Despite great progress in antiretroviral treatment (ART) access in recent decades, HIV incidence remains high in sub-Saharan Africa. We investigated the role of individual and healthcare supply-related factors in HIV transmission risk in HIV-positive adults enrolled in 19 HIV services in the Centre and Littoral regions of Cameroon.Factors associated with HIV transmission risk (defined as both unstable aviremia and inconsistent condom use with HIV-negative or unknown status partners) were identified using a multi-level logistic regression model. Besides socio-demographic and behavioral individual variables, the following four HIV-service profiles, identified using cluster analysis, were used in regression analyses as healthcare supply-related variables: 1) district services with large numbers of patients, almost all practicing task-shifting and not experiencing antiretroviral drugs (ARV) stock-outs (n = 4); 2) experienced and well-equipped national reference services, most practicing task-shifting and not experiencing ARV stock-outs (n = 5); 3) small district services with limited resources and activities, almost all experiencing ARV stock-outs (n = 6); 4) small district services with a wide range of activities and half not experiencing ARV stock-outs (n = 4).Of the 1372 patients  reporting sexual activity in the previous 12 months, 39% [min-max across HIV services: 25%-63%] were at risk of transmitting HIV. The final model showed that being a woman , not having an economic activity , having at least two sexual partners , reporting disease symptoms at HIV diagnosis , delayed ART initiation  and not being ART treated  were all associated with HIV transmission risk. Conversely, longer time since HIV diagnosis was associated with a lower risk of transmitting HIV . Patients followed in the third profile had a higher risk of transmitting HIV  than those in the first profile.Healthcare supply constraints, including limited resources and ARV supply chain deficiency may impact HIV transmission risk. To reduce HIV incidence, HIV services need adequate resources to relieve healthcare supply-related barriers and provide suitable support activities throughout the continuum of care. Despite great progress in implementing innovative HIV programs over the last two decades, HIV incidence remains high in many countries worldwide . In 2020There is wide consensus on the effectiveness of combined HIV prevention initiatives, which comprise various behavioral, biomedical and structural interventions, to prevent HIV transmission (2). One key biomedical intervention is early antiretroviral treatment (ART) initiation which has been shown to dramatically reduce HIV-related mortality and morbidity as well as HIV transmission risk –7. The bIn order to enhance efforts to reduce HIV transmission, in 2014 UNAIDS launched the “90-90-90” care continuum targets which aimed to diagnose HIV infection in 90% of all people living with HIV (PLHIV), initiate ART in 90% of diagnosed PLHIV, and achieve VLS in 90% of PLHIV treated with ART, all by 2020 . In 2021However, several barriers continue to hamper the achievement of these targets in low- and middle-income countries (LMIC), including weaknesses in healthcare supply in health systems, primarily a lack of equipment and of human resources . AchieviIn order to assess the capacity of national programs to reach the third UNAIDS 95 target, it is essential to monitor two key related indicators: VLS and HIV transmission risk. To date, most studies measuring HIV transmission risk in various populations and contexts have used behavioral characteristics as outcome variables (mainly knowledge of one’s own HIV status and inconsistent condom use) –16. OnlyAccordingly, we aimed to: i) measure the prevalence of HIV transmission risk in HIV-positive patients enrolled in the Cameroonian ART access program in the country’s Centre and Littoral regions, and ii) investigate the roles of both individual and healthcare supply-related factors in HIV transmission risk.Cameroon is an LMIC in Central Africa affected by a generalized HIV epidemic with a mean estimated prevalence rate in 2018 of 2.7% in adults (aged 15–49 years), and large disparities according to gender, region and urban area . The higCreated in 2001, the national ART access program initially led to the implementation of Accredited Treatment Centers, first in the national reference hospitals in Yaoundé and Douala, and then in the regional hospitals of the country’s eight other regions. From 2005 onwards, ART delivery services were extended to the district hospital setting, with the setting up of HIV Management Units. The Cameroon national authorities decided to provide ART for free in 2007 and to remove all user fees for HIV care in 2019 . Thanks In its latest National Strategic Plan for HIV/AIDS and STIs for the period 2018–2022 , CamerooWe used data from the cross-sectional survey EVOLCam (ANRS 12288) which was conducted in 19 HIV services in Cameroon’s Centre (n = 11) and Littoral regions (n = 8) between April and December 2014 to study evolutions in the national ART program through a comparison with the 2006–2007 ANRS-12116 EVAL survey . The EVOBriefly, eligible PLWH (≥21 years old and HIV diagnosed >3 months) attending an outpatient consultation in one of the 19 participating HIV services were randomly selected and informed about the study. Patients willing to participate provided written informed consent before data collection. First, a standardized medical questionnaire was completed during the consultation by healthcare providers. The following clinical data were obtained from patient examinations and retrospective medical files: dates of HIV diagnosis and ART initiation, WHO clinical stage of HIV infection at ART initiation and at the time of the study, CD4 count at ART initiation, drug regimen at the time of the study, body mass index and any history of tuberculosis and hepatitis B co-infection and related diagnosis date. Second, patients answered a face-to-face questionnaire administered in a private room by trained independent interviewers which collected data on demographic, socioeconomic, behavioral, psychosocial and domestic information. More specifically, a series of questions were asked on adherence to ART, perceived health and HIV-related stigma as well as alcohol consumption and sexual behaviors during the 12 months prior to the study . The questionnaire is available as supplementary material in Fiorentino et al., 2021  for continuous variables. We also described the variability of the outcome overall across all 19 HIV services, and according to each HIV service and each HSP.σ2(u0) = 0.09; p = 0.032), confirming the relevance of using a multilevel model. We also computed the estimated intra-class correlation coefficient (ICC), which represents the proportion of the inter-class variance compared to the total variance . It was estimated at 0.027 indicating that 2.7% of the outcome’s variance was due to differences between HIV services. Individual-related factors in the level-1 model were then selected using a stepwise backward procedure. Only significant individual variables with a p-value <0.05 were retained, except age which was kept in order to control for key demographic characteristics. Finally, the HSP variable was introduced as a level-2 variable to obtain the final multilevel model. Model fit was assessed using Akaike’s Information Criterion (AIC). Analyses were performed using SAS (version 9.4), RStudio (version 1.1.453) and Stata/SE version 14.2 .We investigated the factors associated with HIV transmission risk using a multilevel logistic regression model. This allowed us to accurately assess the effects of individual characteristics (level 1) and of structural characteristics, that is to say the healthcare supply-related characteristics described in the four HSP categories above and summarized by the HSP variable (level 2) . The mulOf the 2130 HIV-positive patients enrolled in the EVOLCam survey, 748 (35%) were excluded from the present analysis because they did not report any sexual partner in the 12 months prior to the survey interview. A further 10 (1%) were also excluded because of missing data on viral load and/or ART adherence. The study population therefore comprised 1372 PLHIV reporting at least one sexual partner during the 12 months prior to the survey, and with complete data for the study’s outcome see .Patients were predominantly women (67%) and median age [Interquartile (IQR)] was 39 –44 yearsOverall, 84.0% (1152/1372) of the study population had unstable aviremia and 45.9% (629) reported inconsistent condom use with at least one negative or unknown HIV status sexual partner See . As a reThe proportions of patients at risk of transmitting HIV varied across HIV services from 25% to 63% . In termIndividual and healthcare supply-related factors associated with HIV transmission risk in univariate and multivariable analyses are presented in With regard to healthcare supply-related characteristics, only patients followed in HSP3 had a higher risk of transmitting HIV than those in HSP1 . .Our study highlighted that a high proportion (39%) of PLHIV attending HIV services in the Centre and Littoral regions in Cameroon had a risk of transmitting HIV, defined here as both unstable aviremia and inconsistent condom use with at least one of the two (if more than one partner reported) most recent partners of negative or unknown HIV status in the previous 12 months. In addition, multivariable models indicated that at-risk patients were more likely to experience a sub-optimal HIV care cascade with late HIV diagnosis (when symptomatic), no ART initiation, and late ART initiation (>2 months after HIV diagnosis) all independently associated with HIV transmission risk. The prevalence of HIV transmission risk also varied greatly across HIV services (from 25% to 63%), suggesting that besides individual factors, healthcare supply characteristics may also play a key role in HIV transmission risk, specifically the implementation of task-shifting practices and support activities, as well as ARV availability.The prevalence of HIV transmission risk in our study (39%) was higher than values found in three previous studies conducted in Sub-Saharan Africa using a relatively similar comprehensive approach which combined viral load with inconsistent condom use to define the risk of HIV transmission , only half (54%) reported consistent condom use with that (those) partner(s). In addition, although most patients (82%) were on ART for at least six months, only a minority were highly adherent (26%) and had detectable viral (28%), resulting in a high proportion (84%) of study patients with unstable aviremia status. These findings reveal the huge challenges still facing HIV services in Cameroon to achieve the third ‘95’ UNAIDS target  and to prevent new HIV infections. They also highlight the need to further develop supportive adherence services  which have been shown to have a positive impact on adherence and viral suppression among PLHIV in LMIC .Our findings also demonstrate the decisive impact of the two first steps of the HIV care cascade  on the risk of HIV transmission and reflect results from other studies which highlighted that early difficulties encountered by HIV-positive patients in the continuum of care may compromise future treatment success –48. In aFinally, using detailed information on resources and management characteristics of the 19 HIV services participating in EVOLCam, we identified four HIV service profiles to explain the differences observed in terms of HIV transmission risk. After adjustment for individual factors, the multivariable analysis showed that patients followed in HSP3 had a greater risk than those followed in HSP1, while no significant differences were found for HSP2 and HSP4. The description of each profile’s structural characteristics enabled us to identify healthcare supply-related factors possibly explaining this higher risk in HSP3 than in HSP1.First, although HIV services in HSP3 had a low number of medical staff (only 1/6 had more than 4 physicians and 2 nurses), task shifting for consultation was rarely practiced. Conversely HSP1 services had slightly more staff  and most practiced task-shifting and educational counselling. Several studies conducted in sub-Saharan Africa, including in Cameroon, have highlighted that task-shifting from physicians to nurses as well as providing educational counselling, brings substantial benefits to the continuum of HIV care , 55, 56.Second, in HSP3, more HIV services reported limited access to CD4 count machines and ARV stock-outs than in HSP1. The detrimental effect of the latter problem on treatment adherence and interruption , 58, andSeveral study limitations should be acknowledged. First, the EVOLCam survey was conducted in only two  of Cameroon’s 10 regions and is therefore not representative of the whole population of PLHIV enrolled in the Cameroonian ART access program. However, these two regions were among those with the highest HIV prevalence rate at the time of the study  and incSecond, adherence and sexual behaviors were assessed using patients’ self-reports, which may be affected by social desirability bias. However, this bias was limited by the administration of face-to-face questionnaires by independent interviewers trained in the use of non-judgmental approaches. In addition, we used a validated ART adherence scale  which haThird, Cameroon’s national ART access program has seen several substantial changes since 2014 (our study period), including the adoption of the test-and-treat strategy in 2016 and the implementation of free HIV care in the public sector in 2019. These two policies have brought about major progress in terms of ART access, with ART coverage standing at 77% in 2020 . HoweverDespite these limitations, our findings have important implications for HIV healthcare policy.First, they suggest that the whole HIV continuum of care should be considered to improve early HIV diagnosis, engagement in care and treatment, with a view to increasing the likelihood of achieving viral suppression and limiting HIV transmission risk. It is also essential to identify patients at risk of transmitting HIV at each step of the continuum to provide them timely support, thereby enabling them to cope with their difficulties and better manage their HIV infection. As viral load measurement is rarely available in routine care in sub-Saharan Africa, targeting patients with low adherence and/or risky sexual behaviors may be an effective approach, especially given that low adherence to ART was found to be predictive of risky sexual behaviors during the first year after ART initiation in Cameroon . Second,We used a multi-level logistic regression model to disentangle effects of demand-side factors from those of supply-side factors in order to gain a better understanding of the role played by each of these factors on HIV transmission risk in the context of the Cameroonian ART access program. Our findings suggest that early HIV testing and rapid ART initiation may positively influence the engagement of HIV-positive individuals in prevention strategies. They also highlight that HIV transmission risk was higher in HIV services which had limited human resources and did not practice task-shifting, as well as in those with low technical capacity to ensure regular supply of ARV. Adequate resources should be allocated to all HIV services to both relieve healthcare supply-related barriers and enable them to provide adequate support activities to patients throughout the continuum of care, in order to optimize the individual and public health benefits of ART on the reduction of new HIV infections.S1 TableAbbreviations: ARV = Antiretroviral drugs HSP: HIV-service profile.(DOCX)Click here for additional data file.S2 TableAbbreviation: NC: not concerned; MD: missing data; ART: antiretroviral treatment; HSP: HIV-service profile.(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file. 21 Sep 2021 <div> PONE-D-21-22603 </div>  <div> Individual and healthcare supply-related HIV transmission factors in HIV-positive patients enrolled in the Cameroonian antiretroviral treatment access program (ANRS-12288 EVOLCam survey) </div>  <div> PLOS ONEDear Dr. Coulaud, </div>  <div>   </div>  <div> Thank you for submitting your manuscript. 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Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. Since the regions of Littoral and Centre are not representative of the entire country, please insert Littoral and Centre Regions in the title of the manuscript.2. Line 6: These are objectives, not aims3. Please rewrite the introduction to update the situation in Cameroon (with HIV figures), and show the link of this work with new HIV national strategic plan.4. The data were collected between April and December 2014, now 7 years back. Please discuss the validity of the results in 2021, because some progresses have been made so far by the Government of Cameroon.5. Briefly describe the EVOLCam survey.6. In line 428, the authors stated the “Due to French law there are restrictions on publicly sharing the data of this study. French law requires that everyone who wishes to access cohorts data or clinical study data on humans must ask the French data protection authority”. However, the study was conducted in Cameroon and received ethical approval from Ethical Board of Cameroon, not French one. How was the data sharing agreement with Cameroon partners? Please clarify this issue.7. Are there any standard definitions of “the risk of transmitting HIV” and “Unstable aviremia”? if so give references otherwise justify.8. Please note that the Ministry of Public Health gives the Admistrative Autorisation while the ethical approval is given by the “Comité National d’Ethique de la Recherche pour la Santé Humaine (CNERSH)”9. The study was in 19 HIV services in Cameroon’s Center and Littoral, please give the total number of HIV services in these two regions.10. The justification of the choice of the two regions is not clear as the South Region has the highest HIV prevalence rate.11. 1 372 should clearly appear in Figure 2, this is because, 10 the number missing data on viral load and/or ART adherence is not included there.12. Please insert the goodness of fit of the final selected multilevel model.13. Interpret the random component summarized by inter-class variance.14. Are there clear reasons why the final multivariable model was adjusted for age only, not for both age and sex as mentioned in line 275?15. In the Abstract, add p-values to CI of Adjusted ORs, and IQR to the median of age.Minor16. Affiliation 4 is « Faculté de Médecine et de sciences biomédicales, Université de Yaoundé 1, Yaoundé, Cameroun »17. Rewrite line 101Reviewer #2: This paper is easy to read and relevant within the epidemiological context of eradicating HIV pandemic in CameroonIt should be good for the authors to update some informations in the study settings on the prevalence of HIV in Cameroon line 94, in accordance with latest DHS 2018.In addition the context , regardless of TASP, should outline the notion of test and treat, and the model of service for ART delivery can be described following line 102...for instanceThe surprising fact in table 1 is the rate of patient not yet treated, assuming Cameroon at this stage was yet in the test and treat approach. Can the authors have a clear idea of why those patients were not under treatment as this can appear to be a confounding risk.Line 299-301 Explain the need of test and treat and not delaying ARTLine 324-326-emphazise and discuss supportive adherence counseling after ART initiationLine 328-331-Discuss test and treat strategy to address HIV care and treatment cascadeFinal discussion and recommendations can be more focused on the importance of test and treat, task shifting and ARV stocks management to prevent ARV stock outsReviewer #3: In terms of content/form, the document seems to me to be well written. The subject addresses an issue never before addressed in Cameroon to the best of my knowledge and worthy of interest for improving the national response against HIV in Cameroon. The ethical aspects are not well described, but all the required authorizations have been obtained. The methodology is simple and clear and the conclusions are linked to it.********** <font color="black"> 6. 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  14 Jan 2022Dr. Pierre-julien Coulaud, PhDAix Marseille University, INSERM, IRD, SESSTIM, Sciences Economiques & Sociales de la Santé & Traitement de l’Information MédicaleFaculté de Médecine Timone, 27 Boulevard Jean Moulin – 13005 Marseille, Francepierre-julien.coulaud@inserm.fr / pierre-julien.coulaud@bccsu.ubc.caEmails: Marseille, 10th December, 2021Manuscript Number PONE-D-21-22603Title: " Individual and healthcare supply-related HIV transmission factors in HIV-positive patients enrolled in the Cameroonian antiretroviral treatment access program (ANRS-12288 EVOLCam survey)"Dear Editor, Thank you very much for the evaluation of our revised manuscript for publication in PLOS ONE and for giving us the opportunity to submit a new revised version. We carefully considered and replied to all the editor and reviewers’ comments (see below). In addition, we made some minor corrections in the text (for English corrections and editing). All revisions made in the new version of the manuscript are highlighted in yellow. More specifically, we addressed the following three major points raised by the editor and reviewers. First, we revised our rational in the Introduction to better explain how our manuscript advances on our previous work . While this previous work was focused on understanding the role of healthcare supply-related factors on ART initiation delay (which correspond to the second UNAIDS target), our present manuscript investigates the association between these structural factors and HIV risk transmission, corresponding to the third UNAIDS target. We did not mention this publication in our first submission to your journal because this previous article was not published when we finalized and first submitted this manuscript in December 2020 . As some parts of our manuscript was overlapping with this previous work, we carefully revised this new version to remove the duplicated text and our references were updated accordingly.Second, we provided additional information regarding the survey and the ethical statement. Given that we used a similar approach for defining our HIV services profiles that in our previous publication , we reorganized our Methods and Results sections to limit similar extracts from our previous work and summarize the main findings. Regarding the access to the study questionnaire, we specified that they have been previously published as supplement materials in Fiorentino et al., 2021. We also revised our ethical statement to clarify that each participant was assigned with a patient identification number that was used to anonymize all data collection tools, including the datasets for conducting this analysis.Third, as suggested by reviewers, the discussion and limits section of our manuscript were modified to highlight the implications of the test-and-treat strategy on the HIV treatment-related outcomes . In addition, as our data were collected in 2014, we better discussed the recent evolutions of the Cameroonian ART Program as well as the relevance of our findings and public health policy recommendations in the current context. Finally, as suggested by the first reviewer, we also modified the title to specify the study area as follows: “Individual and healthcare supply-related HIV transmission factors in HIV-positive patients enrolled in the antiretroviral treatment access program in the Centre and Littoral regions in Cameroon (ANRS-12288 EVOLCam survey)”.We hope that this new version will be suitable for publication in PLOS ONE and remain at your disposal for any request you may have. Yours faithfully, Pierre-julien Coulaud, on behalf of the co-authors ANSWERS TO REVIEWERS' COMMENTSReviewer #1.1. Since the regions of Littoral and Centre are not representative of the entire country, please insert Littoral and Centre Regions in the title of the manuscript.   As suggested, we modified the title by inserting “Littoral and Centre regions” as follows: �“Individual and healthcare supply-related HIV transmission factors in HIV-positive patients enrolled in the antiretroviral treatment access program in the Centre and Littoral regions in Cameroon (ANRS-12288 EVOLCam survey)”2. Line 6: These are objectives, not aims   As suggested, we revised this sentence  as follows: �“In a previous study conducted in Cameroon, we showed that HIV service profiles, built using a cluster analysis of a wide range of healthcare supply-related characteristics, had different performances in terms of time to ART initiation [1]. In the present study, we used a similar approach to provide a better understanding of the role of supply-related factors, beside individual factors, on achieving the third UNAIDS target in Cameroon.”3. Please rewrite the introduction to update the situation in Cameroon (with HIV figures), and show the link of this work with new HIV national strategic plan.   We revised the Introduction section to update the last estimations and references available regarding the HIV epidemic and the 2025 UNAIDS testing and treatment targets. A detailed study context is also presented in the sub-section “Study setting” at the beginning of the Methods section (p. 5/6). This one has been revised to include the most recent HIV prevalence rate . �As suggested, we also clarified in the Introduction how our study objectives linked to the national HIV strategic plan priorities in Cameroon and provided in the “Study setting subsection” additional information on the priority strategies and interventions promoted by the national health authorities within this plan. Introduction : “In a previous study conducted in Cameroon, we showed that HIV service profiles, built using a cluster analysis of a wide range of healthcare supply-related characteristics, had different performances in terms of time to ART initiation [1]. In the present study, we used a similar approach to provide a better understanding of the role of supply-related factors, beside individual factors, on achieving the third UNAIDS target in Cameroon. This study also provided the opportunity to highlight challenges related to the implementation of the 2018-2022 Cameroonian National Strategic Plan for HIV/AIDS and STIs which aims to reduce the number of new HIV infections by 60% and achieve VLS in 92% of PLHIV on ART in Cameroon by 2022 [2].”Study setting section, Methods :“The Cameroon national authorities decided to provide ART for free in 2007 and to remove all user fees for HIV care in 2019 [3]. Thanks to this continuously developing HIV strategy, the total number of PLHIV on ART increased from 17,940 in 2005 to 145,038 in 2014 and 367,871 in 2020 .In its latest National Strategic Plan for HIV/AIDS and STIs for the period 2018-2022 [2], Cameroonian health authorities promote a set of priority strategies and interventions including i) strengthening HIV prevention, ii) scaling-up HIV testing and ART access through the decentralization of HIV care, task shifting, and the involvement of community-based organizations, iii) ensuring the permanent availability of laboratory equipment, antiretroviral drugs and medicines of opportunistic infections.” 4. The data were collected between April and December 2014, now 7 years back. Please discuss the validity of the results in 2021, because some progresses have been made so far by the Government of Cameroon.   We acknowledge that our study is dated and that some progresses have been made so far by the Government of Cameroon, including the adoption of the test-and-treat strategy and the gratuity of all HIV care. However, our policy recommendations seems to be still relevant in the current Cameroonian context for the following two main reasons: first, the financial resources of the Cameroonian ART programme have not sufficiently increased between 2014 and 2020 [6] to fill the structural gaps highlighted in our manuscript. Second, the test-and-treat strategy and the gratuity of HIV care adopted by the Government led to a large increase in the numbers of PLHIV initiating ART. The consequence is a greater burden on the health system, especially on human resources which have less time to devote to patients [7]. HIV services are thus expected to initiate ART quickly in all newly diagnosed HIV-positive patients while continuing to provide clinical follow-up and psychosocial support to a growing number of patients already being treated. �These challenges have been recently documented in HIV services in Cameroon. In 2018, a qualitative study on patients’ satisfaction with ART services indicated that healthcare providers were facing a rapid increase in the volume of patients on ART, especially an increased number of asymptomatic patients who were more hesitant toward the benefits of ART [8]. During the interviews, PLHIV also expressed barriers to engage in HIV services such as experiencing long waiting time, poor reception and attitudes of some healthcare providers , poor coordination between HIV testing and treatment services, and lack of flexibility of the drug delivery system to patients’ specific needs [8]. A retrospective study also reported mixed results regarding the impact of the test and treat policy on HIV care continuum outcomes. Although higher ART uptake, and earlier ART initiation were found among patients who were enrolled after the implementation of the test and treat policy, lower ART retention was reported in this group compared to those who initiated before the implementation of this strategy [9].While major progress have been made to improve access to ART among PLHIV in Cameroon  [10], recent data  showed that approximately 70% of PLHIV were virally suppressed in 2020, suggesting that the risk of HIV transmission is still high in Cameroon and that the challenges highlighted in our study are still very much a reality. We therefore revised the Limitation section of the Discussion  to better discuss the recent evolutions in the Cameroonian ART programme and the validity of our results in this context, as follows: “Third, Cameroon’s national ART access program has seen several substantial changes since 2014 (our study period), including the adoption of the test-and-treat strategy in 2016 and the implementation of free HIV care in the public sector in 2019. These two policies have brought about major progress in terms of ART access, with ART coverage standing at 77% in 2020 [12]. However, this rapid and large increase in the number of ART-treated patients constitutes a huge burden on Cameroon’s healthcare system, especially in terms of human resources and drug supplies [13]. Recent studies in the country documented important patient-reported barriers to accessing HIV services, including long waiting times, poor patient reception in centers, poor coordination between HIV testing and ART services, long delays before ART imitation [8], and a higher risk of loss-to-follow-up among patients who initiated early ART [9]. In addition, the country’s “free access” policy generates a loss of income for healthcare facilities  which may negatively affect healthcare quality  when not offset by government subsidizing . Finally, the ongoing COVID-19 pandemic may result in financial resources being diverted, which may further exacerbate human resources shortages and inadequate ART supplies [10]. Recent literature [11] and the latest estimation of the proportion of PLHIV achieving VLS in Cameroon (approximately 70% in 2020), [12]) suggest that the risk of HIV transmission is still high in Cameroon and that the challenges highlighted in our study are still very much a reality.” 5. Briefly describe the EVOLCam survey.   As suggested, we described in greater details how and which data were collected in the EVOLCam survey in the sub-section “Study design and data collection” (see p. 6/7):�“We used data from the cross-sectional survey EVOLCam (ANRS 12288) which was conducted in 19 HIV services in Cameroon’s Centre (n=11) and Littoral regions (n=8) between April and December 2014 to study evolutions in the national ART program through a comparison with the 2006-2007 ANRS-12116 EVAL survey . The EVOLCam (ANRS 12288) study protocol is described in detail elsewhere . Briefly, eligible PLWH (≥21 years old and HIV diagnosed >3 months) attending an outpatient consultation in one of the 19 participating HIV services were randomly selected and informed about the study. Patients willing to participate provided written informed consent before data collection. First, a standardized medical questionnaire was completed during the consultation by healthcare providers. The following clinical data were obtained from patient examinations and retrospective medical files: dates of HIV diagnosis and ART initiation, WHO clinical stage of HIV infection at ART initiation and at the time of the study, CD4 count at ART initiation, drug regimen at the time of the study, body mass index and any history of tuberculosis and hepatitis B co-infection and related diagnosis date. Second, patients answered a face-to-face questionnaire administered in a private room by trained independent interviewers which collected data on demographic, socioeconomic, behavioral, psychosocial and domestic information. More specifically, a series of questions were asked on adherence to ART, perceived health and HIV-related stigma as well as alcohol consumption and sexual behaviors during the 12 months prior to the study . The questionnaire is available as supplementary material in Fiorentino et al., 2021 [20]. Third, a blood sample was taken to measure HIV viral load (only for patients ART treated >6 months) and CD4 cell count. All blood samples were analyzed by a reference HIV laboratory in Yaoundé. Finally, detailed data on the characteristics of the participating 19 HIV services were collected through interviews with hospital staff, in situ observations, and cross-validation with data recorded in HIV service activity reports. Specifically, the information obtained included: i) hospital’s general characteristics , ii) human resources working in the HIV service , iii) activity , iv) HIV service organization , v) technical resources . Furthermore, after having checked the issue of data access for people / institute(s) who are not member of the research team, we confirmed that there is no need to receive previous approval from the CNIL in France if data are fully anonymized. We therefore modified the “Availability of data and materials statement” (see p. 23) as follows: �Laurent.vidal@ird.fr and Christopher Kuaban at ckuaban@yahoo.fr). “Fully anonymized data are available on request made to the study investigators , various definitions were used in the existing literature to define HIV transmission risk. It is also important to note that this definition is constantly evolving with the progress in HIV care, the development of new prevention strategies , and the availability of individual HIV clinical characteristics . �Most of previous studies were focused on behavioural characteristics  [21–23] as information on viral load was more difficult to collect. In order to take into account the fact that multiple factors may influence the potential risk of transmission, our definition of HIV transmission risk was based on both behavioural characteristics  and biomedical factors  . This comprehensive approach of the risk of transmitting HIV allowed us to provide a more accurate estimation of the prevalence of HIV transmission risk in our study sample.A similar approach was used to define unstable aviremia. Previous studies often used the number of viral load copies (undetectable versus detectable) as the main indicator to consider a participant with unstable aviremia . However, it’s well known in the HIV literature that the level of adherence to ART play a critical role to help maintain a low viral load and be undetectable [27]. To capture this important aspect that reflects the engagement of PLHIV in HIV care, we considered as “unstable aviremia”: i) participants on treatment with a detectable viral load and ii) participants on treatment with an undetectable viral load but non-adherent to ART. For both definitions, we included references in this new version. We also specified our comprehensive approach in great details to highlight the importance of combining both behavioral and biomedical factors. Below are the changes made in the “Outcome” subsection in the Methods section (p. 8): “The study outcome was a binary variable describing the risk of transmitting HIV (yes versus no). No standard method exists to define the risk of HIV transmission. Accordingly, in order to define the outcome, we used the literature to develop a comprehensive approach which included both biomedical  and behavioral factors ) . More specifically, we defined the risk of transmitting HIV as a combination of both unstable aviremia and reporting inconsistent condom use either with the most recent  sexual partner (if only one partner declared), or with at least one of the two most recent sexual partners (if more than one partner declared) of negative or unknown HIV status. Unstable aviremia was defined as not currently being treated or on treatment for less than six months or on treatment for more than six months but with a detectable viral and/or poor adherence to ART [20]. The latter was defined as taking <80% of the prescribed drug doses or reporting treatment interruptions for at least two consecutive days in the four weeks prior to the survey [28]. Participants on treatment for more than six months with an undetectable viral load who were highly adherent to ART (defined as taking >80% of the prescribed drug doses in the four weeks prior to the survey) were considered to have stable aviremia.”8. Please note that the Ministry of Public Health gives the Admistrative Autorisation while the ethical approval is given by the “Comité National d’Ethique de la Recherche pour la Santé Humaine (CNERSH)”   Thank you for this comment. We revised our ethical statement (see p. 23) accordingly:�“This study was conducted in compliance with international and national regulations on ethics and research on people. It received administrative authorisation from the Ministry of Public Health in Cameroon and was approved by the Cameroonian National Ethics Committee . All participants were informed about the study’s objectives and its modalities and all provided written consent to participate. All individual data collected in the research were anonymized using a patient identification number; only this number was reported in the data collection tools and the databases used for analyses.”9. The study was in 19 HIV services in Cameroon’s Center and Littoral, please give the total number of HIV services in these two regions.   As suggested, we clarified the number of HIV services in each region  as follows: �“We used data from the cross-sectional survey EVOLCam (ANRS 12288) which was conducted in 19 HIV services in Cameroon’s Center (n=11) and Littoral regions (n=8) between April and December 2014 to study evolutions in the national ART program through a comparison with the 2006-2007 ANRS-12116 EVAL survey .”10. The justification of the choice of the two regions is not clear as the South Region has the highest HIV prevalence rate.   There are two main reasons of the choice of conducting the EVOLCam survey in the Centre and Littoral regions of Cameroon. First, those two regions were among those with the highest HIV prevalence rate at the time of the study  and include the country’s two main cities  which both also have a prevalence rate higher than the mean prevalence rate of the country. Second, these two regions had a relatively high number of HIV services , following the largest PLHIV populations in Cameroon at the time of the survey [30]. The justification of the choices of the two regions selected for the study is now provided in the limitation subsection in the Discussion  as follows:�“First, the EVOLCam survey was conducted in only two  of Cameroon’s 10 regions and is therefore not representative of the whole population of PLHIV enrolled in the Cameroonian ART access program. However, these two regions were among those with the highest HIV prevalence rate at the time of the study  and include the country’s two main cities . Accordingly, they were the most populated regions with the largest PLHIV populations [30]. Moreover, we selected a representative sample of existing HIV services in both regions and used a random selection procedure for participant inclusion. We therefore obtained quite a comprehensive picture of the Cameroonian ART access program in two key regions of the country.”11. 1372 should clearly appear in Figure 2, this is because, 10 the number missing data on viral load and/or ART adherence is not included there.   As suggested, we specified the total number of patients included in our study analysis and we simplified the presentation of the number of patients with missing data on viral load and/or ART adherence in the revised Figure 2.�12. Please insert the goodness of fit of the final selected multilevel model.   The goodness of fit of our final multilevel was assessed using Akaike’s Information Criterion (AIC). We provided this information in the sub-section “Statistical Analysis” of the Methods  as well as in the notes of the Table 1 (see p.15).�Sub-section “Statistical analysis”, Methods: “Model fit was assessed using Akaike’s Information Criterion (AIC)”Table 1: “AIC of the final selected model: 1746.74”.13. Interpret the random component summarized by inter-class variance.   As suggested, we provided in the sub-section “Statistical Analysis” of the new version of the manuscript further details regarding the interpretation of the inter-class variance. We also computed and interpreted the intraclass correlation coefficient , as follows: �“Initially, we estimated the empty model (without any explanatory variables) to provide an estimation of the inter-class variance, which was small but significantly different from 0 , confirming the relevance of using a multilevel model. We also computed the estimated intra-class correlation coefficient (ICC), which represents the proportion of the inter-class variance compared to the total variance . It was estimated at 0.027 indicating that 2.7% of the outcome’s variance was due to differences between HIV services.”14. Are there clear reasons why the final multivariable model was adjusted for age only, not for both age and sex as mentioned in line 275?   Our final multivariable model was adjusted for both age and gender. As described in the sub-section “Statistical Analysis” , only significant individual variables with a p-value <0.05 were retained in our final model, which was the case for gender (p<0.001) but not for age (p>0.05). Given that age is a key demographic characteristic, we forced this variable in the model. We therefore specified in the text (lines 266-267) that our final model was adjusted by age but gender is also included in the final model as it is significantly associated with the HIV transmission risk.�15. In the Abstract, add p-values to CI of Adjusted ORs, and IQR to the median of age.   The Results section of the Abstract (see p. 1/2) has been revised as suggested: �“Results: Of the 1372 patients  reporting sexual activity in the previous 12 months, 39% [min-max across HIV services: 25%-63%] were at risk of transmitting HIV. The final model showed that being a woman , not having an economic activity , having at least two sexual partners , reporting disease symptoms at HIV diagnosis , delayed ART initiation  and not being ART treated  were all associated with HIV transmission risk. Conversely, longer time since HIV diagnosis was associated with a lower risk of transmitting HIV . Patients followed in the third profile had a higher risk of transmitting HIV  than those in the first profile.” 16. Affiliation 4 is « Faculté de Médecine et de sciences biomédicales, Université de Yaoundé 1, Yaoundé, Cameroun »   This error has been corrected in the revised version (see Title page): �“Faculté de Médecine et de sciences biomédicales, Université de Yaoundé 1, Yaoundé, Cameroun”17. Rewrite line 101   As suggested, we rewrote this sentence (see p.5) as follows:�“The Cameroon national authorities decided to provide ART for free in 2007 and to remove all user fees for HIV care in 2019 [3].”Reviewer #2.18. This paper is easy to read and relevant within the epidemiological context of eradicating HIV pandemic in Cameroon. It should be good for the authors to update some information in the study settings on the prevalence of HIV in Cameroon line 94, in accordance with latest DHS 2018.   As suggested, we updated the estimation of HIV prevalence rate according to the 2018 Demographic and Health Survey conducted in Cameroon and provided additional estimations on the HIV prevalence according some key characteristics  : �“Cameroon is an LMIC in Central Africa affected by a generalized HIV epidemic with a mean estimated prevalence rate in 2018 of 2.7% in adults (aged 15-49 years), and large disparities according to gender, region and urban area [31]. The highest prevalence rates are observed in women (3.4%), in urban areas (3.9%), and in the South (5.8%), East (5.6%), Adamaoua (4.1%), North-West (4.0%) and Centre (3.5%) [31] regions.” 19. In addition the context, regardless of TASP, should outline the notion of test and treat, and the model of service for ART delivery can be described following line 102...for instance   As suggested, we revised the Introduction (see p.3) to highlight the importance of the test-and-treat strategy as a key component of the U=U movement to reduce HIV infections and discussed the implications of such strategy on HIV risk transmission and quality of HIV services in the Discussion section (see p. 18-21).�Introduction :“One key biomedical intervention is early antiretroviral treatment (ART) initiation which has been shown to dramatically reduce HIV-related mortality and morbidity as well as HIV transmission risk . The beneficial effect of early ART on viral load suppression (VLS) led to the establishment of the U=U  movement, which is widely recognized for its importance in controlling the HIV epidemic [27]. This treatment as prevention approach evolved into the test-and-tread strategy, that is to say ART initiation immediately after HIV diagnosis, irrespective of CD4 count [35].”Discussion section:See p.18/19, lines 324-332:“This highlights the relevance of the test-and-treat strategy currently being rolled out in LMIC, including in Cameroon. In particular, the positive impact of this strategy on the third step of the HIV care cascade has recently been highlighted in four randomized population-based trials [36]. Furthermore, several studies have shown that ART initiation, including early initiation through the ‘test-and-treat’ strategy, leads to a significant decrease in sexual risk behaviors  thanks to greater opportunities for counselling and psychological support arising out of more frequent contact with HIV services [40]. Such a decrease may also contribute to lower the risk of HIV transmission.”See p.20/21, lines 372-389:“Third, Cameroon’s national ART access program has seen several substantial changes since 2014 (our study period), including the adoption of the test-and-treat strategy in 2016 and the implementation of free HIV care in the public sector in 2019. These two policies have brought about major progress in terms of ART access, with ART coverage standing at 77% in 2020 [12]. However, this rapid and large increase in the number of ART-treated patients constitutes a huge burden on Cameroon’s healthcare system, especially in terms of human resources and drug supplies [13]. Recent studies in the country documented important patient-reported barriers to accessing HIV services, including long waiting times, poor patient reception in centers, poor coordination between HIV testing and ART services, long delays before ART imitation [8], and a higher risk of loss-to-follow-up among patients who initiated early ART [9]. In addition, the country’s “free access” policy generates a loss of income for healthcare facilities  which may negatively affect healthcare quality  when not offset by government subsidizing . Finally, the ongoing COVID-19 pandemic may result in financial resources being diverted, which may further exacerbate human resources shortages and inadequate ART supplies [10]. Recent literature [11] and the latest estimation of the proportion of PLHIV achieving VLS in Cameroon (approximately 70% in 2020), [12]) suggest that the risk of HIV transmission is still high in Cameroon and that the challenges highlighted in our study are still very much a reality.” 20. The surprising fact in table 1 is the rate of patient not yet treated, assuming Cameroon at this stage was yet in the test and treat approach. Can the authors have a clear idea of why those patients were not under treatment as this can appear to be a confounding risk.   At the time of the EVOLCam survey , the test-and-treat strategy was not yet implemented in Cameroon (this strategy was adopted in June 16 [41]). Therefore, a subset of participants  were not treated because they may not be eligible for ART initiation at the time of the survey. Those patients were considered at risk of transmitting HIV if they reported inconsistent condom use with at least one partner of negative or unknown HIV status. In addition, specific sub-categories for patients not treated were created for the “time between HIV diagnosis and ART initiation” and “CD4 count at ART initiation” variables in our univariate and multivariable analysis . We specified in the Discussion section that the “test-and-treat strategy” was adopted in 2016 and discuss its potential impact on the study findings (see p.18-21). See also our responses to the comments #4 and #19 of the reviewer #1.�21. Line 299-301 Explain the need of test and treat and not delaying ART   As suggested, the importance of the test and treat strategy to improve the HIV care cascade has been emphasized in the Discussion, as follows : �“This highlights the relevance of the test-and-treat strategy currently being rolled out in LMIC, including in Cameroon. In particular, the positive impact of this strategy on the third step of the HIV care cascade has recently been highlighted in four randomized population-based trials [36]. Furthermore, several studies have shown that ART initiation, including early initiation through the ‘test-and-treat’ strategy, leads to a significant decrease in sexual risk behaviors  thanks to greater opportunities for counselling and psychological support arising out of more frequent contact with HIV services [40]. Such a decrease may also contribute to lower the risk of HIV transmission.”22. Line 324-326-emphazise and discuss supportive adherence counseling after ART initiation   As suggested, we provided more details about the implications of our findings regarding the low level of adherence to ART , as follows: �“These findings reveal the huge challenges still facing HIV services in Cameroon to achieve the third ‘95’ UNAIDS target  and to prevent new HIV infections. They also highlight the need to further develop supportive adherence services  which have been shown to have a positive impact on adherence and viral suppression among PLHIV in LMIC [42].” 23. Line 328-331-Discuss test and treat strategy to address HIV care and treatment cascade   As suggested, we outlined in the Discussion section the positive impact of the “test-and-treat” strategy on the two last steps of the HIV care cascade  as follows : �“In particular, the positive impact of this strategy on the third step of the HIV care cascade has recently been highlighted in four randomized population-based trials [36].”However, as the implementation of this public health strategy results in a rapid and large increase in the number of ART-treated patients in HIV services, human resources shortages and inadequate ART supplies may be exacerbated when funding is not sufficient to face the increased demand. In our Limitations section , we therefore discussed the negative indirect effects of implementing “test-and-treat” on the delivery and quality of HIV care services. Two recent surveys in Cameroon indicated that the test-and-treat strategy may increase the risk for PLHIV of experiencing structural barriers to access HIV services  and being loss-to-follow-up after early ART initiation . These findings provide a more nuanced picture of the impact of the “test-and-treat” strategy on the HIV care continuum which is important to surface in order to better understand the critical role played by healthcare supply-related factors. 24. Final discussion and recommendations can be more focused on the importance of test and treat, task shifting and ARV stocks management to prevent ARV stock outs   We revised the Discussion section by providing additional information on the test and treat strategy (see responses to comments #23). We also provided further details on how to best adapt ARV stocks management in HIV services using previous findings from Cameroon , as follows:� “A previous study in Cameroon recommended implementing a set of harmonization and coordination procedures, as well as continuous quality control monitoring throughout the whole ARV supply chain to ensure permanent availability of these drugs for PLHIV [43].”Reviewer #3.25. In terms of content/form, the document seems to me to be well written. The subject addresses an issue never before addressed in Cameroon to the best of my knowledge and worthy of interest for improving the national response against HIV in Cameroon. The ethical aspects are not well described, but all the required authorizations have been obtained. The methodology is simple and clear and the conclusions are linked to it.   Thank you for your comment. Our Methods section has been revised with further details regarding the study design and data collection as well as with some ethical issues : �“Briefly, eligible PLWH (≥21 years old and HIV diagnosed >3 months) attending an outpatient consultation in one of the 19 participating HIV services were randomly selected and informed about the study. Patients willing to participate provided written informed consent before data collection. First, a standardized medical questionnaire was completed during the consultation by healthcare providers. The following clinical data were obtained from patient examinations and retrospective medical files: dates of HIV diagnosis and ART initiation, WHO clinical stage of HIV infection at ART initiation and at the time of the study, CD4 count at ART initiation, drug regimen at the time of the study, body mass index and any history of tuberculosis and hepatitis B co-infection and related diagnosis date. Second, patients answered a face-to-face questionnaire administered in a private room by trained independent interviewers which collected data on demographic, socioeconomic, behavioral, psychosocial and domestic information. More specifically, a series of questions were asked on adherence to ART, perceived health and HIV-related stigma as well as alcohol consumption and sexual behaviors during the 12 months prior to the study . The questionnaire is available as supplementary material in Fiorentino et al., 2021 [20]. Third, a blood sample was taken to measure HIV viral load (only for patients ART treated >6 months) and CD4 cell count. All blood samples were analyzed by a reference HIV laboratory in Yaoundé.” We also revised our ethical statement to provide additional details about the access to the data (see p.24), as follows: “This study was conducted in compliance with international and national regulations on ethics and research on people. 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Babesia and Theileria) and rickettsia (Anaplasma) of cattle in Jammu region (North India) using microscopy and Polymerase Chain Reaction (PCR). Hematology, microscopy and PCR based prevalence studies were undertaken with 278 whole blood samples from cattle. Molecular prevalence studies were followed by genetic characterization of the isolates of Babesia, Anaplasma and Theileria spp. based on 18S rRNA, 16S rRNA and Tams1 gene, respectively. The data related to metrology and epidemiological variables like temperature, rainfall, season, age and type of livestock rearing was analyzed and correlated with occurrence of disease by statistical methods.The present study was aimed at establishing the prevalence, epidemiology and molecular characterization of major haemoprotozoons , Anaplasma marginale.  and Theileria annulata (MT113479) provided a representative data of the isolates circulating in the region and their proximity with available sequences across the world.The prevalence based on microscopy was 12.9% (36/278) whereas PCR recorded 30.22% (84/278) animals positive for haemoparasitic infections. All the samples found positive by microscopy were also recorded positive by PCR. Thus the study revealed prevalence of Babesia bigemina, Anaplasma marginale and Theileria annulata circulating in the region.Despite holding much significance to the animal sector, comprehensive disease mapping has yet not been undertaken in several parts of India. The present study provides a blue print of disease mapping, epidemiological correlations and genomic diversity of The online version contains supplementary material available at 10.1186/s12917-021-02915-9. Babesia bovis and Babesia bigemina [Babesia bigemina, transmitted by brevirostrate tick, Rhipicephalus (Boophilus microplus) is usually associated with high parasitemia. Anaplasmosis is a rickettsial disease transmitted biologically through ticks mainly by Rhipicephalus (Boophilus) microplus [A. marginale infection has been recorded in livestock [Theileria species that infect bovines in India are T. annulata and T. orientalis, and both species are transmitted by Hyalomma anatolicum. The most common clinical signs observed in TBDs are fever, inappetence and mortality, with peculiar hemoglobinuria and anemia for bovine babesiosis [India is primarily an agricultural country and the losses incurred as a result of tick borne diseases (TBDs) to livestock in India are huge, estimated to be 8.7 million USD, predominantly affecting the small and marginal farmers. Among the various tick transmitted diseases babesiosis, theileriosis, anaplasmosis and ehrlichiosis are the significant ones that drive attention. Babesiosis is considered as economically important tick-borne haemoprotozoan disease in tropical and subtropical countries, the most prevalent species being bigemina . Babesiaicroplus . It is eicroplus , 4 whileivestock –7. The TConventional microscopy is often used for the diagnosis of these diseases  but, cryThe present study was aimed to generate baseline data about the prevalence of tick borne haemoprotozoan and rickettsial infections in cattle in Jammu province of North India, based on conventional and molecular techniques, assess the risk factors and haematological alterations associated with the disease. Moreover, phylogenetic relatedness and genetic diversity of the isolates infecting cattle was also investigated to aid in epidemiological surveillance.χ2 23.475, odds ratio, OR = 2.911, 95% CI = 1.886–4.491) as compared to microscopy. The prevalence of A. marginale was (46/278) 16.5% followed by Babesia bigemina  and T. annulata . Concurrent haemoprotozoan and rickettsial infections were detected in 9 (3.2%) animals   of animals positive for haemoparasites. Decreased milk production was found in 72/84 (85.71%) animals. Among 31 animals showing symptoms of pica, 30 were found positive for anaplasmosis.p = 0.629) between male and female animals. Unorganised farms without proper flooring, cracked walls and poor sanitation showed significantly (p = 0.007) higher prevalence of infection  as compared to organised farms  compared to indigenous animals (15.3%), reiterating the fact that cross bred animals are at higher risk than native breeds. The probability of occurrence of infections in monsoon was 16.56 times higher than winter. The correlation of environmental variables with the prevalence presented a clear picture of epidemiological interventions in the disease occurrence  followed by 1–3 years  and < 1 year of age . The overall prevalence did not vary significantly  were compared with healthy control (n = 10) animals. A significant decrease in the Hb (p = 0.013), PCV (p = 0.001) and RBC (p = 0.027) count of animals with babesiosis was recorded  of Babesia spp. was 99.8% identical to Kathua (MN567603), Udhampur (MN566924.1) and Meghalaya (KF606864.1) isolates of Babesia bigemina and had marked similarity with B. bigemina from Argentina (HQ688688.1) and USA (MH050387.1). At position 363 C was replaced with G in Udhampur and Kathua isolates , revealed 100% nucleotide identity with published sequences of A. marginale of French (MN317256.1), Iranian (MK310488.1) and Vietnamese (MH686047.1) origin. The only sequence of T.annulata from Jammu region (MT113479) was found to be 99% similar to Indian isolates from Izatnagar (MF346013.1) and Hissar (AF214840.1). In Jammu isolate at position 137 C was replaced by A, A by G (607) and C by A (611) in comparison to Izatnagar isolate  on the phylogenetic tree  forming separate Indian subclade , A. marginale  and low prevalence of Theileria annulata  can be attributed to Rhipicephalus (Boophilus) microplus being the only tick which infests the bovines of Jammu region [Anaplasma is also associated with a long term/life long carrier state [H. anatolicum [T. annulata [H. anatolicum), considerably low prevalence of theileriosis was found in union territory of Jammu and Kashmir [In developing countries like India considerable economic losses occurs in large and small holding livestock productivity farming system due to TBDs . Prevaleu region . A thorou region –15. Anaper state  and so t 23.74%, 6/278 ander state . The aniatolicum  and T. a Kashmir . Livesto Kashmir  particulThere were no uncharacteristic clinical signs in the animals found positive for haemoprotozoan and rickettsial infection. High temperature, pale mucous membranes and decrease in milk production were however prominent signs. The endogenous pyrogens released in the blood due to cellular lysis lead to fever and consequently, inappetence . Anemia p = 0.007), latter showing increased prevalence. These farms are Kuchha houses (non cemented) or made of bricks only, having cracks and crevices, with improper drainage and poor ventilation, favouring survival and breeding of vector ticks. Moreover, the owners of unorganized farms are comparatively uneducated, so lack of awareness regarding use of acaricidals and sustainable managemental practices against ticks was observed at the time of sample collection. The other determinants recorded for disease occurrence are age and breed of animal. Inverse age resistance shields the young population from clinical outbreak of diseases [The study suggests that outbreaks are likely to occur in the rainy and post rainy season due to interplay of epidemiological factors. During rainy season the epizootiological determinants such as ambient temperature and atmospheric humidity and microclimate of grazing lands are favourable for growth and development of ticks. This is unlikely to differ in other parts of country as well, based on earlier reports viz. 29.31%  and 58.5diseases . The higdiseases , 28. Alsdiseases , 29. Somdiseases , 31.Babesia spp. has been reported earlier using sequence information of 18S rRNA gene [B. bigemina isolates from North Eastern India with Argentina and Kenya rather than with China. Sequence information of A. marginale isolates from the present study revealed a marked divergence from A. capra, A. central and A. ovis, although, small length of nucleotide sequence couldn’t reveal any marked genetic heterogeneity from isolates of A. marginale across the world. In a study from south India, minimal heterogeneity was revealed within 16S rRNA and msp4 genes among the field isolates from Kerala [Intracellular haemoprotozoons are under constant pressure from the immune system of their hosts, leading to emergence of genetic variants . The genRNA gene , 34. In Theileria spp., is recombination during sexual reproduction [T. annulata parasites [T. annulata showed 99% nucleotide homology with Indian isolates and clustered together with an Indian isolate in the phylogenetic clade. Parasite diversity reports from India suggests that the Indian isolates were distributed into two groups along with other countries like Spain, Italy, Tunisia, Iran, Bahrain, Turkey and Iraq [The key mechanism responsible for genetic diversity among oduction . The Tamarasites , 37, howarasites –38. In tand Iraq , 40. TheIn conclusion the study provided a holistic picture about the prevalence of haemoprotozoon and rickettsial infection in cattle and its correlation with the environmental variables and epidemiological determinants. In addition, the hematological data may be presumed as a marker of health status of animal and its implications on the productivity of animal. The present study is probably the first report of molecular characterization of the haemoprotozoon and rickettsial infection from Jammu region of North India. It provides pioneering information about the circulating genotypes and possible diversities which are quintessential for developing vaccine and diagnostic strategies in future.oN 74.87 °E. To collect maximum number of samples, the expected prevalence of 20% with confidence limits of 95% and a desired absolute precision of 5% was considered. The number of samples thus calculated was adjusted for finite population [The study was conducted in Jammu province of Union Territory of Jammu and Kashmir, India, located at 32.73 According to Indian meteorological department Pune, Ministry of earth sciences, Government of India, seasonal variability in the region for the year can be classified into summer (March to June), monsoon/rainy (July to September), postmonsoon/post rains (October to November) and winter (December to February). Hence study period was divided as per climatic variables and correlations were drawn with environmental variables like temperature, relative humidity and rainfall. Metrological data was obtained from the metrology observatory installed at Division of Agrometeorology, SK University of agricultural sciences and technology of Jammu.3/μl), lymphocytes (%), monocytes (%), granulocytes (%), red blood cell count (RBC) (106/mm3), haemoglobin (Hb) (g/dl), platelet count (× 103/μl), mean corpuscular haemoglobin (MCH) (picogram), mean corpuscular haemoglobin concentration (MCHC) (g/dl), mean corpuscular volume (MCV) (femtolitre) and packed cell volume (PCV) (%).The various blood parameters of clinically suspected blood samples were measured with automatic blood analyser . The various attributes included white blood cell count (WBC)  following the manufacturer’s protocols. Concentration of isolated DNA was measured using spectrophotometer  and purity was checked in agarose gel electrophoresis (1.5% gel). Aliquots of extracted DNA were stored at − 20 °C until further use.Babesia spp., A. marginale and T. annulata targeting 18S rRNA, 16S rRNA and Tams1 gene, respectively [Each blood sample was subjected to three PCR reactions for detection of ectively  with somBabesia spp. initial denaturation at 94 °C for 5 min, followed by 35 amplification cycles . For A. marginale, initial denaturation at 94 °C for 5 min, followed by 35 amplification cycles . For T. annulata, initial denaturation at 95 °C for 5 min, followed by 37 amplification cycles . A final extension step at 72 °C for 15 min was followed after amplification cycles in all three reactions. Known positive genomic DNA of Babesia spp., A. marginale and T. annulata was used as positive control, while nuclease free water was used as negative control. The PCR products were electrophoresed in 1.5% agarose gel (Tris-borate-EDTA), incorporated with ethidium bromide (0.5 μg/ml) and visualized under transilluminator .The PCR reaction was carried out in 25 μl total volume containing 4 μl of template DNA, 2.5 μl of 10× PCR Green buffer , 0.5 μl of 10 mM dNTP, 0.5 μl of each forward and reverse primer (20 pmol/μl), 0.2 μl Dream Taq DNA polymerase and nuclease free water to make the volume 25 μl. Amplification was performed using a S1000 thermal cycler  under following conditions: For n = 6) generated in the study were primarily analysed using BioEdit software and submitted to GenBank. The sequences were compared with the available sequences in GenBank using BLAST program of NCBI. The sequences were aligned with clustal W programme of MEGA 6 software using gap opening penalty of 10 and gap extension penalty of 0.1 and 0.2 for the pair wise and multiple alignments, respectively. Phylogenetic trees were constructed using maximum parsimony (MP) with the tree–bisection–regrafting (TBR) algorithm and tested at 1000 bootstrap replications. The sequences were initially truncated at both ends, so as to obtain sequences that started and ended at the homologous nucleotide positions. Plasmodium falciparum (JQ627149), Rickettsia rickettsii (U11021.1) and Theileria lestoquardi (KY965146.1) sequences were used as outgroup members to root the respective trees.PCR amplified product of each selected isolate was gel purified using PCR clean up system . The eluted product was commercially sequenced in an automated DNA sequencer at Agrigenome Pvt. Ltd., Kochi, Kerala. Nucleotide sequences  using SPSS 16 software .Additional file 1.Additional file 2.Additional file 3.