This is a retrospective cohort study on patients living with HIV-1 infection (PLWH) followed at the Division of Infectious Diseases of the San Raffaele Hospital, with cirrhosis and HCC diagnosed between 1999 and 2018 and with an available AFP value at HCC diagnosis. The area under the receiver operating characteristic curve (AUC) was used to estimate the accuracy of baseline AFP in predicting death. Factors associated with the risk of death were identified using multivariable Cox proportional-hazards regression models. P = 0.003]. Other factors were HBV infection  and treatment allocation .  Overall, 53 PLWH were evaluated: 18 patients received a curative treatment  and 35 a noncurative treatment . Baseline AFP was predictive of death , and the optimal cut-off was 28.8 ng/mL. At multivariable analysis, BL AFP ≥28.8 ng/mL was associated with death [adjusted hazard ratio (aHR) 7.05, 95% CI 1.94–25.71  Our findings suggest that in PLWH AFP serum levels ≥28.8 ng/mL, HBV coinfection and treatment allocation represent predictive markers for death at the time of HCC diagnosis. Hepatocellular carcinoma (HCC) is the fourth most common cause of death worldwide.The high prevalence of coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV), the hepatotoxicity associated with antiretroviral therapy and alcohol abuse has made HCC a rapidly increasing cause of morbidity and mortality, currently accounting for up to 50% of liver-related deaths in PLWH –3.Nevertheless, the clinical course of HCC in an HIV-1-infected setting is not well defined yet, probably because of the heterogeneity in the characteristics of the studied groups, such as, for instance, differences in HCC aetiology, use of ART, and use of anticancer therapy.It has been shown that alpha-fetoprotein (AFP) levels correlate with mortality in patients with HCC in the nontransplant setting, across all aetiologies, and specifically in patients with chronic HCV infection . Alpha-fIt remains controversial if AFP can be used as an independent prognostic factor, with some authors believing that AFP is a prognostic marker affecting the long-term survival of people with HCC.The actual prognosis effect of AFP incorporated into a score or as independent marker is either unclear or limited in the PLWH population.Given that AFP is an inexpensive, simple, reliable, and widely available tool, it is important to examine the prognostic properties of AFP in an HIV-1-infected setting. Therefore, we decided to evaluate the effect of serum AFP at HCC diagnosis on survival in PLWH and to determine the factors associated with the risk of death.This is a retrospective cohort study on 84 PLWH with incident HCC diagnosed between November 1999 and May 2018; of these 84 PLWH, 53 had alpha-fetoprotein levels available at HCC diagnosis (baseline (BL) evaluation).Data were collected as part of routine clinical care and recorded in the database of the Division of Infectious Diseases of the San Raffaele Hospital (CSLHIV Cohort). The CSLHIV Cohort was approved by the ethics committee of the San Raffaele Hospital, Milan, Italy. On their first visit, patients provide written informed consent on the use of their data in scientific analyses. Recorded data are anonymized and managed according to the Good Clinical Practice.Serum AFP levels were required within 60 days of HCC diagnosis. All the other data were also collected at the time of HCC diagnosis. HCC diagnosis was based on imaging or on histologic criteria according to international guidelines [The individuals were staged according to Child–Turcotte–Pugh (CTP) functional class and Barcelona clinic liver cancer (BCLC) algorithm following standard prepublished methodology , 8.Treatment was differentiated in curative  and noncurative . Patients who underwent more than one type of treatment were categorized according to the following hierarchy: LT, LR, RABL, CRE, SOR, and BSC.In the analyses, the main exposure variable of interest was serum AFP levels at HCC diagnosis. The main outcome variable was time to death after HCC diagnosis.Results were expressed as median  or frequency (%). Continuous variables were compared using the Wilcoxon rank-sum test; categorical variables were compared using the chi-square or the Fisher's exact test, as appropriate. Imputation for missing data was not performed.The ability of baseline AFP in predicting death was determined by the area under the curve (AUC) of receiver operating characteristics (ROC) curves. The optimal cut-off value, predicting the presence or absence of death, was determined on the highest Youden index value (sensitivity + specificity-1). The diagnostic accuracy of this parameter was evaluated in terms of sensitivity, specificity, negative predictive value, and positive predictive value with the corresponding 95% confidence intervals (95% CIs) either at the optimal cut-off or at the 200 ng/mL cut-off levels according to EASL guidelines for HCC management . The 400Total accuracy was also assessed by the percentage of patients that were correctly classified according to the estimated optimal cut-off.Kaplan–Meier curves were calculated to estimate time to death according to baseline AFP ; curves were compared by the log-rank test.Two multivariable Cox proportional-hazards models were applied to compare strata of baseline AFP  with respect to death. Models included also tumour-related factors, treatment allocation, hepatic reserve, and clinical parameters related to the severity of HIV infection; the choice of covariates was based on current knowledge on factors with a suspected or an established effect on death.The results of these analyses were reported as adjusted hazard ratios (aHR) with the corresponding 95% confidence intervals (CIs). The assumption of the proportional hazards was examined.P values were considered statistically significant if less than 0.05. Statistical analyses were performed using SAS Software, release 9.4 .Two-sided n = 45 (85%)), and the main risk factor for HIV-1 infection was past or actual drug use (n = 26 (49%)).The baseline characteristics of the 53 individuals included in this study are summarized in At baseline, HIV viral load was suppressed (<50 copies/mL) in 43 (81%) PLWH.The majority of PLWH were hepatitis C carriers (36 (68%)) with HCV genotype (GT) available in 23 patients: GT3a was the predominant infecting virus , and the other GT were 1a in 5 patients, 1b in 2 patients, and 4 in 3 patients. Of these 36 patients with HCV infection, 12 were treated for chronic hepatitis C before HCC diagnosis: 8 received interferon-ribavirin with no response in 4 of them, while the remaining 4 patients received direct acting antivirals (DAAs) with sustained virological response.Of 17 patients with hepatitis B virus (HBV) infection, 3 patients had concomitant HCV and one coinfection with hepatitis Delta virus. Fourteen/17 (82%) HBV-infected patients were under treatment with nucleot(s)ide analogs with undetectable HBV-DNA levels (limit of detection 10 IU/mL).Child–Turcotte–Pugh (CTP) score was A in 40 (75.5%) PLWH, B in 9 (17%), and C in 4 (7.5%); BCLC stage was 0/A in 18 (34%) PLWH, B in 11 (21%), and C/D in 24 (45%) PLWH. The treatment allocation with regard to BCLC stage is summarized in In particular, 6 patients with terminal BCLC stage received sorafenib: one patient (CTP stage B) stopped treatment for liver decompensation, 3 patients (2 in CTP B and one CT P C) discontinued treatment for HCC progression, 1 patient (CTP C) was lost to follow-up, and the remaining 1 patient with CTP C stopped sorafenib for toxicity (thrombocytopenia).Overall, median AFP value was 41.8 ng/mL (IQR 15.1–256.0), while, according to BCLC stage, median AFP levels were 24.5 ng/mL (IQR 8.3–174.3) in patients with very early/early stage, 45.7 ng/mL (19.4–347.7) in patients with intermediate/advanced stage, and 40.3 ng/mL (IQR 27.8–367.9) in patients with terminal stage.Baseline AFP values ≥28.8 ng/mL were deemed the best performing for identifying high-risk people for death .Characteristics of patients according to the estimated BL AFP cut-off are summarized in Baseline AFP was ≥28.8 ng/mL in 34 (64%) PLWH. The median values of BL AFP were 136 ng/mL (IQR 45–645) in patients with AFP ≥28 ng/mL and 11.3 ng/mL (IQR 6.9–18.3) in those with AFP <28/ng/mL.P = 0.006). Pseudocholinesterase value was found significantly lower in patients with AFP ≥28 ng/mL, with respect to those with AFP< 28 ng/mL (P = 0.016). Total bilirubin levels were slightly higher in patients with AFP ≥28 ng/mL, with a trend toward significance (P = 0.09).The HCC patients with BL AFP ≥28 ng/mL had more frequently a multinodular disease with respect to those with BL AFP <28 ng/mL (P = 0.039). The other variables attaining tumor-related factors, hepatic reserve, and clinical parameters related to the severity of HIV infection were similarly distributed in patients with AFP ≥28 or <28 ng/mL.The HCC patients with BL AFP ≥28 ng/mL less frequently received a curative treatment for HCC with respect to those with AFP <28 ng/mL . The presence of extrahepatic metastasis tended to be higher in patients with AFP >200 ng/mL, showing a trend toward significance (P = 0.084).The intrahepatic burden of disease assessed by the number and size of lesions was similar in patients with AFP >200 or ≤200 ng/mL. Patients with AFP >200 ng/mL had a shorter median follow-up (0.79 years (IQR 0.43–1.1)) compared to those with AFP ≤200 ng/mL (2.57 years (IQR 0.67–4.95); P = 0.046). The other variables were similarly distributed in patients with AFP > or ≤200 ng/mL.HCC patients with BL AFP >200 ng/mL less frequently received a curative treatment with respect to patients with BL AFP ≤200 ng/mL ; 12 (80%) deaths occurred among patients with BL AFP ≥200 ng/mL compared to 20 (53%) with BL AFP ≤200 ng/mL (P = 0.067).During a median follow-up of 20 months (IQR = 8–42), 32 (60.4%) PLWH died. Overall, the 1- and 2-year cumulative probabilities of death were 30.2% (95% CI 19.4%–45.1%) and 47.4% (95% CI 34.4%–62.3%), respectively. According to the estimated BL AFP cut-off, 25 (74%) deaths occurred among patients with BL AFP ≥28 ng/mL compared to 7 (37%) with BL AFP <28 ng/mL . The 1-year cumulative probability of death was 11.2% (95% CI 2.9%–37.9%) among PLWH with BL AFP <28.8 ng/mL and 40.6% (95% CI 25.9%–59.5%) among those with BL AFP ≥28.8 ng/mL; at 2 years, the probabilities of death were 23% (95% CI 9.3%–50.4%) vs. 60.9% (95% CI 44.3%–77.8%) among individuals with BL AFP <28 and ≥28 ng/mL, respectively.Time to death was significantly different between PLWH with BL AFP <28 and ≥28 ng/mL (log-rank test: P = 0.067).Twelve (80%) deaths occurred among patients with BL AFP ≥200 ng/mL compared to 20 (53%) with BL AFP ≤200 ng/mL (P = 0.0008): the 1-year cumulative probability of death was 20.0% (95% CI 10.0%–37.4%) vs. 55.0% (95% CI 32.2%–80.6%), respectively; at 2 years, the estimated probability was 32.2% (95% CI 19.2%–50.6%) vs. 85% (95% CI 62.2%–97.5%), among people with BL AFP ≤200 ng/mL or >200 ng/mL, respectively.Time to death was significantly different even when considering the cut-off of 200 ng/mL for BL AFP (log-rank test: P = 0.001).P < 0.0001).The cumulative probability of death according to the use of a curative treatment is shown in P = 0.003 with BL AFP ≥28.8 as compared with BL AFP <28.8. Other predictive factors were HBV coinfection and type of HCC treatment .P = 0.031. One other predictive factor was the type of HCC treatment  was associated in the multivariate analysis with the risk of death and 47.4% of patients with AFP <28.8 ng/mL had BCLC 0/A, while 26.5% of patients with AFP ≥28.8 ng/mL had BCLC 0/A. Altogether, these our findings suggest that a number of patients with AFP <28.8 ng/mL may have HCC at early stage.We showed that HBV infection but not HCV was associated with worse survival, while none of the parameters indicative of the severity of HIV disease  was revealed as predictors of poor survival, probably as a consequence of good HIV control in our group of patients. The finding of a negative effect of HBV infection on survival in our series of PLWH with HCC is in line with previous reports , 21 indiIn our group of PLWH, the survival during a median follow-up of 20 months was 60.4%.Overall, the 1- and 2-year cumulative probabilities of death were 30.2% and 47.4%, respectively.Although we did not compare PLWH with a group of PNLWH, we showed that the survival in PLWH is similar to that of HIV-negative individuals, –24 when Studies comparing groups matched for HIV-1 status reported a significantly shorter survival in HIV-1-infected patients with respect to HIV-1-negative patients. In the report by Puoti et al. , 60% of Therefore, the different characteristics of patients as well as clinical end point between our study and that by Pinato are likely responsible for the discrepancy in the survival rate.P = 0.16) with 33% of HIV-infected patients showing BCLC = 0/A vs. 56% of HIV uninfected. In our group of PLWH, we found a higher cumulative probability of survival at one year (68%) with a similar prevalence of BCLC = 0/A (34%).In the study by Merchante et al.,  1- and 3P = 0.44) with BCLC stages C or D present in 50% of HIV-positive and in 58.4% of HIV-negative patients. Median survival was also no different between HIV-positive and HIV-negative subjects when separating those who did receive potentially curative therapy  and those who did not .Brau et al.  showed sInterestingly, the multicentre study by Berretta et al.,  which reSo, the different characteristics of study population, regarding BCLC stage and treatment allocation, may be responsible for discrepancy in survival.Nonadherence to the original BCLC guidelines , 30 is cAlthough most current guidelines recommend sorafenib for CTP A patients, in real-world practice also CTP B patients are frequently treated with sorafenib. In this regard, a recent report investigating the benefit of sorafenib in patients with HCC and CTP B cirrhosis  suggesteOnly one previous study on sorafenib treatment in PLWH  evaluateIn the present study, of 6 patients with BCLC stage D who received sorafenib, 3 were on CTP B and 3 on CTP C: only one of these 6 patients stopped treatment for decompensated cirrhosis.Therefore, our findings suggested that HIV-positive patients with HCC could be treated with a more aggressive approach with respect to that proposed by the BCLC staging algorithm.Notably, the median survival in our patients under noncurative treatment was 12.8 months see . This ouOur study acknowledges a number of limitations. First, this study design is retrospective, which might be associated with misclassification or information bias; although data collection was not specifically planned for this study, the CSLHIV Cohort database prospectively collects many detailed information on all the individuals followed in our centre, so that we are confident that such biases are limited and our data are reliable. Second, as the study was conducted in a single centre, the generalizability of our findings may be limited; however, a single-centre study is more homogeneous with respect to multicohort studies in term of access to care, availability, and adherence to surveillance programms, anticancer treatment, and quality of palliative care support after HCC diagnosis. Third, although AFP levels seem to affect survival, the study might not be sufficiently powered for this outcome due to the small sample size.Another limitation relies on the lack of a control group; we tried to overcome this limitation by comparing our outcomes with historical studies on PLWH or on HIV-1-negative individuals with similar or different characteristics.In conclusion, we showed that AFP levels at a precise cut-off at HCC diagnosis are predictive of subsequent mortality in PLWH. Other related factors were viral aetiology (HBV) found at the basis of HCC, as well as the type of HCC treatment, but not parameters related to the severity of HIV infection.Testing for AFP at HCC diagnosis is inexpensive, widely available, easily interpretable, and may help to clarify prognosis. We believe that it could be of clinical utility to incorporate AFP levels at a precise cut-off in a staging system including also aetiology at the time of HCC diagnosis and that such system could be relevant for the management of patients with HIV-associated HCC.Future studies on a larger group of PLWH with HCC are needed to better validate our conclusions.


Retention of patients who did not initiate antiretroviral therapy (ART) has been persistently low compared to those who initiated ART. Understanding the temporal trends in clinical outcomes prior to ART initiation may inform interventions targeting patients who do not initiate ART immediately after diagnosis.A retrospective cohort analysis of known HIV-infected patients who did not initiate ART from healthcare facilities in Central Kenya was done to investigate temporal trends in characteristics, retention, and mortality outcomes. The data were sourced from the Comprehensive Care Clinic Patient Application Database (CPAD) and IQ care electronic patient-level databases for those enrolled between 2004 and 2014.A total of 13,779 HIV-infected patients were assessed, of whom 30.7% were men.There were statisitically significant differences in temporal trends relating to marital status, WHO clinical stage, and tuberculosis (TB) status from 2004 to 2014. The proportion of widowed patients decreased from 9.1 to 6.0%. By WHO clinical stage at enrollment in program, those in WHO stage I increased over time from 8.7 to 43.1%, while those in WHO stage III and IV reduced from 28.5 to 10.8% and 4.0 to 1.1% respectively. Those on TB treatment during their last known visit reduced from 8.3 to 3.9% while those with no TB signs increased from 58.5 to 86.8%. Trends in 6 and 12 month retention in the program, loss to follow-up (LTFU) and mortality were statistically significant. At 6 months, program retention ranged between 36.0% in 2004 to a high of 54.1% in 2013. LTFU at 6 months remained around 50.0% for most of the cohorts, while mortality at 6 months was 7.5% in 2004 but reduced to 3.8% in 2014. At 12 months, LTFU was above 50.0% across all the cohorts while mortality rate reached 3.9% in 2014.Trends in pre ART enrollment suggested higher enrollment among patients who were women and at earlier WHO clinical stages. Retention and mortality outcomes at 6 and 12 months generally improved over the 11 year follow-up period, though dipped as enrollment in asymptomatic disease stage increased. In 2018, there were 36.9 million people living with HIV (PLHIV) globally, and 1.8 million new HIV infections [Trends in HIV programs suggest higher enrollment numbers among women, the less symptomatic, and a reduction in enrollment of children over time , 4. The The global community has embraced the strategy to treat all PLHIV regardless of clinical stage or CD4 count, also known as Universal Test and Treat (UTT) . Since iPatients not on ART have lower retention in HIV care, compared to those on ART , 7. A reThis analysis aims to describe trends in patient characteristics and clinical outcomes for pre -ART patients, such as 6 and 12 month program retention, loss to follow-up (LTFU), and mortality over an 11 year period in Central Kenya. This analysis focuses on the period prior to UTT, where eligibility criteria for initiation of treatment varied from CD4 counts of 350–500 cells/μl effected in 2014.Center for Health Solutions—Kenya (CHS) received funding through the US Centers for Disease Prevention and Control (CDC) to support the implementation of the HIV prevention and treatment programs in the former Central Province of Kenya since 2010. The package of care for patients was standardized across 47 health facilities and was provided by a multidisciplinary team (MDT) composed of clinical officers, nurses, lay counselors, peer educators, nutritionists, social workers, pharmaceutical technologists/pharmacists and laboratory technologists. Quality of care assessments and continuous quality improvement (CQI) projects were routinely conducted to optimize service delivery. The model of care for patients not initiated on ART included same day enrollment, comprehensive clinical evaluation with appropriate management of any diseases, tuberculosis screening using a symptom questionnaire administered during clinic visits, three sessions of pre-ART education and counselling and based on assessed readiness, initiation of ART, baseline CD4 count, weekly follow-up visits, psychosocial support group meetings, peer support, and tracing of those LTFU.This analysis focused on 13,779 patients enrolled but not initiated on ART between 2004 and 2014 with 6 and 12 month follow up data documented in the Comprehensive Care Clinic Patient Application (CPAD) electronic and IQ care medical records systems. The patients had not initiated ART by 12 months but may have gone on to initiate ART later on. Patients documented as having transferred to other facilities were excluded.Patient level variables collected included age, sex, marital status, baseline World Health Organization (WHO) clinical stage, and tuberculosis (TB) status. CD4 variable was excluded due to large percentage of missing values. Outcome variables were LTFU, program retention, and mortality at 6 and 12 months. LTFU was defined as not having attended a clinic for more than 90 days and retained patients were defined as patients enrolled, still alive, and active in the review period. Determination of mortality and transfers out were based on documentation available in the patient charts.Routine data collected between January 2004 and 2014, were extracted from CPAD and IQ care electronic medical record (EMR) systems. Data entry from the paper-based Kenyan ministry of health (MOH) longitudinal patient level data tool (MOH 257), was updated daily after patients’ clinical visits into the EMR by trained facility data clerks. Data were routinely reviewed for errors and completeness by the data manager and subsequently cleaned by monitoring and evaluation teams after verification by the clinical teams as per standard CHS monitoring and evaluation procedures.Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC.).Demographic and baseline clinical characteristics were described and presented as counts and respective percentages for all the variables. A nonparametric test for trend across ordered groups was done and corresponding p-values reported to assess for temporal trends in outcomes and baseline characteristics across the pre-ART cohort years. Line graphs were used to visually display the trends in the outcomes. Statistical significance was evaluated at the 5.0% level. All the analyses were done in Stata version 15.1 . Table Pre-ART enrollment increased from 254 to 2396 patients between 2004 and 2008. This could have been explained by the adoption of broader diagnostic interventions, particularly, the implementation of Provider Initiated Testing and Counselling (PITC) in 2008 to augment the initial client-initiated approaches . This waThe majority of pre-ART patients on follow-up were women during the 11 year period with a non-significant change in gender trends. More women than men were infected with HIV as reported in the Kenya AIDS Indicator Survey (KAIS) 2012 . This isThere was no statistically significant difference in the proportions of children, adolescents and adults enrolling into HIV care over the years. This is in contrast to the KAIS 2012 report that suggested a change in the peaks in age specific prevalence in men and women from 2003 to 2012 [Our study demonstrated a reduction in the number of patients enrolled with advanced HIV disease. This is similar to trends seen in several studies in sub Saharan Africa (SSA). A study in Rwanda reported marked improvement in the immune status of patients at enrollment into care . EnrollmEnrollment in early stage HIV disease can be explained by the scale up of PITC initiatives in the country resulting in earlier identification prior to onset of AIDS defining illnesses . FurtherThis study reported a reduction in HIV patients on TB treatment over time while those screening positive for TB signs increased over time. This is similar to findings from the 2016 Kenya TB prevalence survey . Similar3 [Pre-ART retention, LTFU and mortality outcomes at 6 and 12 months improved from the year 2013 onwards compared to years 2006–2012 even though mortality slightly increased from 2012 to 2014. The improvement in later cohorts could be due to improved health systems and revision of the Kenya HIV treatment guidelines in line with emerging evidence such as replacement of stavudine (D4T) with tenofovir (TDF) as the preferred first line resulting in better toxicity profile and hence better adherence as well as raising the CD4 cut-off at ART initiation from 350 to 500 cells/mm3 , 32. Des3 . In cont3 . Lower rLTFU was noted to be a major contributor to attrition averaging at 50.0% for most cohort years at both 6 and 12 months. The high LTFU reported in our study is similar to data reported by other studies conducted in SSA. A prospective cohort study of 530 clients registering for HIV care between July 2008 and August 2009 at Kilifi District Hospital in Kenya and followed up for 6 months recorded a LTFU rate of 33.6% . In EthiThe high rates of LTFU among ineligible patients may suggest the difficulty of retaining asymptomatic patients in care especially due to lack of motivation to attend clinic in absence of ART. A study in Western Kenya reported higher rates of disengagement from care among those on pre-ART who reported feeling well and therefore not requiring care. Among the same group, re-engagement to care was limited by ack of locator information hampering tracing efforts . FurtherThe other contributor to attrition was mortality. Our study demonstrated a reduction in mortality over time from earlier cohorts to later cohorts. The mortality rate also generally remained low throughout the cohorts years. The low mortality points towards earlier diagnosis of HIV disease as well as earlier treatment. Similar to our study, a study conducted in Nigeria reported that 5.9% of pre-ART patients died during the first 12 months of follow-up . A studyThe main advantage of this study was the use of a large dataset with patients data collected over a long period of time which allows for better inference. However there were some limitations which included poor documentation for patients who may have self-transferred or died. This may have resulted in an overestimation of LTFU outcome. Another limitation was missing data where no WHO staging and TB status was documented during the study period which could have affected the results. There is also a possibility that the results may have been influenced by the natural maturation of the program and improvements in HIV service delivery over time. Data were routinely collected and entered into the electronic system and could have included transcription errors. Lack of unique patient identifiers limited the capacity to track patients through various service delivery points and even across various healthcare facilities. Lack of a comparative group among those who initiatied treatment also limits inference of the findings in this study.Patients were enrolled at earlier WHO clinical stage over time. This suggested improvements in diagnosis during the follow-up period through interventions such as PITC scaled-up in 2008/2009. Retention and mortality outcomes at 6 and 12 months improved over the 11 year follow-up period. However, as the proportion of asymptomatic patients increased, retention decreased in the later cohorts. The general trends showed a decrease in mortality outcomes at 6 and 12 months with a slight increase in later years mirroring the reduction in retention. There was also a notable decrease in the proportions of children aged 0–4 years enrolled during the period suggesting the contribution of PMTCT interventions in the region. Overall, our findings demonstrate the challenges of retaining asymptomatic patients during program scale-up. It is expected that pre-ART patient numbers will reduce with the scale-up of UTT in Kenya since 2016. We recommend that programs continue to monitor outcomes for patients who fail to initiate ART in the UTT era.


At a multivariate analysis, a longer time of virological suppression  and having experienced a previous virological failure  resulted protective against DTG discontinuation. Most discontinuations (84.0%) happened within the first 12 months of DTG initiation, in particular, 92.2% of discontinuations due to neuropsychiatric toxicity were observed in the first year. Our data confirm the overall good tolerability of DTG in clinical practice, with a low rate of discontinuations. CNS toxicity resulted the main reason for DTG discontinuation, with most related interruptions happening in the first year from DTG introduction.Dolutegravir (DTG) is currently one of the most used Integrase inhibitors (INI) in antiretroviral therapies (ARV) in both naïve and experienced people living with HIV (PLWHIV). We analyzed a multicenter cohort of PLWHIV, both naïve and experienced, starting an ARV including DTG. We enrolled 3775 PLWHIV: 2763 (73.2%) were males, with a median age of 50 years. During 9890.7 PYFU, we observed 930 discontinuations (9.4 per 100 PYFU). Estimated probabilities of maintaining DTG at three and five years were 75.1% and 67.2%, respectively. Treatment-naïve pts showed a lower probability of maintaining DTG at three and five years compared to treatment-experienced PLWHIV (log-rank  Dolutegravir (DTG), a second-generation integrase inhibitor (INI), has shown high efficacy and safety in both naïve and treatment-experienced (TE) people living with HIV (PLWHIV) ,2, in boWe analyzed data from a multicenter cohort  of adultWe enrolled 3775 PLWHIV: 2763 (73.2%) were males, with a median age of 50 years (IQR 42–56). Naïve PLWHIV were 702 (18.6%), of whom 128 (18.2%) were AIDS-presenters. As to experienced PLWHIV, median time from HIV diagnosis was 17.0 years (9.0–24.8) while median time on ARV was 13.4 years (6.7–19.9); 393 (12.8%) had an HIV-RNA > 50 cp/mL at baseline. Full population characteristics are available in During 9890.7 PYFU, we observed 930 discontinuations, a rate of 9.4 per 100 PYFU. The reasons for DTG discontinuation were: toxicity , simplification to a Single Tablet Regimen , treatment intensification (17.0%), death due to non-HIV-related issues (3.7%), drug–drug interaction (1.9%), pregnancy (1.1%), concern of cardiovascular toxicity (1.1%), other/unknown (22.8%).Regarding discontinuations due to toxicity, the most frequently reported causes were: Central Nervous System (CNS) toxicity , gastrointestinal (GI) toxicity , hypersensitivity , and renal toxicity . We further investigated neuropsychiatric events leading to TD. In detail, there were cases of: insomnia , headache , depression , anxiety , nightmares/hallucinations  and vertigo . PLWHIV experiencing TD due to neuropsychiatric events were predominantly males , with a median age of 49 years (IQR 40–55). No significant differences were observed in terms of baseline characteristics between PLWHIV who experienced CNS toxicity and those who did not.p < 0.001). In particular, we observed that both virologically suppressed experienced PLWHIV  0.50–0.68, p < 0.001) and experienced PLWHIV with a detectable HIV-RNA at baseline  had a lower risk of discontinuing DTG compared to naïve PLWHIV. At a multivariate analysis, a longer time of virological suppression  and having experienced a previous virological failure  resulted protective against DTG discontinuation, after adjusting for CDC stage, peak HIV-RNA and nadir CD4+ cell count. The vast majority of discontinuations  happened within the first 12 months of DTG initiation. Estimated probabilities of maintaining DTG at 144 and 240 weeks were 75.1% (SD ± 0.8%) and 67.2% (SD ± 0.9%), respectively. Treatment-naïve PLWHIV showed a lower probability of maintaining DTG at 144 and 240 weeks  compared to treatment-experienced PLWHIV  and a previous episode of VF  were associated with a higher risk of failing DTG while months of virological suppression at baseline  resulted protective against VF.In our cohort, we observed 318 VF over 9499.8 PYFU, a rate of 3.3 VF per 100 PYFU. Estimated probability of not experiencing VF was 96.1% (SD ± 0.3%) at 48 weeks, 90.1% (SD ± 0.6%) at 144 weeks and 87.0% (SD ± 0.8%) at 240 weeks. In our regression analysis, we found that a peak HIV-RNA over 500,000 copies/mL . Discontinuations due to CNS toxicity showed an overall rate of 1.3 per 100 PYFU and they were observed almost entirely in the first year since DTG initiation .In a specific survival analysis, the probability of not discontinuing DTG due to neuropsychiatric toxicity was 88.5% at one year, 77.7% at three years, and 75.4% at five years of follow-up. No differences in this regard were observed between naïve or experienced pts. In our regression analysis, concomitant abacavir use was not a predictor of discontinuations due to CNS toxicity  resulted protective against TD. These findings are similar to those observed by Penafiel et al.  and suggData from our cohort show a lower rate of neuropsychiatric events leading to TD compared to other studies ,12,13. IRegarding TD due to pregnancy, new data from the Tsepamo study re-evaluate the potential effects of DTG on neural tube development and may change clinicians’ perspective on DTG use in young women .Even if our study was not specifically designed to investigate efficacy, we observed a very low rate of VF, in line with what we expected from the results of previous studies . As prevOur study’s main limitations are its retrospective design and the fact that low-grade toxicity not requiring treatment interruption was not registered in the cohort’s database. Instead, the long follow-up time and the large sample size represent the main strengths of our work. In conclusion, our work confirms the high tolerability of DTG in a clinical-practice setting, both in naive and TE PLWHIV. There has been significant controversy regarding the potential role of DTG in inducing neuropsychiatric toxicity and, with our study, we tried to evaluate the “real-life” impact of DTG-based strategies on neuropsychiatric toxicity events.


HIV-1 DNA persists in infected cells, forming viral reservoirs. Pre-antiretroviral treatment (ART) HIV-1 DNA load was reported to predict ART success in European severely immunocompromised patients. The aim of this study was to determine whether HIV-1 DNA levels are associated with virological success in less severely immunocompromised patients who receive early ART in sub-Saharan Africa.The association between pre-ART HIV-1 DNA and the virological response after 30 months on ART was studied in multivariate logistic regression in patients randomised to immediate ART groups in the Temprano trial, which assessed the benefits of early ART in HIV-infected adults in Côte d’Ivoire. HIV-1 DNA was quantified in peripheral blood mononuclear cell (PBMC) using real-time PCR.3, 4.7 [4.0–5.3] log10 copies/ml and 2.9 [2.5–3.2] log10 copies/million PBMC, respectively. Pre-ART HIV-1 DNA was significantly correlated with pre-ART HIV-1 RNA . In multivariate analysis, HIV-1 DNA < 3 log10 copies/million PBMC was significantly associated with virological success at M30 after adjustment for other key variables .HIV-1 DNA levels were measured in 1013 patients. Their medians [IQR] of pre-ART CD4 count, HIV-1 RNA and HIV-1 DNA levels were 465 [379–578]/mm3. HIV-1 DNA could become a useful tool for guiding some therapeutic decisions in the test-and-treat era.Low HIV-1 DNA was statistically associated with virological success in this population of sub-Saharan African adults who started treatment with a median pre-ART CD4 count at 465/mmTrial registration TEMPRANO ANRS 12136 ClinicalTrials.gov, number NCT00495651, date of registration 03/07/2007.The online version contains supplementary material available at 10.1186/s12879-022-07082-2. It has been widely demonstrated that HIV-1 related morbidity and mortality rates have declined dramatically since antiretroviral treatment (ART) was introduced. It is notable that highly active ART reduces HIV-1 viral load and limits viral heterogeneity, and this has improved the life expectancy of people living with HIV-1 . AlthougThe Temprano trial, a randomised controlled trial which we carried out to evaluate the benefits and risks of early ART and isoniazid preventive therapy (IPT) in West African HIV-positive adults, took place between 2008 and 2015. The participants were monitored for 30 months and the primary endpoint was severe morbidity.In the present study, we looked for an association between pre-ART HIV-1 DNA levels in PBMC and M30 virologic success in HIV-infected adults who were randomised to early ART groups in the Temprano trial [3 and no criteria for starting ART according to the most recent WHO guidelines. Participants were randomised into one of four arms: Arm-1 (Deferred-ART), in which ART was deferred until the WHO criteria for starting ART were met; Arm-2 (Deferred-ART + IPT), in which ART was deferred and a 6-month IPT prescribed; Arm-3 (Immediate-ART), in which ART was started immediately; Arm-4 (Immediate ART + IPT), in which ART was started immediately and a 6-month IPT prescribed.Temprano was a 2 × 2 factorial RCT conducted in Côte d’Ivoire. The trial design and results have been reported previously . To summAfter randomisation, all participants had a series of blood tests, including CD4 count and plasma HIV-1 RNA. Plasma and whole blood samples were also frozen at − 80 °C. All participants were monitored for 30 months. The primary endpoint was severe morbidity, defined as a combination of all-cause deaths, AIDS diseases, non-AIDS malignancies and non-AIDS-invasive bacterial diseases. The main result of the Temprano trial at month 30 (M30) was that early ART and IPT both reduced severe morbidity significantly.10 copy number per million PBMC.PBMC HIV-1 DNA levels were measured on whole blood samples frozen at baseline. The HIV-1 DNA real-time PCR was carried out in the virology laboratory at the Necker University Hospital in Paris. The technique was based on amplification of the Long Terminal Repeat (LTR) gene, well adapted to HIV-1 non-B subtypes, and reference to the 8E5 cell line containing one HIV copy per cell  . The thrPlasma HIV-1 RNA was measured semestrially in all participants, using a real-time polymerase chain reaction assay  , 14  plus efavirenz  Patients with contraindications to efavirenz  received tenofovir/emtricitabine plus lopinavir–ritonavir (100 mg/400 mg tad), or tenofovir/emtricitabine plus zidovudine (300 mg tad). The latter regimen was abandoned in December 2008 due to increased side effects in the upper digestive tract .All Temprano participants randomised to the immediate ART strategy and having a baseline HIV1-DNA quantification were included in the analysis. Univariate logistic regression models were used to look for an association between virological success at M30 and the following variables: baseline PBMC HIV-1 DNA, baseline HIV-1 RNA, baseline WHO clinical stage, first-line ART regimen prescribed at baseline, IPT prescribed at baseline, sex, age, and baseline CD4 count. Virological success at month 30 was defined as HIV-1 RNA < 100 copies/mL. HIV-1 DNA, CD4 count  and other variables with a p < 0.25 in univariate analysis were included in the multivariate model. The primary analysis included all patients with available plasma HIV-1 RNA measurement at M30. We then carried out a sensitivity analysis considering missing HIV-1 RNA measurement at M30 as failures. The interaction between variables was tested. Statistical analyses were carried out using SAS® 9.3 software.Between March 2008 and July 2012, 2076 patients were enrolled in the Temprano trial and randomly assigned to one of four treatment arms. 20 (1%) were subsequently excluded and 2056  were included in the Temprano analysis at month 30. Of the 1033 patients included in the Immediate-ART arms, 20 (2%) were further excluded from the present analysis due to absence of a HIV-1 DNA value at baseline, and 1013 were included in the present analysis  patients died and 30 (3%) were lost to follow-up.p < 0.0001)  Fig. .Fig. 2Co10 copies/106 cells. 711 (83.9%) had achieved virologic success (VL undetectable < 100 copies/ml).At the end of the trial, 847/1013 (83.6%) patients had an HIV-1 RNA viral load available at M30, including 481/847 (57%) patients with baseline PBMC HIV-1 DNA < 3 and 366/847 (43%) with baseline PBMC HIV-1 DNA ≥ 3 log10 copies/million PBMC was significantly associated with virological success at M30 . There was no significant interaction between variables, including between CD4 count and HIV-1 DNA (p = 0.16).In the primary analysis Table , HIV-1 D10 copies/million PBMC remained significantly associated with treatment success in patients with baseline CD4 < 500 cells/mm3 , but not in those with baseline CD4 ≥ 500 cells/mm3 .For the missing-equal-failure sensitivity analysis Table , there wThe objective of this study was to look for an association between PBMC HIV-1 DNA levels and virological success in HIV-infected West African adults enrolled in the Temprano trial and randomised to receive ART immediately regardless of the CD4 count.We found that patients with low HIV-1 DNA level at baseline had a higher probability of virological success at 30 months than other patients, irrespective of plasma viral load and CD4 cell count.3 have a higher risk of reaching this combined enpoint, and this risk is associated with the size of the reservoir. In people who start ART with a higher CD4 count, the reservoir may be less influential on the risk of virological failure, death or loss to follow up at 30 months.Our results are obtained in a population with a low clinical stage and a relatively high median CD4 count. In the main analysis, the association between DNA and virological failure is significant, adjusted for CD4 count. In missing = failure, however, the interaction between CD4 and DNA led to stratification of the analyses. The association between DNA and the combination of virological failure, death or loss to follow up remained significant in people with less than 500 CD4, but was no longer significant in people with more than 500 CD4. This may illustrate the value of early ART. People who start ART with less than 500 CD4/mmOther studies previoulsy reported the association between HIV-1 DNA and virological success at 12–16 months of ART. These studies were conducted in Europe, and involved relatively low numbers of participants, having low CD4 counts . This isHIV-1 DNA may provide complementary information to standard markers, CD4 count and plasma HIV-1 RNA . In our As long as current treatments are unable to significantly decrease the reservoir level, HIV-1 DNA is not likely to be a useful tool for monitoring the efficacy of ART. However, it can be useful in guiding the choice of specific therapeutic strategies. In the MONARK therapeutic lightening trial, ART-naïve patients with low HIV-1 DNA had a higher rate of success on ritonavir-boosted lopinavir monotherapy compared with other patients, suggesting that simplified one-drug treatments may be safer in patients with low reservoir levels .Our study has several limitations:Firstly, the association of a marker with prognosis in a cohort study should be interpreted with caution, as there may be one or more confounding factors that are not accounted for in the analyses.Secondly, HIV-1 DNA in peripheral blood mononuclear cells is a reflection of the reservoir, but do not represent the entire reservoir in view of its heterogeneity , 21. HowCellular HIV-1 DNA is an interesting tool, complementary to plasma viral load. As a marker of the viral reservoir, it may be proposed as an efficacy endpoint in trials of therapies aimed at eradicating the virus in the future. In the present time, it may also help identify individuals at higher risk of virological failure, and thus allow for the adaptation of monitoring or the choice of treatments in these patients.Additional file 1. Methods for HIV-DNA quantification.


Aflatoxin suppresses cellular immunity and accentuates HIV-associated changes in T- cell phenotypes and B- cells.This prospective study was conducted to examine the association of aflatoxin levels with CD4 T-cell count and antiretroviral therapy uptake over time.1 albumin adducts, hepatitis B surface antigen, and HIV viral load. Participants were separated into high and low aflatoxin groups based on the median aflatoxin B1 albumin adduct level of 10.4 pg/ml for data analysis.Sociodemographic and food data were collected from antiretroviral therapy naïve HIV-infected patients. CD4+ counts were collected from participants’ medical records. Plasma samples were tested for aflatoxin B1 albumin adduct levels had lower mean CD4 at baseline and at each follow-up period. Adjusted multivariable logistic regression analysis showed that higher baseline aflatoxin B1 adduct levels were associated with statistically significant lower CD4 counts . Not starting ART and low/middle socioeconomic status were associated with higher CD4 counts  and , respectively.Participants with high aflatoxin B1 adduct levels with lower CD4 over time indicate that there is an independent early and prolonged effect of aflatoxin on CD4 even with the initiation of antiretroviral therapy. The prospective study design, evaluation of baseline and follow-up measures, extensive control for potential confounders, and utilization of objective measures of aflatoxin exposure and CD4 count provide compelling evidence for a strong epidemiologic association that deserves careful attention in HIV care and treatment programs.Consistent correlations of higher aflatoxin B Aspergillus fungi, namely A. flavus and A. parasiticus .Two hundred and ninety-five participants who had complete data on baseline AF-ALB levels are included in the current analysis. Participants were divided into high and low AF-ALB levels based on the median AF-ALB level of 10.4pg/mg. We describe sociodemographic characteristics, food consumption patterns, and clinical variables of participants by high (≥10.4 pg/mg) and low (<10.4 pg/mg) AF-ALB levels at baseline using chi-square tests for categorical variables, fisher’s exact test for cell counts <5, and t-tests to compare group means for continuous variables. Principal components analysis (PCA) was conducted to assess lasting household indicators such as housing type, plumbing, water, and electricity to determine the socioeconomic status (SES). An SES score was attained from the PCA analysis by weighting each indicator by the coefficient of the first principal component, with each member of the household being assigned the same SES. Further, SES was categorized into tertiles ranging from lowest to highest. Mixed methods analysis was employed in Statistical Analysis System  to analyze this longitudinal data to assess statistically significant predictors of CD4 levels over time from baseline to 5 years. Initial adjustments included sociodemographic variables , season (dry and rainy), baseline AF-ALB levels and knowledge of HIV-positive status. A second model was additionally adjusted for alcohol intake and food consumption patterns. A final model included further adjustment for health status, viral load, HBV status, and ART. For all analyses, p-values ≤0.05 were considered as statistically significant. All statistical analyses were performed with SAS 9.4.The mean AF-ALB level for the participants in this study was 14.75 pg/mg (standard deviation ±15.61); median = 10.37, range = 0.20–109.87, and inter-quartile range = 4.67–19.56 pg/mg. The median AF-ALB was used to separate participants into high and low AF-ALB groups . A majorth year (533.1±41.5 vs. 755.0±38.8). This study was conducted in a major metropolitan area of Kumasi in the Ashanti Region of Ghana where people are at high risk for exposure to aflatoxin in food. Maize and groundnuts are staple crops in Kumasi, with maize being the principal crop and these crops having the highest aflatoxin contamination –28. MostAF-ALB adduct levels in our study participants ranged from 0.20–109.87 pg/mg. AF-ALB is a reliable biomarker that has been used as a standard for assessment of population exposures. It represents accumulation of adducts from repeated (chronic) exposure to aflatoxin over a 2–3 month period and has been used in epidemiological and clinical intervention studies on aflatoxin exposure in humans in different countries. Studies conducted in regions of the world at high risk of aflatoxin exposure show that more than 95% of people are positive for AF-ALB adducts with concentrations ranging from 3–5 pg/mg albumin to >1000 pg/mg .The most significant finding from this prospective study is that adjusted multivariable logistic regression analysis showed that higher baseline AF-ALB was associated with significantly lower CD4 counts. The unadjusted data showed that higher AF-ALB was associated with lower mean CD4 at baseline and at each follow-up time point over the 5-year period, with the lowest mean level during the fifth year. This indicates that the effect of aflatoxin occurs early in HIV infection and remains consistent over time even with the initiation of ART. In the third and fourth years of the study as more participants initiated ART, the mean CD4 levels increased but the increases were less among those with high AF-ALB compared to those with low AF-ALB and the difference was much greater at year 5.Participants reporting higher SES had lower mean CD4 counts at all time-points. This was a significant finding in all of the multivariable logistic regression models. One possible explanation could be that these higher SES participants were diagnosed with HIV after a longer period of HIV infection and/or took charge of their own health care for a longer period before attending the public clinic for HIV care. We observed that in the early to mid-2000s, HIV infected people with better economic means would attend private clinics/hospitals to avoid disclosure of their HIV-positive status. However, there was an agreement at the United Nations General Assembly High-Level Meeting on AIDS in 2006, to scale up HIV prevention, treatment, care, and support services. In response, the Ghana AIDS Commission developed the National Strategic Framework outlining targets to increase ART coverage to 60% and increase the number of persons receiving HIV care by 200% by 2013 . This reWhen adjusted multivariable logistic regression models were run with AF-ALB as a continuous variable, male gender was found to be significantly associated with lower CD4 count. Lower CD4 counts could be due to a longer time of HIV infection before diagnosis among males who access healthcare facilities less frequently than females . HoweverIt is understandable that participants who initiated ART during the study had significantly lower baseline mean CD4 counts than those who did not initiate ART and that by year 5 of follow-up, the mean CD4 count was higher among those on ART when compared to those who did not initiate ART. Although ART is available in most countries of the developing world where people are chronically exposed to aflatoxin, the number of people who need ART globally has increased as the WHO guidelines change and have become more difficult to meet. In 2018, 62% of HIV-positive people globally and 64% in the WHO Africa Region who needed ART were able to receive it . TherefoFurther, HIV-positive people in these high aflatoxin exposure countries who are not diagnosed early, as well as those who do not receive ART, will die faster. It has been shown that HIV-positive people in high aflatoxin-exposure countries progress to AIDS and die more rapidly than those living in high income countries with strict regulation of aflatoxin levels . The medThe WHO has set global targets for the elimination of HIV as a public health threat by 2030 . HoweverThis study has certain limitations that should be considered in interpreting the results. First, a convenience sampling method was used that is prone to inherent bias in representation. As such, the sample may not be representative of the study population and the results may not be generalizable to the population being studied. However, convenience sampling enabled us to study the population in a relatively expedient way and to obtain novel results that would not have been possible otherwise. Secondly, we were unable to evaluate aflatoxin levels in food consumed by study participants, examine their nutritional status, or assess their exposure to other mycotoxins or environmental toxins. In addition, the survey data on food grown, maize storage, and food consumption is likely to have recall bias. However, there are several strengths of this research as well. Foremost, the prospective design allowed us to observe changes in CD4 levels over time and to examine CD4 levels by sociodemographic  and seasonal factors that could not be done using a cross-sectional design. Additionally, the use of the AF-ALB biomarker is a direct and valid measure of aflatoxin exposure in participants over the previous two to three months. Finally, the relatively homogenous dietary pattern among study participants allows for greater generalization of our findings to others in the population.This study advances previous work by revealing significant consistent correlations between higher AF-ALB and lower CD4 counts every year over a 5-year period, in a unique cohort of ART-naïve HIV infected asymptomatic individuals, from across all SES. This strongly indicates that the effect of aflatoxin occurs early in HIV infection and remains consistent over time even with the initiation of ART. Our prospective study design, evaluation of baseline and follow-up measures, extensive control for potential confounders, and utilization of objective measures of AF-ALB and CD4 counts provide compelling evidence for a strong epidemiologic association that deserves careful attention. Guidelines and assessments to minimize chronic exposure to aflatoxin among people living with HIV should be incorporated into HIV care and treatment programs for optimum effect of ART and the healthiest survival of those affected.The finding of the association of significantly higher AF-ALB levels with lower CD4 counts at baseline and over time is a distinctive contribution to the literature on the effect of aflatoxin on CD4 levels in HIV-infected people. The results indicate that aflatoxin has an immunological effect that contributes to decrease in CD4 and that the effect of aflatoxin occurs early in HIV infection and remains consistent over time even with the initiation of ART. Although the mean CD4 levels increased when patients initiated ART, after year four the CD4 dropped among those with high AF-ALB compared to those with low AF-ALB.S1 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file. 6 May 2021PONE-D-21-00637Association of Aflatoxin B1 Levels with Mean CD4 Cell Count and Uptake of ART among HIV Infected Patients: A Prospective StudyPLOS ONEDear Dr. Jolly,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In particular, patient's description and statistical analysis as well as a number or other points that may be of importance.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 3 months. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.  If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:   http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols  . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at   https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols  .  We look forward to receiving your revised manuscript.Kind regards,Isabelle Chemin, PhDAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and  https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf  2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained . If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.3. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.4. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works.https://moam.info/mycotoxins_5a3c12051723dd42662aa122.html- https://doi.org/10.3390/toxins7124868- https://doi.org/10.2217/fmb.13.166- We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable, even for works which you authored. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: No**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: No**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********  5. Review Comments to the Author  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: This clinical prospective study was among ART naïve HIV+ patients in Ghana over 5 years between 2009-2013 with relatively high baseline CD4 count with mean in the 600s (wide range of 300-1616). Participants were grouped into two groups, high AF-ALB vs. low AF-ALB levels. The authors observed that higher AF-ALB group at each time point was associated with lower CD4 count and even with multivariable analysis, this association was significant. Data on diet and sociodemographics were based on questionnaires and HIV disease and diagnosis (including CD4 and ART initiation) were based on medical record. Blood was collected for measurement of AF-ALB level (quantifies level in the past 2-3 months) through HPLC, as well as HIV-1 RNA viral load.The study placed participants into high (63%) and low (37%) AF-ALB groups. The results showed that higher level AF-ALB group, there was a greater proportion of males compared to females; higher mean VL and lower CD4 count observed compared to lower group. Higher level also was correlated with storing maize for longer period of time, buying food.Overall, the hypothesis and data supporting it is intriguing; reporting a significant observation that higher AF-ALB group associated with lower CD4 count at baseline and continued through year 1-4. If validated, this could be clinically important in guiding food consumption/storage guidelines to minimize exposure to high levels of AF-ALB exposure among PWH.The study had several important observations:• The cohort was unique, in that all were treatment naïve and across socieoconomic status with good spread in age groups. There was some uneven distribution in low/middle SES group between the two groups, with more in the high AF-ALB group (67% vs. 60%) which may effect the endpoint measured, like CD4, access to ART and baseline VL.• Lower CD4 count significantly observed in those with baseline high AF-ALB levels which persisted at each collection period gives a consistent correlation between the two factors.• Interesting finding of lower CD4 count observed at baseline and throughout years during dry season, which previous study had showed higher AF-ALB levels in dry season. This is intriguing, but could be confounded by many factors not accounted for in this study.Minor criticisms:• Survey data about food grown, maize storage and food consumption likely has significant recall bias and unclear if these questionnaire had not been previously validated to be accurate measures.• More lower + middle SES in the high AF-ALB group may bias results although comparison of SES between groups was not statistically different based on authors analysis. However, if the group was divided into low+middle vs. high there is likely difference between the two groups based on AF-ALB levels. For example, baseline VL was 200K in high vs 83K in low AF-ALB group. This could be biased by SES and related assess to care and ART which is suggestive of greater proportion of high socioeconomic status in the low AF-ALB group (28 vs. 34%). Similarly, lower SES is related to high AF-ALB exposure, but low SES likely means less access to ART or care and so allows, therefore, augmenting the CD4 difference.• Data needs to be presented on proportion in each group on ART vs. not on ART at each year of follow up as this ART frequency in each group is unclear and could certainly be biasing the CD4 observations. Figure 3 shows ART frequency but not broken down by frequency in each AF-ALB group, which is the basis of all the comparisons.• Figure 3: Unclear why CD4 dropping in this cohort at year 5 when each year more people were started on ART, this should continue to go up, thus suggesting groups of patients are not being started on ART and therefore will dropping CD4 counts.• Discussion mentioned many participants were females in “antenatal clinics”; female hormone and pregnancy is known to affected CD4 and VL count and therefore may again bias data. It would be important to know how many of participants were pregnant and recent post-natal because of effects of pregnancy hormones on CD4 count.Major criticism:Overall, the study reports a significant association between high AF-ALB level and lower CD4 baseline and recovery. It is purely a correlative study and does not establish causality even though it has the samples and cohort to do so. A major issue with the study is the heterogeneity of the two groups in terms of HIV status  and the unclear rationale for dividing into two groups based on cutoff of 15pg/mg of AF-ALB. I am concern of the strengths of the associations found in this study as these are all important confounders which could have biased results to show an effect when in fact, biologically they may not be.For example, conclusion that males had lower CD4 count at baseline and then at each follow up timepoint. If they started at lower T cell, they will continue to be lower regardless of AF-ALB level. Although this difference can be due to AF-ALB effect, this observation could simply be due to the fact that males in this cohort were infected for longer periods. Again, showing that duration of HIV infection of each participant (or marker such as nadir CD4 count) would need to be considered in the analysis. Similarly, the observation that age was associated with lower mean CD4 count in the study could again be simply due to difference in duration of HIV infection . The two groups would have to matched or controlled for differences in baseline CD4 count.The second major criticism is that the study treated AF-ALB levels as a categorical or dichotomous variable (high vs. low) instead of continuous variable which would have been more informative as I would argue that seems more clinically relevant as we expect in real-life that AF-ALB levels would run a wide range and there will be a concentration effect if there is indeed an immunosuppressive effect on CD4 cells. It is unclear why the 15pg/mg was used as the cut-off to determine the high vs. low categories as the author never described the significance of this chosen level. It would be interesting to run the analysis with AF-ALB levels as a continuous variable and assess for correlation with CD4 count, but need to control duration of HIV infection or nadir CD4 count.The third major criticism is that the study does not control or separate out analysis of those on ART, many started during study period. They observed that HIV VL was higher and CD4 cell count lower in the high AF-ALB group at baseline but this could be accounted for by the greater proportion of those NOT on ART (69% vs. 66.3%). Looking at CD4 level change over time is important, but not as a group (AF-ALB high vs. low). The difference proportion in each on ART and/or ART initiation in the middle of study is not accounted for and still treated as two groups. It would be more accurate to look at trajectory of CD4 count each group . Making this comparison with a heterogenous and unevenly distributed groups  is not accurate.Fourth major criticism of the study is that this is a correlative study and does not establish or even suggest causality. To make a more casual association, authors could assess change in level of AF-ALB in each patient to change in T cell frequency or even test for function  over time. This could have been done with this prospective longitudinally followed cohort of 5 years.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. 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,  15 Jul 2021Isabelle Chemin, PhDAcademic EditorPLOS ONERe: PONE-D-21-00637Association of Aflatoxin B1 Levels with Mean CD4 Cell Count and Uptake of ART among HIV Infected Patients: A Prospective StudyPLOS ONEDear Dr. Chemin,Thank you for sending the comments from the reviewers of our paper submitted to PLOS ONE. We have made the corrections requested and have attached the revised paper with highlights for further consideration. We thank the reviewers for their careful review and believe that the changes have significantly improved the quality of the manuscript. This is a point-by-point response detailing the revisions that have been highlighted in the manuscript.COMMENTS FOR THE AUTHOR:http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: Response: The laboratory protocols used in this paper have been published previously; the references for the publications are given in the paper. https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at Response: We read the PLOS ONE style requirements at the websites above and ensure that our manuscript meets the style requirements.2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained . If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.Response: We have specified that written informed consent was obtained from each participant in the ethics statement in the Methods and in the online submission information. This study did not include minors. 3. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.Response: We have explained that we developed a questionnaire as part of this study. A copy has been included as Supporting Information file S1_File.pdf.  4. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works. https://moam.info/mycotoxins_5a3c12051723dd42662aa122.html- https://doi.org/10.3390/toxins7124868- https://doi.org/10.2217/fmb.13.166- We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable, even for works which you authored. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.Response: We have revised the manuscript to completely rewrite the duplicated text, cite the sources, and provide details as to how the current manuscript advances previous work (pages 18-19). Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Response: We have revised the data analysis and the manuscript in response to the reviewer’s critiques and suggestions below to ensure that it is technically sound and that the data support the conclusions.________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Response: We have re-run and conducted additional statistical analyses in response to the reviewer’s comments below. Tables 1 and 3 have been re-done completely using the median aflatoxin value to divide the study participants into high and low aflatoxin groups. Based on this change, the first two rows on Table 2 have been revised and highlighted. Figure 3 has been changed to into Figures 3a and 3b to show CD4 counts and ART initiation for the low and high AF-ALB groups separately over time. A Supplemental Table has been added for the multivariable logistic models with aflatoxin as a continuous variable. ________________________________________3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: NoResponse: The data for this study involves human research participant information  and therefore will be made available upon reasonable request.________________________________________4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Response: We thank the reviewer. ________________________________________ 5. Review Comments to the AuthorPlease 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: Reviewer’s description of the study and findings: This clinical prospective study was among ART naïve HIV+ patients in Ghana over 5 years between 2009-2013 with relatively high baseline CD4 count with mean in the 600s (wide range of 300-1616). Participants were grouped into two groups, high AF-ALB vs. low AF-ALB levels. The authors observed that higher AF-ALB group at each time point was associated with lower CD4 count and even with multivariable analysis, this association was significant. Data on diet and sociodemographics were based on questionnaires and HIV disease and diagnosis (including CD4 and ART initiation) were based on medical record. Blood was collected for measurement of AF-ALB level (quantifies level in the past 2-3 months) through HPLC, as well as HIV-1 RNA viral load.The study placed participants into high (63%) and low (37%) AF-ALB groups. The results showed that higher level AF-ALB group, there was a greater proportion of males compared to females; higher mean VL and lower CD4 count observed compared to lower group. Higher level also was correlated with storing maize for longer period of time, buying food.Overall, the hypothesis and data supporting it is intriguing; reporting a significant observation that higher AF-ALB group associated with lower CD4 count at baseline and continued through year 1-4. If validated, this could be clinically important in guiding food consumption/storage guidelines to minimize exposure to high levels of AF-ALB exposure among PWH.The study had several important observations: • The cohort was unique, in that all were treatment naïve and across socioeconomic status with good spread in age groups. There was some uneven distribution in low/middle SES group between the two groups, with more in the high AF-ALB group (67% vs. 60%) which may effect the endpoint measured, like CD4, access to ART and baseline VL. Response: There was some uneven distribution in AF-ALB in the low/middle vs high SES group (70% vs 63% using the median), therefore our analytical strategy included covariate adjustment for SES to account for potential confounding. We created an SES score using Principal Components Analysis based on household indicators such as housing type, plumbing and electricity, similar to approaches used in studies of LMIC populations. We observed that low/middle SES status was associated with higher CD4 counts. A possible explanation for this is that higher SES participants were diagnosed with HIV after a longer time of HIV infection and/or took charge of their own health care for a longer period before attending the public clinic for HIV care. We observed that in the early to mid-2000s, HIV infected people with better economic means would attend private clinics/hospitals to avoid disclosure of their HIV-positive status. However, in accordance to scale up HIV prevention, treatment, and support services , the Ghana AIDS Commission established programs to provide ART in public hospitals and health centers in districts in all ten regions of Ghana and removed of the availability of ART from private clinics/hospitals [34]. HIV infected people of higher SES may have taken a longer time to attend the public facilities for HIV care. Access to ART was not a factor in this study as ART was available free of cost to ALL participants during the time of the study regardless of SES. Baseline viral load was significantly higher in the high AF-ALB group. It is established that high viral load is associated with low CD4. This explanation is included in the discussion on pages 15-16. • Lower CD4 count significantly observed in those with baseline high AF-ALB levels which persisted at each collection period gives a consistent correlation between the two factors.• Interesting finding of lower CD4 count observed at baseline and throughout years during dry season, which previous study had showed higher AF-ALB levels in dry season. This is intriguing, but could be confounded by many factors not accounted for in this study.  Response: We have removed the discussion on season from the paper since season was not significant in any of the multivariable models. Minor criticisms:• Survey data about food grown, maize storage and food consumption likely has significant recall bias and unclear if these questionnaire had not been previously validated to be accurate measures.Response: We agree with the reviewer that there could be recall bias in the survey data on food grown, maize storage and food consumption, and have included this as a limitation in the paper (page 18). The questionnaire was developed for the study and has been included as supporting information. Eight staff members  from the hospitals reviewed it for understanding, clarity and cultural appropriateness after which it was revised. It was then pilot tested among six clinic patients similar to the ones recruited for the study and revised before use in the study.• More lower + middle SES in the high AF-ALB group may bias results although comparison of SES between groups was not statistically different based on authors analysis. However, if the group was divided into low+middle vs. high there is likely difference between the two groups based on AF-ALB levels. For example, baseline VL was 200K in high vs 83K in low AF-ALB group. This could be biased by SES and related assess to care and ART which is suggestive of greater proportion of high socioeconomic status in the low AF-ALB group (28 vs. 34%). Similarly, lower SES is related to high AF-ALB exposure, but low SES likely means less access to ART or care and so allows, therefore, augmenting the CD4 difference. Response: In re-running multivariable model 1 using the median AF-ALB to separate participants into high and low groups, SES is significant. However, contrary to the reviewer’s comments that the findings “could be biased by SES and related access to care and ART which is suggestive of greater proportion of high SES status in the low AF-ALB group (now 30 vs. 37%). Similarly, lower SES is related to high AF-ALB exposure, but low SES likely means less access to ART or care and so allows, therefore, augmenting the CD4 difference”, we found that low/middle SES was associated with higher CD4 counts. We explained on page 4 above in our response to the reviewer that the likely reason for this finding is that HIV infected people of higher SES may have taken a longer time to attend the public hospitals and clinics for HIV care. Access to ART was not a factor in this study as ART was available free of cost to ALL participants during the time of the study regardless of SES. Ghana Government hospitals and clinics provided free HIV care and treatment to all HIV infected patients. • Data needs to be presented on proportion in each group on ART vs. not on ART at each year of follow up as this ART frequency in each group is unclear and could certainly be biasing the CD4 observations. Figure 3 shows ART frequency but not broken down by frequency in each AF-ALB group, which is the basis of all the comparisons.Response: We have added Figures 3a and 3b that present the proportions of participants in the low and high AF-ALB groups on ART vs. not on ART at each year of follow-up. A higher number of participants in the low AF-ALB group went on ART in the first two years. • Figure 3: Unclear why CD4 dropping in this cohort at year 5 when each year more people were started on ART, this should continue to go up, thus suggesting groups of patients are not being started on ART and therefore will dropping CD4 counts.Response: The mean drop in CD4 for the total study group  was 40 cells (from 680 at year 3 to 640 at year 4). The new Figures 3a and 3b show that the drop in CD4 at year 5 occurred among the high AF-ALB group. There could be a number of reasons for this besides access to ART. As a result of their high AF-ALB status, the participants could have developed poorer health over time, developed less ability to tolerate ART regimen, or decreased adherence to ART. ART was available to ALL participants. • Discussion mentioned many participants were females in “antenatal clinics”; female hormone and pregnancy is known to affected CD4 and VL count and therefore may again bias data. It would be important to know how many of participants were pregnant and recent post-natal because of effects of pregnancy hormones on CD4 count.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898601/. This “drop” is only temporary and is not believed to be a real reduction in CD4 cells but the result of the same amount of cells in a larger amount of blood (the amount of blood increases in pregnancy). In our study, we did not collect pregnancy or recent postnatal data for the women. However, our results show that women had higher CD4 counts than men at ALL time points. Response: It has been shown that “CD4 counts were an average of 56 cells/mm3 lower during pregnant compared to non-pregnant periods and 70 cells/mm3 lower during pregnant compared to postpartum periods” : 231-236). Major criticism:Overall, the study reports a significant association between high AF-ALB level and lower CD4 baseline and recovery. It is purely a correlative study and does not establish causality even though it has the samples and cohort to do so. Response: Typical epidemiologic studies outside of clinical trials are unable to establish causality in any context. However, our prospective study design, evaluation of baseline and follow-up measures, extensive control for potential confounders, and utilization of objective measures of AF-ALB and CD4 counts provide compelling evidence for a strong epidemiologic association that deserves careful attention. A major issue with the study is the heterogeneity of the two groups in terms of HIV status  and the unclear rationale for dividing into two groups based on cutoff of 15pg/mg of AF-ALB. I am concern of the strengths of the associations found in this study as these are all important confounders which could have biased results to show an effect when in fact, biologically they may not be. Response: In the original paper submitted the mean AF-ALB level of 15pg/mg was used to divide the participants into high and low AF-ALB groups. Since AF-ALB is skewed, we went back and ran all of the analyses using the median AF-ALB level of 10.4pg/mg and present entirely new Tables and Figures. For example, conclusion that males had lower CD4 count at baseline and then at each follow up timepoint. If they started at lower T cell, they will continue to be lower regardless of AF-ALB level. Although this difference can be due to AF-ALB effect, this observation could simply be due to the fact that males in this cohort were infected for longer periods.Response: We removed the discussion on gender from the paper since gender is not significant in the adjusted models using the median. However, when the models were run with AF-ALB as a continuous variable, female gender was significantly associated with higher CD4 count in all of the adjusted models. Again, showing that duration of HIV infection of each participant (or marker such as nadir CD4 count) would need to be considered in the analysis. Response: We included knowledge of HIV positive status in the new analysis. Similarly, the observation that age was associated with lower mean CD4 count in the study could again be simply due to difference in duration of HIV infection . The two groups would have to matched or controlled for differences in baseline CD4 count. Response: We agree with the reviewer, we have removed the discussion on age from the paper. The former discussion was based on unadjusted data. Age is not significant in the adjusted models. The second major criticism is that the study treated AF-ALB levels as a categorical or dichotomous variable (high vs. low) instead of continuous variable which would have been more informative as I would argue that seems more clinically relevant as we expect in real-life that AF-ALB levels would run a wide range and there will be a concentration effect if there is indeed an immunosuppressive effect on CD4 cells. It is unclear why the 15pg/mg was used as the cut-off to determine the high vs. low categories as the author never described the significance of this chosen level. It would be interesting to run the analysis with AF-ALB levels as a continuous variable and assess for correlation with CD4 count, but need to control duration of HIV infection or nadir CD4 count. Response: We thank the reviewer for this critique and based on the skewedness in AF-ALB levels, we used the median level of 10.4 pg/mg (instead of the mean level of 15pg/mg) to separate participants into low and high groups and re-analyzed the data. We also ran the adjusted multivariable models with AF-ALB as a continuous variable and obtained results similar to that obtained using the median AF-ALB. Additionally, we obtained significant results for gender with females having higher CD4 levels than males in all models . We controlled for the time when participants knew of their HIV positive status in the new analysis. The third major criticism is that the study does not control or separate out analysis of those on ART, many started during study period. They observed that HIV VL was higher and CD4 cell count lower in the high AF-ALB group at baseline but this could be accounted for by the greater proportion of those NOT on ART (69% vs. 66.3%). Looking at CD4 level change over time is important, but not as a group (AF-ALB high vs. low). The difference proportion in each on ART and/or ART initiation in the middle of study is not accounted for and still treated as two groups. It would be more accurate to look at trajectory of CD4 count each group . Making this comparison with a heterogenous and unevenly distributed groups  is not accurate. Response: We have separated CD4 counts and ART initiation for the high and low AF-ALB groups over time and presented the results in Figures 3a and 3b as the reviewer suggested. Fourth major criticism of the study is that this is a correlative study and does not establish or even suggest causality. To make a more casual association, authors could assess change in level of AF-ALB in each patient to change in T cell frequency or even test for function  over time. This could have been done with this prospective longitudinally followed cohort of 5 years. Response: Please see response on causality above. Observational studies are not methodologically able to establish causality, however our study design approach enabled rigorous control for potential confounders that taken together indicate strong epidemiologic associations. We thank the reviewer for the suggestion about evaluating AF-ALB in relation to T-cell frequency over time. Our goal with the current analysis and manuscript is to present initial findings that serves as the basis for future studies examining T cell function over time. This area of research is markedly understudied, and our findings will shed more light on the biology of AF-ALB exposure in this population. We will explore funding opportunities to conduct additional analysis of immune cell function as a next step.Thank you for your kind consideration.Respectfully,Pauline Jolly, PhD, MPHProfessor Emerita,Director, UAB Minority Health International Research Training ProgramRecipient, 2014 Ellen Gregg Ingalls/UAB National Alumni Society Award for Lifetime Achievement in Teaching 2018 Fulbright Specialist Scholar, Institute of Public Health, Ho Chi Minh City, VietnamAttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 19 Nov 2021Association of Aflatoxin B1 Levels with Mean CD4 Cell Count and Uptake of ART among HIV Infected Patients: A Prospective StudyPONE-D-21-00637R1Dear Dr. Jolly,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Isabelle Chemin, PhDAcademic EditorPLOS ONEAdditional Editor Comments :The answers provided and changes in the manuscript are sound and improved the quality of the manuscript.Reviewers' comments: 14 Jan 2022PONE-D-21-00637R1 Association of Aflatoxin B1 Levels with Mean CD4 Cell Count and Uptake of ART among HIV Infected Patients: A Prospective Study   Dear Dr. Jolly:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofMrs Isabelle Chemin Academic EditorPLOS ONE