP primer sequences and positions in the target gene regions. In this figure, we outline the EHV-1 gene target, where previously described primers are indicated in orange, while the newly designed primers are indicated in purple.Additional file 3. Workflow for development, evaluation and application at the POC for the equine isothermal assays. In this diagram we present the utilised workflow.Additional file 4. Isothermal assays sensitivity and specificity comparisons. In this table, time to amplify (min:ss) and HRM (oC) melts, and colour change results are presented for serially diluted C. psittaci (sheet 1) and EHV-1 (sheet 2) DNA to evaluate detection limits of isothermal assays. On sheet 3, we denote results for rtLAMP amplification using a range of bacterial and viral DNA.Additional file 5. Stability and reproducibility of the isothermal assays with components kept on 4 °C. In this table, we present results for real-time and colorimetric isothermal amplification using same DNA templates over 6-week period when reagents were kept on 4 °C.Additional file 6. Rapid swab processing. A: Clinical swabs available for testing, from left: placental and foetal swabs; Mare nasal, vaginal and cervical swabs; B: Vortexed swabs in 300 μl sterile MilliQ water; C: Heat lysis of the swab suspension; D: Heat lysed swab suspension used as a template in isothermal testing. The images were taken with iPhone SE. In this image, we present step-by-step rapid swab processing method.Additional file 7. Validation of LAMP testing (reported as time to amplify (min:ss), and signature melt in (°C) using diagnostic laboratory tested samples. In this table, we present (paired) results of reference diagnostic assay and our isothermal assays when testing the same samples.Additional file 8. Clinical samples testing. In this table, we present (paired) results of our three isothermal assays compared to reference qPCR assays when testing the same samples. On sheet 1 are results for C. psittaci, and on sheet 2 for EHV-1 detection, respectively.Additional file 9. Testing at the POC. In this table, we present (paired) results of C. psittaci isothermal assays, performed at the POC in Scone Equine Hospital on two separate occasions, compared to reference qPCR assay.


By analyzing an unprecedentedly large, longitudinal HIV-1 CRF07_BC sequence dataset collected from China in the past two decades, we sought to build CRF07_BC lengthwise transmission networks, and understand its transmission dynamics. We divided CRF07_BC into two clusters based on phylogenetic analysis and an estimation of the pairwise genetic distance at 0.7%. Of 6213 sequences, 3607 (58.1%) linked to ≥1 other sequence. CRF07_BC was divided into two clusters: 07BC_O and 07BC_N. The 07BC_O is the original CRF07_BC, circulating in people who inject drugs (PWID) and heterosexuals, predominantly in southwestern and northwestern provinces of China. The 07BC_N is a new cluster, identified mostly in men having sex with men (MSM) in the northern provinces of China. Bayesian analysis indicates that CRF07_BC has experienced two phases of exponential growth, which was first driven by 07BC_O then 07BC_N. Compared to 07BC_O, the proportion of the parameter of population transmission risk (TR) of 07BC_N has risen constantly. The power-law function analyses reveal that 07BC_N has increased over years with higher degree. In 07BC_N, only 13.16% of MSM were linked to other risk groups, but these links represent 41.45%, 54.25%, and 55.07% of links among heterosexual females, heterosexual males, and male PWID respectively. This study indicates that CRF07_BC has evolved into two clusters in China, and their distributions are distinct across risk groups and geographical regions. 07BC_N shows a greater risk of transmission, and has gradually replaced 07BC_O. Furthermore, the results show that strengthening the MSM interventions could lower the rapidity of 07BC_N transmission in all risk groups. The CRF07_BC is a variant of a recombination of the HIV-1 B’ and C among people who inject drugs (PWID) in southwest China in the 1990s , CRF07_BAccording to the latest nationwide HIV-1 molecular epidemiological survey in 2018 , CRF07_BThe huge number of HIV-infected population in China has been causing much pressure on HIV prevention and control . With thThe sequences of 5099 patients for the nationwide cross-sectional HIV-1 molecular epidemiological surveys prior to 2017 were extracted from the National Center for AIDS/STD Control and Prevention (NCAIDS). Sampling locations were grouped into seven regions  according to their proximity and socio-economic status in accordance with the guidelines from the National HIV-1 Surveillance system of China. Transmission categories include MSM, PWID , and heterosexuals . The missing data in transmission categories were designated as unknown. Sequences from NCAIDS have not been uploaded to any databases (e.g. GenBank) and only one sequence (the earliest) per individual was included.http://www.HIV.lanl.gov). Only one sequence (the earliest or the longest) from the same individual was included. The methods are illustrated step by step in Supplementary Figure S2.Thereafter, additional 1114 HIV-1 CRF07_BC sequences, which were sampled in China prior to 2017, were collected from Los Alamos HIV-1 sequence database  – associated with information such as sampling year, transmission route, gender, age at diagnosis, provinces, and marital status – were included. The study was approved by the institutional review boards of NCAIDS, China CDC.The viral sequences containing ≥5% ambiguities were excluded. The sequences less than 920 nucleotides in length were also excluded. Eventually, 6213 sequences  for all pairs of sequences identified as CRF07-BC using the HyPhy package. The network was constructed under the threshold of 0.7% genetic distance by linking two individuals (nodes) using cytoscape_v3.7.0. CRF07_BC was divided into two clusters in the transmission networks when the gene distance was 0.7%: 07BC_O and 07BC_N. We described the characteristics of the network including the number of sequences (nodes), links (degrees/edges), and clusters (groups of linked sequences) . The degWe compared the two clusters from the number of individuals, links, links/individuals, and the largest cluster in cumulative transmission networks (CTNs).Individuals of each CTNs were divided into three population groups by quartiles according to degrees which were defined as low-level , medium-level , and high-level . To evaluate the change of transmission risk between 07BC_N and 07BC_O under the different levels of CTNs, we put forward the parameter of population transmission risk (TR). TR of one cluster was defined as the sum of the degrees of all individuals of this group for medium-level and high-level, and the number of individuals for low-level. Then, we calculated constituent ratios of TR of different clusters under each of the three levels from 1997–2008, 2009–2010, 2011–2012, 2013–2014, to 2015–2017 transmission networks.y = cx^(−r). To compare the trends of the degree distributions in 07BC_N and 07BC_O, their degree distributions from 1997–2008, 2009–2010, 2011–2012, 2013–2014, to 2015–2017 transmission networks were fitted into curves of power-law functions respectively.The degree distribution of a network is a long-tailed distribution, which indicates that a large majority of nodes have a low degree but a small number have a high degree ,16. SuchBecause the number of sequences sampled from each province does not correlate well with the estimated number of new CRF07_BC infections in each province in 2015 , we randomly sampled sequences according to the proportion of CRF07_BC in each province in the 2015 nationwide cross-sectional survey data. To retain the full early samples, all samples prior to 2007 were retained in the subsets. In order to retain the full geographic distribution, provinces with less than 25 sequences available were not subsampled. Finally, a subset of 993 and 645 sequences were sampled from 07BC_N and 07BC_O datasets, respectively.TempEst was used to validate that there were sufficient temporal signals in the subsets to estimate molecular clock phylogenies. Time phylogenies for the 07BC_N and 07BC_O subsets were constructed in BEAST 1.8.4, using an uncorrelated log-normal relaxed clock, a Bayesian skygrid coalescent model, and a GTR+ G4 substitution model ,18.To understand the extent to which HIV-1 transmission occurs between individuals of the same or different transmission categories of 07BC_O and 07BC_N, the characteristics of potential transmission partners were analysed. To account for the bias arising from individuals with multiple links, we followed the protocol outlined by Oster and colleagues . Each pop values<0.05 were considered as significant.Univariate and multivariable logistic regressions were used to evaluate the factors associated with clustering in the network compared to the entire case with a genotype. All analyses were conducted in SPSS 22.0 software package and Overall, 6213 HIV-1 nucleotide sequences from 6213 persons were analysed. Among them, 3607 (58.06%) were in the network, 2946 (47.42%) were MSM, 5101 (81.10%) were male, 1702 (27.39%) were from the southwest, 2359 (37.97%) were aged 21–30 years old, and 1654 (26.62%) were unmarried .Table 1The maximum-likelihood phylogeny identified two well supported  and distinct clusters of CRF07_BC strains . Under tp < 0.001), sampling year during 1997–2010 (p < 0.001), MSM (p = 0.004), heterosexual males (p = 0.044), northern provinces (p = 0.044), and individuals over 60 years old (p = 0.016) .The highest proportions of clustering were observed in 07BC_N (72.48%), the sampling year 2011–2014 (70.55%), MSM (71.89%), southern provinces (73.71%), and individuals below 20 years old (58.72%) (Supplementary Table S1). Some factors enhanced the odds of clustering significantly including 07BC_N  in 2017  . The division of the phylogenetic relationships between the two CFR07_BC clusters using transmission networks under the threshold of 0.7% genetic distance was consistent with those inferred using the phylogenetic tree. Each cluster appears to have a unique role in the Chinese HIV-1 CRF07_BC epidemic with distinct associations in particular risk groups and geographic regions. 07BC_O predominantly stems from heterosexual populations, PWID, and married individuals, with most cases reported in northwestern and southwestern China. By contrast, 07BC_N was mainly found in MSM and unmarried individuals among northern provinces. The characteristics of 07BC_N are consistent with previous observations that a large and strongly supported sub-cluster of CRF07_BC strains was observed among young MSM in Jilin, Liaoning, Beijing, and Shijiazhuang (northern provinces) within the CRF07 radiation ,19,20.Our analysis showed that the major risk cluster for CRF07_BC transmission in China at an early stage was 07BC_O and the first wave of population growth of the CRF07_BC epidemic was driven by 07BC_O. Firstly, 07BC_O was the main cluster in the low and medium-level of TR before 2006. Then, other results showed that individuals, links, density , and the largest cluster of 07BC_O in the cumulative network accounted for the vast majority before 2006. Finally, the Bayesian skyline plot analysis indicates that 07BC_O had undergone significant growth during 1991–2005. Our findings are in line with previous studies that the origin and early epidemic of CRF07_BC in China was in the early 1990s among PWID in Yunnan (southwest) and Xinjiang (northwest) , and alsThe Bayesian skyline plot analysis indicates the epidemic of 07BC_O had been declining in population size after 2006 and was finally overtaken by 07BC_N. This development coincides with Chinese countrywide policies on universal access to HIV/AIDS prevention, treatment, and care services. In fact, the needle and syringe programme for PWID, free distribution of condoms, and antiretroviral treatment programmes have been rolled out since 2003, and China proclaimed a People’s War on Drugs in 2005 . This leFurther analysis of the longitudinal network revealed that 07BC_N had gradually replaced 07BC_O, and became the major risk cluster for current CRF07_BC transmission in China. TR takes the connectivity of individuals and the number of individuals in each group into account synthetically. A previous study reported that a high degree was more likely to result in a greater risk of HIV-1 transmission , and theThe reason why 07BC_N has a greater transmission risk and spread so rapidly may be associated with the population it circulates in. Previously, we demonstrated that CRF07_BC with relatively lower net charges in the V3 loop exclusively utilizes the CCR5 co-receptor for infection and exhibits slow replication kinetics in the primary target cells. These suggest that CRF07_BC may be superior to other HIV-1 subtypes in initiating infection in high-risk populations in China . This inOur results further suggest that the interventions for the two clusters of CRF07_BC should target various risk groups across the country. More than 90% of risk groups except MSM link to PWID and heterosexuals within 07BC_O, showing that PWID and heterosexuals play a major role in the spread of 07BC_O. The proportion of links with PWID and heterosexuals  among 07BC_N is lower compared to 07BC_O across all risk groups. This indicates that the needle and syringe programme for PWID and free distribution of condoms for heterosexuals rolled out countrywide in China since 2003 has slowed the spread through injections and heterosexual behaviour. In terms of 07BC_N that has continued to increase rapidly in recent years, we found that a minority of MSM were involved in transmission with other risk groups, including heterosexual males, heterosexual females, and male PWID, but these transmissions represented a substantial proportion of HIV-1 acquisitions by these risk groups. Our finding that MSM became disproportionately connected in the network of 07BC_N implies a concentrated uncontrolled HIV-1 epidemic among MSM. Therefore, this finding underscores the importance of focused interventions on MSM to retard the rapid transmission of 07BC_N because efforts that reduce transmissions involving MSM will not only reduce HIV-1 acquisition in MSM but also lessen HIV-1 acquisition among other risk groups.In summary, with an unprecedentedly large, longitudinal and detailed HIV-1 CRF07_BC sequence dataset in China, we identified two distinct clusters of HIV-1 CRF07_BC strains spreading in China and revealed that the two clusters are distinct among different risk groups and geographical regions. The first wave of population growth of the CRF07_BC epidemic was driven by 07BC_O before 2005. 07BC_N has a greater risk of transmission, gradually replaced 07BC_O, and caused the second wave of the CRF07_BC epidemic in China after 2005. The divergent transmission risk of the two CRF07_BC clusters highlights the importance of monitoring the genetic evolution and the phenotypic shift at the phylogenetic cluster level to identify and control clusters with greater transmission risk like CRF07_N. We further argue that the rapid increase of 07BC_N since 2005, especially after 2010, was associated with chemsex and online dating. Across CRF07_N, we also revealed that interventions that reduce transmissions involving MSM will also reduce HIV-1 acquisition among other risk groups. This study makes several contributions. First, it helps to grasp the long-term dynamics of CRF07_BC epidemic and factors of CRF07_BC transmission. Second, it provides guidance to target potential risk groups, thus a more effective prevention strategy is proposed to lower HIV-1 CRF07_BC transmission in China.Limitations of our work open avenues for future research. First, a proportion of our data from LANL may give rise to sample selection bias. Second, some relevant clinical information, such as CD4 count, HIV stage, accurate diagnosis time and the number of sexual partners were not collected. The inclusion of such data in future studies can improve the accuracy of the model to better fit the population distribution.Click here for additional data file.


Verticillium dahliae , is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by  Verticillium dahliae (Kleb.). Fungal inoculum consists of long-lasting microsclerotia, which remain infective in soil from 10 to 15 years1. V. dahliae (Vd) is a polyphagous pathogen affecting over 350 dicotyledonous hosts, thus making its management difficult through common agricultural practices5. The wilting caused by SVW exerts up to 30% yield reductions in susceptible commercial sunflower hybrids7 and up to 73% in susceptible materials grown in highly infested fields8. Yield losses have a direct relationship with symptom level (i.e. foliage necrotic surface)9.Sunflower Verticillium Wilt and Leaf Mottle (SVW) is a monocyclic vascular disease whose causative agent is the soil-borne fungal pathogen 10. Historically, this has been the most prevalent disease in Argentina and it has high impact on extensive regions of Canada and USA, where new pathogenic races have arisen12. Moreover, it has recently become a serious threat for European sunflower producing countries of temperate regions with increasing prevalence rates in France, Italy, Spain and countries around the Black Sea14.SVW occurs in most sunflower producing areas of the world16, Vd is an endemic pathogen with a variety of local races19. Vd inoculum is spread over 1.2 million hectares, affecting over 70% of the country’s sunflower growing region20. In the south of Buenos Aires Province, the main area for sunflower production in Argentina, the SVW-prevalence levels have shown an average of 45% (± 14%) over the last 8 years21.In Argentina, the third sunflower producer and edible-oil exporter worldwide4, genetic resistance is still the most effective strategy to cope with this affection. SVW resistance was first reported as a qualitative trait governed by a single dominant locus22. Later contributions have described different inbred lines (ILs) with dominant, additive and recessive sources of resistance to North American races24. Since then, the V1 locus, which was identified on the maintainer IL HA8925, has been the main source of resistance for hybrid development worldwide12.Despite the relevance of the pathogen, the disease-management tools available to date are limited. Although no-tilling is known to be a useful tool for lowering disease incidenceVd races overcoming V1 locus resistance, first in Argentina and later in the USA and Europe27. In Argentina, two local races, VArg1 and VArg2, were identified among the isolates affecting sunflower18. Consequently, researchers of the private sector have reported pairs of differential inbred lines bearing specific resistance to these races, giving rise to SVW-resistant hybrids28. However, further reports described less-frequent new races overcoming these resistance sources as well19.During the last 40 years, various studies have reported Even though some commercial hybrids currently display SVW-resistance, the lack of public research on this subject has prevented disentangling the genetic architecture of the trait. Exploring large sets of germplasm and characterizing their behavior against SVW are the first steps towards identifying novel resistance sources to contribute to sunflower breeding. However, no large-scale surveys have been conducted and little is known about the importance of the different DDs  or their heritability, up to this date.32. The AMP was extended for this study by incorporating ILs from the INTA breeding program with good behavior for SVW-resistance formerly identified by breeders on the EEA-INTA-Pergamino using artificial inoculation33. The AMP contains a representative sample of the genetic diversity held in INTA’s sunflower breeding program and comprises 164 ILs. The genetic diversity of the AMP is comparable to that of other sunflower inbred panels from public breeding programs from France, Canada and USA, while containing the singularities of the Argentinian germplasm32.In this study, we evaluated 301 ILs of two mapping populations (MPs) developed by the INTA for SVW Broad Spectrum Resistance (BSR) against Argentinian local races. The first MP is the result of the biparental crossing between two restorer public-ILs PAC2 × RHA439 . The second is the sunflower association mapping panel of INTA (AMP)The aims of the present study were: (1) to assess the diversity held in INTA´s genetic resources for SVW-resistance, (2) to identify inbred lines representing new SVW-resistance sources, (3) to understand the inheritance and (iv) to estimate the heritability of the trait.The phenotypic evaluation for SVW resistance of the two MPs retrieved robust results for 162 ILs from the AMP  and 139 RILs from the BMP (4 FTs in one location). SVW was scored at four Evaluation Dates (EDs). The DDs recorded to evaluate ILs at each ED were: disease incidence (DI), disease severity (DS), binomial disease severity (bDS) and disease intensity (DInt). Estimation of the Area under the Disease Progress Curve for disease incidence (AUDPC.DI) and for disease intensity (AUPDC.DInt) integrated all EDs. The combination of DDs and EDs rendered 18 SVW-DDs to be modeled in the statistical analysis. We selected four of those to illustrate IL’s behavior on the basis of their potential impact on yield components. The selected combinations were DI at flowering (DI.Flw or DI.3), AUDPC.DI, DS at grain filling (DS.Gf or DS.4) and bDS at grain filling (bDS.Gf or bDS.4).The analysis of the raw DI.Flw and DS.Gf values showed similar overall patterns in both MPs, with DI.Flw and DS.Gf ranging from 1.6 to 7.3 plants per plot (p.p.p.) and from 2.9 to 3.9 symptomatic p.p.p., respectively  held values of DI.Flw and AUDPC.DI markedly above the overall mean in season 2016/17 and below it in 2014/15 and 2015/16. Altogether, AUDPC.DI and DI.Flw showed a similar pattern , except for DInt at the first evaluation date in the AMP (DInt.1). The selected model for further analysis was the Row-Column design with Genotype × Season interaction  or DS  derived descriptors  ranked in the bottom ten of the four DDs. The highest level of DI.Flw and bDS.Gf occurred in IL PMA70 . As seen in the resistant group, some ILs exclusively ranked at the bottom of a particular DD. None of the parental lines of the BMP, both present in the AMP, were ranked on the extremes of any DD , we calculated Pearson’s correlation coefficients between Best Linear Unbiased Predictors (BLUPs) of individual FTs, and between these and the G × E adjusted BLUPs, for the main four DDs , whereas the lowest was between the first and the last FTs (0.541)  of the 18 DDs scored in this study, we computed Pearson’s correlations among the IL’s G×E adjusted means on each MP. All coefficients were significant, positive and rather high. Values were always above 0.5 or 0.6 for the AMP and BMP, respectively. Coefficients above 0.95 occurred between some contiguous evaluation dates for DI, DInt and bDS  explained 86.1% of the total variance  in both cases. Further hierarchical and non-hierarchical clustering analyses based on distances and cluster number allowed the distinction of a fourth phenotypic group that was not apparent when analyzing the data on a single variable basis (DGC procedure detected between 2 and 3 classes). The K-mean method, presented here, generated more evenly represented groups for both MPs . In comparison to the remaining DDs, DI.Flw showed the lowest average H2. Furthermore, DS.Gf (H2 = 53.2) and AUDPC.DI (H2 = 43.87) had the highest heritability coefficients for the AMP and for the BMP, respectively. As expected, the AMP showed higher levels for each H2 estimate and larger standard deviations than the BMP. The BMP denoted a progressive increase in the H2 estimates for most of the variables along FTs. In the AMP, this response was evident only for DI.Flw, considering only FTs performed in Balcarce. In general, the H2 estimates obtained at Coronel Suarez were lower than those of Balcarce on the same year. For most DDs, the environments with higher H2 coefficients were FT-2016 and FT-2017B for the AMP, and FT-2015 and FT-2016 for the BMP.The study of broad sense heritability . Since the critical stages for potential grain-yield definition are flowering and grain filling onset, three DDs focused on these stages assessing DI, DS and the combination of both relative to a tolerance threshold . AUDPC.DI, while integrating all DI scores through time, gives a cumulative resistance profile from early stages of each IL, giving higher values to ILs presenting symptomatic plants earlier. The bDS.Gf descriptor seemed to be most suitable for quick disease assessment since it provided a weighted estimation of DS.Vd pathogenic races have underscored the need for BSR sources in sunflower41. The inoculum load measured in the infested field of EEA INTA Balcarce . This notion supports the BSR displayed by the best performing ILs of the AMP. Despite the significant G×E effects, the high correlation coefficients observed among FTs indicate that the ranking of ILs was consistent across years for both MPs. Furthermore, the alternation of the groups of ILs occupying the top ranks for each DDs  described in Filippi et al.32, with the addition of 29 new accessions, thus reaching 164 ILs , from the growing seasons 2013/14 (F6) to 2016/17 (F10). The AMP was evaluated in five FTs, four at EEA-INTA Balcarce (2014/15 to 2017/18) and a fifth (2017/2018) at “El Cencerro” seed company, Coronel Suárez, Buenos Aires .ILs from both MPs were grown under field conditions in artificially infested fields containing a diverse set of Vd inoculum titter of EEA-INTA Balcarce’s infested field was estimated around 900 CFU per gram of soil in season 2015/16  infections. Mechanical and manual controls were implemented for weed management.Plots of each FT consisted of a single 5 m row (± 0.5 m) disposed at an inter-row spacing of 0.7 m with 20 ± 5 plants. Each plot corresponds to an experimental unit. An Alpha-Lattice design in two replications was implemented for all FTsThe DDs recorded to evaluate ILs were DI, DS, bDS, DInt, AUDPC.DI and AUPDC.DInt. DI and DS were scored at individual plant level. DI was assessed as the overall count of symptomatic plants per plot, whereas DS was scored using an ordinal scale of six levels, as follows: “0”, non-symptomatic plant, “1” plant with symptoms at basal leaves , “2” symptoms below middle leaves (20–40% t.l.a.), “3” symptoms reaching middle leaves (40–60% t.l.a.), “4” symptoms in upper leaves (60–80% t.l.a.) and “5”: a totally wilted plant  corresponds to the time elapsed between two observations (days).DInt, bDS and AUDPCs were also calculated according to DI and DS scores. DInt is the per-plot weighted mean resulting from the multiplication of the frequencies obtained for each severity level and the score-level. The descriptor bDS accounts for infected plants with DS above “2” and any plant below this threshold is considered tolerant. AUDPC is the integration of the scores of a particular variable across the evaluation dates, according to the formula described by Shaner and Finney60 Eq. .1\docum39. Therefore, each ED represents a particular phenological stage as follows: ED-1 = Floral initiation to Early floral bud growth (R1–R2); ED-2 = Floral growth to Pre-Flowering (R3–R4); ED-3 = Flowering ; ED-4 = Grain filling 39. DDs are named both with number or acronym indistinctly (i.e.: DI.3 = DI.Flw). These combinations yielded 18 variables: 16 DD × ED combinations and two AUDPCs (for DI and DInt).Disease evaluation was performed weekly from early reproductive stages to grain filling onset, covering four evaluations per FT. Each ED was centered on the most frequent phenological stage among ILs at the time61. The selection of the right linking function when modelling was determined according to the statistical properties of each DDs. DI and bDS are discrete binomial variables and were modeled by logistic linear regressions. DS was transformed to a continuous variable by the Normal Scores method62, and modelled as a normally distributed variable by Linear Mixed Models (LMM). DInt and AUDPCs are continuous variables that were converted to relative proportions of the maximum score reached per plot and per FT, respectively, and modeled as gamma distributed variables. Several models were employed for obtaining IL’s adjusted phenotypic means and BLUPS63 for each DD, in both single and combined FTs . These approaches allowed the comparison between them and the variance components analysis of random-effect models for DDs’ H2.For the estimation of IL’s mean phenotype for each SVW-DD, the statistical analysis at each evaluation date was boarded by Generalized Linear Mixed Models (GLMM)GS” (Genotype-Season) interaction factor. The same models were tested for Alpha-Lattice design, which was obtained by excluding the factor “Column” (C) on (2) and (3).All of these models were fitted after the best model for each DD was found by comparing four models considering two experimental designs, Alpha-Lattice or Row–Column, and the inclusion of a random effect factor term considering G × E interaction under each design. For the Row–Column design, models were compared without (2) and with (3) the “i, in Season j, under Replicate k, within Block m and Column n; μ is the overall mean of observations; Gi is the Inbred Line fixed effect (as random for BLUPs and H2 determination); Sj is the random effect of Season j; k within Season j; m within Replicate k; Cn is the random effect of Column n within Replicate k; and Models 1 and 2 are defined as follows:35. BLUPs were calculated for each IL on every DD both, on each FT and across FTs, to evaluate the phenotypic stability of ILs across FTs and locations. Pearson’s correlation coefficients were used to compare BLUPs among FTs and models. Broad-sense heritability estimates were assessed for each DD considering the individual FT data. For binomial variables, residual variance components were estimated as described in Snijders and Bosker64. LMM and GLMM were built with the lme4 package65 for the R statistical software66. Pearson’s correlations were estimated and visualized using the GGpairs R-package67.The adjusted means were then subjected to multiple comparison tests using the DGC procedureOf the combination of DDs and EDs modeled, mainly four were considered to characterize ILs regarding SVW-BSR in a single-variant fashion. The criterion for selecting them accounted for agronomical issues and took into consideration SVW impact on yield components and the maximization of each DD descriptive capability. These four DDs were: DI.Flw (DI.3), AUDPC.DI, DS.Gf (DS.4) and bDS.Gf (bDS.4).prcomp function of the stats package for the R statistical software66. Both MPs were analyzed with 18 DDs . Results were visualized using the ggfortify R-package68.PCA were carried out using the standardized adjusted means of DDs for each mapping population and the get_dist of package vegan for R software69. The most likely number of clusters was defined using the function fviz_nbclust from the factoextra R-package70. Non-hierarchical clusters were estimated for each MP using the function “kmeans” of the R-package stats66. Finally, a hierarchical approach was performed by using the “hclus” function from the same package, by implementing different linkage methods and comparing them by their co-phenetic correlation coefficients with original distance matrices.The clustering analysis was performed by estimating distance matrices for each MP from the adjusted means of DDs across FTs using the function Supplementary Information 1.Supplementary Information 2.


HGD) showed four novel variants c.16-2063 A > C, p.(Thr196Ile), p.(Gly344AspfsTer25), and p.(Gly362Arg) in six mutated alleles (83.3%). RNA sequencing revealed that c.16-2063 A > C activates a cryptic exon, causing protein truncation p.. A literature search identified another 6 patients with alkaptonuria in East Asia; including our cases, 13 of the 18 mutated alleles have not been reported elsewhere in the world. Alkaptonuria is rare in Taiwan and East Asia, with HGD variants being mostly novel and private.Alkaptonuria is a rare autosomal recessive inherited disorder of tyrosine metabolism, which causes ochronosis, arthropathy, cardiac valvular calcification, and urolithiasis. The epidemiology of alkaptonuria in East Asia is not clear. In this study, patients diagnosed with alkaptonuria from January 2010 to June 2020 were reviewed. Their clinical and molecular features were further compared with those of patients from other countries. Three patients were found to have alkaptonuria. Mutation analyses of the homogentisate 1,2-dioxygenase gene ( HGD gene, catalyzes the conversion of homogentisic acid (HGA), a metabolite of tyrosine, to 4-maleylacetoacetate. Alkaptonuria  is a rare autosomal recessive disease caused by tissue accumulation and urinary excretion of HGA1. The hallmark of the disease is dark urine, which is caused by the oxidization of HGA. Associated morbidities include ochronosis, ochronotic arthropathy, cardiac valvular calcification, and urolithiasis in the 4th to 6th decades of life2. Although the life expectancy of patients with AKU is not reduced, their quality of life deteriorates mainly due to painful joint diseases3. The disease is not yet curable, and symptomatic relief management, such as physiotherapy, painkillers, and joint replacement therapy, is necessary4. Recently, nitisinone, a potent inhibitor of HGA production, was shown to decrease urinary HGA excretion and decelerate disease progression5.Human homogentisate 1,2-dioxygenase, a homogentisic acid oxidase encoded by the 6, but the disease is more prevalent in Slovakia, Jordan, the Dominican Republic, and India4. HGD p.(Gly161Arg) is a common disease-causing variant in Slovakia and the Czech Republic; HGD p.(Cys120Trp) is common in the Dominican Republic7. The most prevalent variants in European countries excluding the abovementioned is p., accounting for 11.2% of all HGD variants, followed by p., p.(Gly270Arg), and p.(Pro230Ser), which together account for ~9.5%8. Because AKU is rarely reported in Asia, we conducted this study to examine its characteristics in this region.The worldwide prevalence of alkaptonuria is estimated to be 1:100,000 to 1:1,000,000Three Taiwanese (Han ethnicity) patients, two males, and one female, with a diagnosis of AKU were identified. Two of the patients had dark urine during infancy or early childhood, and one had dark urine at the age of 13 years. Their current ages are 10–20 years  and c.473 C > T p.(Pro158Leu). p.(Trp97Ter), a nonsense variant, is reported as pathogenic in ClinVar, and other pathogenic variants at this amino acid p.(Trp97Arg) and p.(Trp97Gly) have also been reported, indicating that it is likely an important site for disease variants9. The variant p.(Pro158Leu), which occurs at CpG dinucleotides, is thought to be a mutation hot spot11. The other four variants have not been reported to date: c.16-2063 A > C, p.(Thr196Ile), p.(Gly344AspfsTer25), and p.(Gly362Arg) , several larger-than-expected fragments were found , allele-specific PCR was designed for c.1084 G > A Fig. . The c.112. We employed structure-based variant characterization tools to identify the molecular consequences of the novel variants in the current study, including mCSM, mCSM-lig, mCSM-PPI2, and HGDiscovery , p.(Glu42SerfsTer69), p., c.342 + 3A > C, p.(Gly152Ala), p.(Thr196Ile), p.(Glu329Cys), p.(Gly344AspfsTer25), p.(Gly362Arg), p.(Ser366_Thr367dup), c.16-2063 A > C, and c.469 + 1 G > C. Therefore, most of the AKU patients in East Asia harbor private variants in HGD. All HGD missense variants were analyzed by the pathogenicity analysis tool MetaDome, and the results revealed that most are located in intolerant parts of the protein; for the Asian variants, tolerant for p., slightly tolerant for p.(Gly362Arg), neutral for p.(Thr196Ile), slightly intolerant for p.(Gln33Arg) and p.(Phe329Cys), intolerant for p.(Pro158Leu) and p.(Glu168Lys), and highly intolerant for p.(Gly152Ala) . However, only 6 patients from East Asia  are reported in PubMedWe only identified three cases in our institute, and there is no previous report of AKU in Taiwan. Although we were not able to calculate the prevalence of AKU in Taiwan, the disease must be very rare, as in all of East Asia. In addition, we were unable to fully elucidate the phenotype of the disease in our four Taiwanese patients because they were still young. Nonetheless, the suspicious ochronosis and arthralgia in a portion of them alert us to the potential for more serious symptoms in the future.HGD variants being mostly novel. More efforts involving molecular analysis are expected to contribute to the diagnosis of new cases in this region.Alkaptonuria is rare in Taiwan and East Asia, with From Jan 2010 to Jun 2020, patients diagnosed with AKU at National Taiwan University Hospital were reviewed. Informed consent was obtained from all subjects or their guardians, and the guardian of patient 2 provided written consent for the publication of the photograph in Fig. HGD (RefSeq: NM_000187.4) were performed by targeted panel sequencing using a SeqCap EZ probe  and MiSeq sequencer  to produce 300-bp paired-end reads, with an average coverage >150 crossing exons and flanking intronic (50 bp) sequences. Sequence alignment to the human reference genome (GRCh37) was performed using Burrows-Wheeler Aligner, and variant calling was performed using Genome Analysis Tool Kit 26. Variants were annotated by ANNOVAR (http://wannovar.wglab.org/)27. DNA and protein sequence variants were described as recommended by Human Genome Variation Society . For missense variants, we employed Sorting Intolerant From Tolerant , PolyPhen-2 , mCSM (http://biosig.unimelb.edu.au/mcsm/)16, mCSM-PPI2 (http://biosig.unimelb.edu.au/mcsm_ppi2/)18 and mCSM-lig (http://biosig.unimelb.edu.au/mcsm_lig/)17 to predict effects on protein function and structure using the experimental crystal structure of HGD (PDB ID: 1EY2). Intramolecular interactions were calculated and visualized using Arpeggio28 and mutational tolerance was calculated using MTR-Viewer19. For intronic variants, we used Human Splicing Finder (http://umd.be/Redirect.html) and Splice AI to predict their splice effects. We also searched the HGD mutation database (http://hgddatabase.cvtisr.sk/home.php) and ClinVar to assess whether variants have been reported. The pathogenicity of variants was classified according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology guidelines29.Variant analyses of GAPDH was used as an internal control. HRM was performed by Rotor-gene Q (Qiagen). Allele-specific PCR for RNA was designed for the c.1084 G > A variant, and the specific lower primers were 1084 G (wildtype) 5’-tcattgtgctgtgtagactccc and 1084 A (mutant) 5’-tcattgtgctgtgtagactcct. Densitometry was performed using ImageJ. All primer sequences are listed in Supplementary Table RNA-Seq was performed on total RNA extracted from peripheral whole-blood samples. After the depletion of ribosomal RNA and globin mRNA, each sample was sequenced with a total output of 50 M reads. Data obtained from RNA-Seq were aligned using STAR and annotated for DNA analysis. RT-PCR was further applied to validate aberrant splicing. The region of exon 1–3 was amplified to demonstrate aberrant splicing. For quantitative PCR using the SYBR Master Mix (Applied Biosystems), http://biosig.unimelb.edu.au/hgdiscovery/submit_prediction)15. Asian HGD variants were illustrated by the tolerance landscape using Metadome Version 1.0.1 webserver (https://stuart.radboudumc.nl/metadome/dashboard)14.Computational analysis to evaluate possible enzyme inactivation of novel missense variants of HGD was used by webserver of HGDiscovery , and the HGD mutation database from Leiden Open Variation Database (LOVD) for the HGD mutations described in the publications retrieved.We searched PubMed publications using the criteria “ AND ((mutation) OR (variant) OR (gene diagnosis))”. We targeted studies published after 1996, and the time the human Further information on research design is available in the Supplementary InformationReporting Summary


Candida vaginitis on this infection. From 11 August 2017 to 11 September 2018, 12,628 outpatient participants ranged from 19 to 84 years old were enrolled and analyzed. HPV DNA was amplified and tested by HPV GenoArray Test Kit. The vaginal ecology was detected by microscopic and biochemistry examinations. The diagnosis of Candida vaginitis was based on microscopic examination  and biochemical testing  for vaginal discharge by experts. Statistical analyses were performed using SAS 9.4. Continuous and categorical variables were analyzed by t-tests and by Chi-square tests, respectively. HPV infection risk factors were analyzed using multivariate logistic regression. Of the total number of participants, 1753 were infected with HPV (13.88%). Females aged ≥ 40 to < 50 years constituted the largest population of HPV-infected females (31.26%). The top 5 HPV subtypes affecting this population of 1753 infected females were the following: HPV-52 (28.01%), HPV-58 (14.83%), CP8304 (11.47%), HPV-53 (10.84%), and HPV-39 (9.64%). Age  and alcohol consumption  were found to be risk factors for HPV infection. However, the presence of Candida in the vaginal flora was found to be a protective factor against HPV infection . Comparing with our previous study of 2016, we conclude that the subtype distribution of HPV infection is relatively constant in Changsha. Our data suggest a negative correlation between vaginal Candida and HPV, however, more radical HPV management is required in this area for perimenopausal women and those who regularly consume alcohol.This cross-sectional study investigated the characteristics of cervical HPV infection in Changsha area and explored the influence of  Nearly 989,000 new cases of CC are diagnosed in China every year, and the overall hrHPV infection rate in mainland Chinese women is 19.0% 2. The top 5 hrHPV subtypes with the highest infection rates in China include HPV-16, HPV-52, HPV-58, HPV-53, and HPV-183. HPV infection is most prevalent among childbearing-age and menopausal women; the prevalence among menopausal women is known as the “second-peak”4. Some of the factors reported as closely correlated with HPV infection include alcohol consumption, smoking, age at first marriage, marital status, vulvovaginal ulcers, and vulvovaginal inflammation6. The biological mechanisms of risk factors on HPV infection are less well understood over the decades. However, these investigations have yielded few results when it comes to the pathogenesis of this infection.Human papillomavirus (HPV) infection is the most prevalent sexually transmitted infection in women. Persistent infection with high-risk HPV (hrHPV) subtypes, but not all HPV infections, can lead to the development of cervical cancer (CC), a disease that adversely affects the health of women worldwide7. Lactobacillus maintains a low vaginal pH value through producing lactic acid to perform HPV protection8, and Candida stimulates T cell proliferation to against HPV infection9. However, Gardnerella, Fusobacteria, Mycobiota, and Chlamydial trachomatis (CT) were associated with HPV infection11, for example, CT plays as the entryway of the cervical epithelium and facilitate HPV infection11.In recent years, scientists have become interested in and made promising progress investigating the mechanisms of vaginal flora. Some studies have reported that vaginal microorganisms might promote or impede HPV infection by infecting a host’s cervical micro-ecological environment, and may further play essential roles in the pathogenicity of HPV and cervical lesions12. However, more evidence and investigation need to be updated to analyze the longitude development of cervical cancer prevention and treatment in Changsha. Many risk factors of cervical cancer include lifestyle such as smoking, HPV type and distribution, and the influence of vaginal flora on HPV need to be updated. We examined patients that attended the Physical Examination Outpatients Center at our hospital and hypothesized that the distribution of HPV might have changed in the few years since this previous study. We also hypothesized that vaginal flora or Candida might play a vital role in influencing this infection.In 2016, Xiao et al. reported on the characteristics of HPV infection in Changsha based on information obtained from the Gynecological Outpatient Center of our hospital Candida infection (P < 0.001), age at first marriage (P < 0.001), age at first childbirth (P < 0.001), and alcohol consumption (P = 0.003). Additionally, there were no significant differences between the two groups in BMI (P = 0.855), white blood cell (WBC) count (P = 0.873), neutrophil percentage (P = 0.298), lymphocyte percentage (P = 0.199), fasting blood glucose (P = 0.199), vaginal H2O2 (P = 0.199), vaginal grades (P = 0.227), vaginal trichomonas (P = 0.482), age of menstruation onset (P = 0.874), whether they had given birth (P = 0.106), diet [including spicy diet (P = 0.224) and sweet diet (P = 0.716)], smoking (P = 0.299), daily sitting time (P = 0.748), and waistline measurements (P = 0.704) . There were significant differences between the HPV-negative and -positive participants in mean age , vaginal pH , vaginal samples positive for galactosidase (P < 0.001), sialidase, leukocyte esterase (P = 0.001), and We analyzed the age distribution of the study participants. The highest positive infection rate of HPV was 31.26% in 40–49.9 years old group , followed by 27.44% in 30–39.9 years old group (n = 481), and 24.59% in 50–59.9 years old group (n = 431). This data showed the HPV distribution of different age groups , HPV-58 , CP8304 , HPV-53 , and HPV-39 , respectively. These results are strongly consistent with previous research performed at our hospital; 28.01%,u) and the adjusted OR (ORa) were found to be consistent. The risk factors of HPV infection were age  and alcohol consumption . However, vaginal Candida infections , age at first marriage , and age at first childbirth  were protective factors against HPV infection in the study. Additionally, the age at first childbirth ≥ 20 to < 30 years  was also found to be protective against HPV infection, but the difference was not significant (P = 0.38) . The AUC values for all these variables were found to be approximately 0.8. Additionally, the ANOVA test and the initial models that included all variables produced results that were not significant, which indicates that the statistics using AUC were reliable. As an example, the largest AUC as a predictive factor for HPV infection was that of vaginal Candida infection. This AUC value was 0.881, the optimal cut off value was 0.164, the sensitivity was 1.000, and the specificity was 0.827  were significant at vaginal 3. In our study, the top five HPV subtypes were the following: HPV-52, HPV-58, HPV-CP8304, HPV-53, and HPV-39. However, the top 5 subtypes found in the study of Xiao et al., performed at the same hospital as our study, were HPV-52, HPV-16, HPV-58, HPV-CP8304, and HPV-5312, and the top 5 HPV subtypes found in China overall were HPV-16, HPV-52, HPV-58, HPV-53, and HPV-183. The detection results of our hospital in recent five years showed that the positive rates of hrHPV and HPV16 were lower than the past and the national average level, indicating that Changsha city has achieved good results in reducing the high risk of HPV16 and related cervical precancerous lesions. The HPV-CP8304 is a popular subtype in our region with high prevalence but low risk. Our data were predominately consistent with the previous investigation12 and mostly in line with the analysis of national data3. These differences may be a result of variable geography, ethnicity, education, and horizontal health care. However, when considering vertical health care, this decrease in HPV infection evident in our study may be explained by improvements in women's health care and the improvements in awareness of women's health in recent years14.The overall HPV-positive rate of this study was lower (13.88%) than that of the overall in mainland Chinese women (19.0%)16. In 2015, Wang et al., confirmed that the hrHPV rate was 91.8% patients with invasive cervical cancer (ICC) in Hunan province17. Also, HPV-16 and HPV-18 are considered the top hrHPV subtypes worldwide20, However, neither HPV-16 (6th) nor HPV-18 was identified as a prevalent subtype in our study. Several factors may account for this, such as regional differences, improvements in women's health care, and the awareness of women's health. Another reason is the participants involvement differences between studies. We investigated women from the Physical Examination Outpatient Center from our study while others from the Gynecological Outpatient Center. Symptomatic or sick patients would generally choose the Gynecological Outpatient Center for CC screening, while the Physical Examination Outpatient Center encounters more asymptomatic or healthy cases. Taking this into account, although our data is reasonably consistent with previous studies, the differences were unavoidable. These screening sample differences also suggest that the CC screening at physical examination outpatient centers, rather than the gynecological outpatient centers, might receive a closer result compared to the rate of HPV infection across the whole country.Persistent hrHPV infection was believed as the causative agent in over 90% CC in early in 2000 by Bosch et al.21. Several factors might contribute to this pattern, including that immunity decreases with age, meaning that older women are at an increased the risk of developing HPV infections22. Besides, the postmenopausal population have elevated pH values due to the decreased estrogen levels that is correlated to the low glycogen levels and Lactobacillus abundance, making them sensitive to HPV infection24. Additionally, older women tend to seek out for routine gynecological care and cancer screenings25, which potentially elevates the HPV-positive rate for this age group. This explains our finding that increased age may be a risk factor for HPV infection  . This might be explained that the sialidase, one of the virulent biomarkers of Gardneralla vaginalis, that through hindering the epithelial biofilm formation, facilitates the infection/co-infection of HPV and other microorganisms , herpes simplex virus (HSV), human cytomegalovirus (HCMV)35. In line with the study of Lili et al., that the leukocyte esterase positive rate is higher in the HPV-positive group when compared with that of the HPV-negative 36. We then figured out that alcohol consumption was correlated with HPV infection. An underlying reason for this could be that alcohol may increase sexual disinhibition, which results in increased unsafe sexual behaviors38. Smoking is another potential risk factor. Multiple studies have posited that both passive and active smoking of cigarettes increase the risk of developing HPV infection, as smoking suppresses innate immunity and can cause structural and functional changes within the respiratory system41. However, our data found no significant correlation between smoking and HPV infection. This is possibly due to limited case numbers of HPV-infected participants and the relatively restricted region within which participants lived.We also investigated potential risk factors of HPV infection. Our data showed that the vaginal pH of the HPV-positive group was significantly elevated than that of the HPV-negative group , this might due to the vaginal dysbiosis, and particularly with the displacement of 6. This phenomenon might be explained by the notion that older women are more likely to engage in safe sexual behaviors6. Additionally, older women are believed to have greater awareness of genital hygiene and healthcare, which reduces the rate of HPV infection42. The vaginal Candida infection was significantly negatively correlated with HPV infection . This might be due to the presenting symptoms of Candida vaginitis, such as leukorrhea, vulvar pruritus, dyspareunia, and dysuria43, encouraging patients to seek timely gynecological screening. Additionally, Candida parapsilosis can serve as a biofilm on the surface of the genital tract, which may act as a shield against invasion by other microorganisms44. Liang et al. also supports this result; they reported similar findings of Candida albicans being protective factor against HPV infection 45. Engberts et al. also reported that infection by Candida does not increase the risk of developing CC46. Additionally, Gonia et al. found that Candida parapsilosis protects premature intestinal epithelial cells from invasion and damage by other microorganisms47. Wang et al. also reported that Candida albicans can enhance T cell proliferation, which might play a vital role in regulating the local vaginal and cervical microenvironments, and further inhibit the pathogenicity of HPV infection and cervical lesions9. However, the mechanisms that mediate the relationship between Candida infections and HPV infection remain controversial. Studies of larger sample sizes and further investigations are required to explore these mechanisms.Meanwhile, the probable protective factors were summarized. Our study found that a marriage age of 20 years or older (OR 0.79) and an age at first childbirth of 30 years or older (OR 0.67) were protective against HPV infection (P < 0.05). Consistent with these findings, Niyazi et al. reported that early marriage might be a risk factor for hrHPV infectionGonorrhea, Syphilis and HIV that might infect the vaginal-cervical microbiota49. There are further constraints in our study that might limit its wider applicability. We neither investigate details of alcohol consumption, nor follow up on infection persistence, progression, or the outcomes of participants. Besides, our documented vaginal microecology narrowly focused on Candida of different fugal types, more preliminary clinical findings need to be further studied, with the rest of vaginal cofounders, such as Gardnerella, Fusobacterium, bacterial vaginosis and aerobic vaginitis50. Furthermore, we excluded the previous positive HPV patients with or without local drug administration which might bias its distribution. Additionally, we did not further investigate the participants infected by multiple subtypes of HPV.Some deficiencies cannot be ignored for this study. Besides the limited number of HPV cases and lack of information regarding participants’ marital status, number of sexual partners, use of oral contraceptives, and the history of sexual transmitted infections (STIs) such as Candida with HPV infection in Changsha, Hunan. Our findings potentially provide valuable information to assist in the improvement of clinical HPV screening and CC prevention in local regions. To improve future research, larger sample sizes, optimization of the participant questionnaire, improved follow-up of participants, and in vitro experimentation might be considered.Despite these limitations, our study is in strong agreement with previous research. Additionally, this study is the first to explore the potential relationship between vaginal Candida infection is a protective factor against HPV infection and that more radical HPV management is required in the local Changsha area for perimenopausal women and who regularly consume alcohol.In conclusion, we found that the prevalence of HPV infection and the distribution of its subtypes is relatively constant in Changsha, Hunan. Our data suggest that vaginal Every female patient that attended the Physical Examination Outpatients Center at the Third Xiangya Hospital of Central South University between 11 August 2017 and 11 September 2018 was asked to complete a questionnaire and voluntarily sign written informed consent. This study was approved by the ethics committee of the Third Xiangya Hospital of Central South University (IRB No. 20017). All methods were carried out in accordance with relevant guidelines and regulations.To be included in this study, participants had to satisfy all of the following inclusion criteria: (1) age ≥ 18 years old and no previous positive HPV results; (2) no sexual intercourse, vaginal douching, or administration of vaginal medications in the 3 days before vaginal samples taken; (3) mentally competent so as to understand the consent form and communicate with study staff. The exclusion criteria: (1) history of no sexual activity; (2) HPV positive results in one year or a history of CC; (3) received a hysterectomy; (4) received treatment for vaginitis in the past 3 months; (5) had sexual intercourse, vaginal douching, or taken vaginal medication in the 3 days before vaginal samples taken; (6) unable understand the consent form and communicate with study staff; (7) the pregnant; (8) withdraw from the study.2O2-negative/positive; Candida-negative/positive; Trichomonas-negative/positive; grades-I/II/III/IV  and low amounts of Lactobacillus species, mainly L. iners. Finally, Grade IV is characterized by the presence of a variety of Streptococcus spp.)52 were carefully recorded and double-checked by two research assistants.Patient data extracted from medical records, including general information  and test results  and vaginal microecology test results which were diagnoses by experienced cytologist  and biochemical testing  for vaginal discharge by experts. In our study, the criteria of bacterial infection diagnosis by Amsel method are as follows: (1) uniform vaginal secretion; (2) pH > 4.5; (3) amine smell in secretion with 10% potassium hydroxide; (4) positive laboratory test results (Gram staining for bacteria in secretion or wet film for clue cells). If any three of the above criteria are met (but the last one is necessary), the diagnosis can be made.The vaginal and cervical samples were collected by gynecologist, while the blood was drawn by nurse. All vaginal samples were collected for the test of vaginal microecology, the cervical samples were collected for HPV screening, and the blood samples were collected for lab testing. The Pentaplex Vaginitis Detection kit  was used for vaginal ecology testing, including galactosidase, sialidase, leukocyte esterase, HA total of 12,628 participants with complete medical records were enrolled in this study and retrospectively analyzed; 10,875 were non-HPV infected females and 1753 were HPV-infected females .53.HPV DNA was amplified by polymerase chain reaction (PCR). Then, HPV genotyping by HybriMax was performed using an HPV GenoArray Test Kit . This assay can determine 21 HPV types, including 14 high-risk HPV types , five low-risk HPV types , and two unknown-risk types (53 and CP8304), by the flow-through hybridization technique using HPV DNA amplified by PCRStatistical analyses were performed using Statistical Analysis System 9.4 . Continuous variables were analyzed by t-tests, and the categorical variables were analyzed by Chi-square tests. The age distribution and subtypes distribution were summarized based on HPV positive cases.The multivariate logistic regression risk model was used for investigating the risk and protective factors of HPV infection. The risk factor was defined as odds ratio (OR) > 1 and the protective factor was OR < 1. The multivariable regression model was established in three steps. Firstly, univariate analyses were performed to demonstrate which patient variables correlated with the presence of HPV infection with a significance of P < 0.05, and the analysis of variance (ANOVA) test and all variables were significantly different in the model (P < 0.05). Next, non-significant variables (P > 0.05) were removed, and stepwise regression was performed using the forward and backward method. Finally, the variables demonstrated as significant (P < 0.05), including age, a vaginal microecology test sample positive for fungal infection, age at first sexual intercourse, age at first childbirth, and alcohol consumption were included in the model. Analyses were performed using tenfold cross-validation, that the data set is divided into 10 parts, including 9 parts as training data and 1 part as test data. The final data is generated area under the curve (AUC) values of the receiver operating characteristic (ROC) curves. This was then used to determine the model's classification ability, and AUCs were compared to assess prediction accuracy. A P value of < 0.05 was considered statistically significant.The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Supplementary Information.