We developed a candidate DNA vaccine called “DNA-4”consisting of 4 plasmid DNAs encoding Nef, Gag, Pol(rt), and gp140 HIV-1 proteins. The vaccine was found to be safe and immunogenic in a phase I clinical trial. Here we present the results of a phase II clinical trial of “DNA-4”. This was a multicenter, double-blind, placebo-controlled clinical trial of safety, and dose selection of “DNA-4” in HIV-1 infected people receiving antiretroviral therapy (ART). Fifty-four patients were randomized into 3 groups . All patients were immunized 4 times on days 0, 7, 11, and 15 followed by a 24-week follow-up period. “DNA-4” was found to be safe and well-tolerated at doses of 0.25 mg and 0.5 mg. We found that the amplitudes of the spontaneous viral load increases in three patients immunized with the candidate DNA vaccine were much higher than that in placebo group—2800, 180,000 and 709 copies/mL, suggesting a possible influence of therapeutic DNA vaccination on viral reservoirs in some patients on ART. We hypothesize that this influence was associated with the reactivation of proviral genomes. Since AIDS was first described in 1981 about 60 million people have been infected with HIV, and about 30 million have died of AIDS. In Russia more than 1.2 million infected individuals  have been detected [Our group has developed a candidate DNA vaccine called “DNA-4” which consists of 4 plasmid DNAs encoding Nef, Gag, Pol(rt), and gp140 HIV-1 proteins of the Eastern European subtype A. The candidate vaccine has passed preclinical studies in laboratory animals  and phasHere we present the results of a phase II clinical trial of the candidate vaccine “DNA-4” in HIV-1 infected people receiving ART. The objectives of the clinical trial were to assess safety and to determine an optimal dose of the vaccine for HIV-positive patients. We also were looking for the possible influence of vaccination on spontaneous increase of the viral load.nef, gag, rt, or gp140 HIV-1 FSU subtype A genes [A candidate DNA-vaccine “DNA-4” has been developed at The Biomedical Center  in collaboration with the Research Institute of Ultra Pure Biologicals . The vaccine contains four plasmid DNA encoding consensus sequences of  A genes . Amino a A genes .Each gene was inserted into the vector pBMC that had been created at The Biomedical center. Inserted genes were expressed in eukaryotic cells under the control of the cytomegalovirus promoter and the bovine growth hormone polyadenylation signal .DNA-4 was manufactured by the production facility of the Research Institute of Ultra Pure Biologicals  in accordance with the existing Russian federal regulations. The plasmids were equally formulated in 0.5 mL of sterile saline solution with overall plasmid concentration of 0.25 mg/mL. No adjuvants were added to the vaccine. Placebo vials contained 0.5 mL of saline solution without plasmids.Phase II clinical trial was a multicenter, double-blind, placebo-controlled study. It was conducted to assess the safety of two “DNA-4” doses (0.5 mg and 0.25 mg) in patients with HIV-1 receiving ART by the analysis of frequency and severity of adverse events.The study was conducted in 7 Centers for the Prevention and Control of AIDS and Infectious Diseases situated in different Russian cities: Moscow region, Kazan, Tolyatti, Volgograd, Lipetsk, Kaluga, Izhevsk. During screening (visit 1) the following data were obtained: medical history, assessment of weight and height, electrocardiography, chest X-ray , laboratory tests of blood and urine were performed, viral load, levels of CD4 and CD8 T cells. For women pregnancy tests were performed. Patients eligible for inclusion were included in the study. The inclusion and exclusion criteria used in the study are listed in Randomization was performed centrally by an unblinded study monitor according to the randomization list and stratum. At screening, each subject was allocated an individual registration. Investigator completed the Inclusion form including following information: screening date, site number, subject number, subject initials, date of birth, and basic ART. At Randomization visit the eligible patients were randomized to one of three treatment groups with the ratio 1:1:1. Trial participants were stratified by basic ART. Investigator indicated basic ART for each subject during randomization: 2NRTI + NNRTI or 2NRTI + PI. Patients with different basic ART were allocated equally to one of three treatment groups.A dose of the studied vaccine was blinded by using two types of packages for each patient (box A and box B). Each package contained 4 ampoules with the DNA-4 vaccine with a dosage of 0.25 mg or with placebo.Patients from 0.25 mg DNA-4 group were immunized with one ampoule from box A with DNA-4 vaccine of 0.25 mg intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with placebo intramuscularly to the deltoid muscle of the left shoulder.Patients from 0.5 mg DNA-4 group were immunized with one ampoule from box A with DNA-4 vaccine of 0.25 mg intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with DNA-4 vaccine of 0.25 mg intramuscularly to the deltoid muscle of the left shoulder.Patients from the placebo group were immunized with one ampoule from box A with placebo intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with placebo intramuscularly to the deltoid muscle of the left shoulder.The candidate vaccine was administered intramuscularly in 1 mL of sterile saline solution in the deltoid muscle of each shoulder. Safety and tolerability were evaluated by the frequency and severity of adverse events (AE) according to subjective complaints from the patient’s diary, vital signs, physical examination, laboratory tests and development of local reactions. The severity of AE was assigned in accordance with the DAIDS scale, Version 1.0, December 2004. Each adverse event was graded using a 4-grade scale: 1—mild, 2—moderate, 3—severe, 4—potentially life threatening.Association of AE with vaccine administration was determined as associated, possibly associated, unlikely associated, or not associated. AE associated with the vaccine injection should meet the following criteria: occurs in a short time after injection, accompanies a known response to the use of the vaccine, terminates after cessation of the vaccination, re-occurs after the resumption of the vaccination.The viral load was assessed at screening and at visits 2 and 6–11 by real-time PCR analysis. “AmpliSense HIV-Monitor-M-FL” kit (Russia) were used to detect transient viral increases above 50 copies/mL (the sensitivity of the kit was 20 copies/mL). The magnitude of viral blips, the number of viral increases as well as the number of patients with viral increases were compared between vaccinated groups and placebo group.The quantity and ratio of CD4 and CD8 T cells were measured by flow cytometry analysis.The viral load and CD4 and CD8 T cell levels at visit 2 were the baselines for assessing the dynamics of the viral load.The study was reviewed and approved by the Ethical Committee of the Ministry of Health of the Russian Federation . The volunteers provided written informed consent following protocol review, as well as discussion and counseling with the clinical study team.54 HIV-1 infected individuals receiving ART participated in the study. All participants were randomized into three equivalent groups: 0.5 mg of vaccine—17 individuals, 0.25 mg—17 individuals, and placebo—20 individuals. Demographic characteristics of trial participants are presented in Vaccination was fully completed in 53 trial participants. One individual from group vaccinated with 0.25 mg of the vaccine was prematurely withdrawn from the study after first vaccine application due to a cold caused by a respiratory virus. There was no temporal association with vaccine administration, so this AE was determined as unlikely to be associated with the vaccination. However, data on safety and tolerability were analyzed in this participant. The diagram describing the course of the study is presented at Adverse events were registered in 17 out of 54 trial participants (31.8%). In the vaccinated groups (0.25 mg and 0.5 mg combined) 35 AE in 12 patients were detected (35.3%), in the placebo group—13 AE in 5 patients (25.0%). In the group receiving 0.25 mg of the vaccine AE were found twice as often as in the group receiving the 0.5 mg dose (47.1% and 23.5% respectively). The total data on the adverse events registered in trial participants are presented in Pain in the left arm and hyperemia at the injection site were associated with immunization with the studied candidate vaccine. Fourteen cases of AE were determined to be possibly associated with vaccination including leukopenia, neutropenia, fever, itching at the injection site, hypersecretion from the genital tract, and menstrual disorders.No deaths were detected. Most adverse events had mild or moderate severity. In the vaccinated groups, 4 cases of 3rd grade AE (3 cases in the 0.25 mg group and 1 case in the 0.5 mg group) and 1 case of 4th grade AE (in the 0.25 mg group) were registered, all of them neutropenias. This did not lead to an interruption of the vaccination. In all cases neutropenias were completely resolved.A viral load was measured at screening and at visits 2 and 6–11 by real-time PCR analysis. Transient viral increases above 50 copies/mL  were analyzed. The relative frequency of the viral blips as well as the number of patients with viral blips in the placebo group and the vaccinated groups did not differ . But theThe number of CD4 and CD8 T cells were measured by flow cytometry at visits 2, 6, 7, 9, and 11. At visit 2 blood donation was performed before the first vaccination. Results at visit 2 present the data on the CD4 and CD8 level at the time of study entry. The results are shown in There was a weak trend to increase in the absolute number of CD4 T cells in the group receiving 0.25 mg of the studied vaccine, but the differences with the placebo group were not statistically significant.Different therapeutic vaccine strategies including tools based on DNA, viral vectors such as modified vaccinia Ankara (MVA) and vesicular stomatitis virus (VSV), RNA, peptide, or protein, Lentiviral vector and dendritic cell have been used in numerous clinical trials. Despite the major advances in our immunological understanding of HIV-1 specific T cell responses and HIV-1 reservoir, we have not been able to achieve a cure and none of these vaccines have proven to be effective . Combinagag, env, rt, and nef [In our previous studies we have developed a candidate DNA vaccine against HIV-1 consisting of four plasmids encoding four HIV-1 subtype A genes:  and nef . The pre and nef ,3. The pThis study was conducted as a multicenter, double-blind, placebo-controlled study of safety and dose selection of a candidate HIV vaccine for HIV-infected people receiving ART. It can be concluded that the DNA-4 candidate vaccine at doses of 0.25 mg and 0.5 mg was safe and well-tolerated by HIV-infected individuals receiving ART. In vaccinated groups, three spontaneous increases of viral load with largest amplitude were detected.The proportion of trial participants who demonstrated adverse events associated or possibly associated with the vaccine administration was 7.4% higher in the vaccinated group than in the placebo group. The frequency of local reactions in group immunized with 0.25 mg of the vaccine and the placebo group was similar, and in the group immunized with 0.5 mg no local reactions were revealed. This is in contrast with other AE, which were highest in the group receiving 0.25 mg of the vaccine. Immunogenicity of the DNA-4 vaccine was performed in Phase I clinical trial using IFNγ-ELISpot, intracellular cytokine staining (ICS) of IFNγ, TNFα and IL-2, lymphocyte proliferation assay (LPA) and ELISA . For speFor complete eradication of the HIV infection the destruction of latent viral reservoirs is necessary, and this cannot be achieved by modern ART. The only example of HIV cure is the so-called “Berlin patient” who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) from a donor carrying homozygous mutation in the HIV coreceptor CCR5 ,8. RecenOne of the approaches used for eliminating viral reservoirs is reactivation of latent proviral genomes during ART treatment by histone deacetylase inhibitors and some cytokines ,11.+ T cells specific for HIV-1 epitopes [Another way is enhancement of cellular immunity in HIV-infected individuals using therapeutic vaccines capable of inducing functional CD8epitopes ,14,15,16epitopes . DNA vacProviral genome reactivation may be caused by TNFα expression. TNFα activates transcription factor NFkB and HIV transcription ,18. DNA-To investigate the possible effects of DNA vaccination on viral reservoirs we analyzed the magnitude and frequency of the blips in the placebo and immunized groups . NeitherThe number of patients is small. However, the largest increases were registered in double-blinded vaccinated groups. The results suggest that the lower DNA concentration (0.25 mg) is more active than 0.5 mg. This is in correspondence with more AE in the group vaccinated with 0.25 mg.These results may have several explanations. The participation of therapeutic DNA-4 vaccination during ART in destruction of latent viral reservoirs in some patients due to the reactivation of a latent provirus by TNFα is possible but is not proved. The destruction of latent cells containing viral RNA can be the source of the viral blips.“Repliclones”, populations of replicating cells with HIV’s genome nested inside them can also produce new virions . The studied vaccine contains Nef protein which has been shown to have an ability to induce viral reactivation. It was demonstrated that exogenous Nef activated virus production in latent cell lines and in peripheral blood mononuclear cells isolated from asymptomatic HIV-infected individuals . Early pNef increases the production of exosomes containing activated ADAM17 , an enzyme that converts pro-TNF-α into its active form. The uptake of ADAM17-containing exosomes by target cells can induce the release of TNF-α, which subsequently binds to TNF receptor type 1 and activates NF-κB and c-Jun N-terminal kinase (JNK) pathways .On the other hand, Nef is able to selectively downregulate surface CD4 and HLA-I molecules that may lead to evade immune surveillance by reactivated cells. Moreover, Nef can counteract multiple apoptotic pathways and promote cell survival could further hinder the clearance of reactivating reservoirs . So, NefThe further studies of these effects are necessary. As far as we know, no systematic studies of blips in vaccinated patients have been performed before. The measurement of the magnitude of spontaneous increases of viral load could become the part of monitoring the results of immunotherapy of HIV-infected patients in the future.In conclusion, we demonstrated safety of the candidate DNA vaccine in HIV-infected patients receiving ART, and detected unusual blips effects in vaccinated individuals, which may be of interest for the future studies.


The long-term treatment outcome of visceral leishmaniasis (VL) patients with HIV co-infection is complicated by a high rate of relapse, especially when the CD4 count is low. Although use of secondary prophylaxis is recommended, it is not routinely practiced and data on its effectiveness and safety are limited.A prospective cohort study was conducted in Northwest Ethiopia from August 2014 to August 2017 (NCT02011958). HIV-VL patients were followed for up to 12 months. Patients with CD4 cell counts below 200/μL at the end of VL treatment received pentamidine prophylaxis starting one month after parasitological cure, while those with CD4 count ≥200 cells/μL were followed without secondary prophylaxis. Compliance, safety and relapse-free survival, using Kaplan-Meier analysis methods to account for variable time at risk, were summarised. Risk factors for relapse or death were analysed.Fifty-four HIV patients were followed. The probability of relapse-free survival at one year was 50% : 53% (30–71%) in 22 patients with CD4 ≥200 cells/μL without pentamidine prophylaxis and 46% (26–63%) in 29 with CD4 <200 cells/μL who started pentamidine. Three patients with CD4 <200 cells/μL did not start pentamidine. Amongst those with CD4 ≥200 cells/μL, VL relapse was an independent risk factor for subsequent relapse or death . Except for one case of renal failure which was considered possibly related to pentamidine, there were no drug-related safety concerns.The relapse-free survival rate for VL patients with HIV was low. Relapse-free survival of patients with CD4 count <200cells/μL given pentamidine secondary prophylaxis appeared to be comparable to patients with a CD4 count ≥200 cells/μL not given prophylaxis. Patients with relapsed VL are at higher risk for subsequent relapse and should be considered a priority for secondary prophylaxis, irrespective of their CD4 count. Achieving parasitological cure at the end of visceral leishmaniasis (VL) treatment in HIV co-infected patients does not assure definitive cure, as the disease will recur within a year in many patients. In this cohort study, the probability of relapse-free survival at one-year was 50% in all patients. The use of monthly pentamidine infusion for those with lower CD4 counts (<200 cells/μL) at the time of VL cure appeared to result in a comparable relapse-free survival rate to those patients with higher CD4 count (≥200 cells/μL) who did not receive secondary prophylaxis. On the other hand, patients with a history of previous VL treatment (VL relapse) remained at high risk of relapse despite achieving CD4 count ≥200 cells/μL at the end of the VL treatment. While all VL patients with HIV co-infection may benefit from secondary prophylaxis, those with CD4 <200 cells/μL and previous history of treatment should be prioritized for secondary prophylaxis. New modalities for prevention of VL relapse in HIV patients should also be explored. When visceral leishmaniasis (VL) occurs in HIV patients, it presents several challenges . These iLeishmania parasites from the body [The anti-leishmanial medicines available cannot completely eradicate the the body . Even ththe body . In immuthe body , which tthe body . Despitethe body .L infantum transmission in Europe [Leishmania parasites may serve as reservoirs of infection. Their need for repeated treatments with the limited available drugs increases the risk that they will be a source of emergent drug-resistant parasites [Hence, it is logical to introduce a maintenance therapy or secondary prophylaxis for this group of patients, as is done for other opportunistic infections in HIV, to continually suppress the multiplication of the parasite. Although secondary prophylaxis has been recommended in some international guidelines ,10, thisn Europe ,12. In aarasites . Thus, fThe use of secondary prophylaxis for VL has not been routinely practiced in high endemic regions such as Northwest Ethiopia. Previous studies have demonstrated that the relapse rate of VL in HIV patients is 60–70% within a year of VL treatment ,15. PatiThis was a prospective cohort study of parasitologically cured VL patients with HIV co-infection. It followed another randomised clinical trial (NCT02011958), that had a non-comparative design to evaluate the efficacy and safety of two treatment regimens for VL in HIV co-infected patients . In the preceding trial, one or two courses of the allocated treatment were given until patients achieved parasitological cure. One course of treatment was defined as the standard dose/duration of therapy with a specified anti-leishmanial drug e.g. a total dose of 40 mg/Kg of Ambisome monotherapy or 30 mg/kg of Ambisome plus 28-day course of miltefosine regimens. Patients who still had not reached cure after two cycles of VL therapy received rescue treatment at the discretion of the treating physician. At the end of VL therapy, patients who had a negative tissue microscopy result for Leishman-Donovan (LD) bodies were eligible for this prospective cohort study.initiative; and the Abdurafi Health Centre Médecins Sans Frontières Leishmaniasis Treatment Centre. Both are referral centres for complicated leishmaniasis cases.The study was conducted in Northwest Ethiopia at two large leishmaniasis treatment centres: the Leishmaniasis Research and Treatment Centre at University of Gondar Hospital, supported by Drugs for Neglected Diseases Each of these centres treats over 400 VL patients every year, of whom around 15–20% are co-infected with HIV. Most are adult seasonal migrant workers who travel from highland areas to work on large lowland farms where VL is endemic.th August 2014, and follow-up ended on 12th August 2016. There were 55 patients who achieved parasitological cure with VL treatment(s) during the trial preceding this study. One patient was subsequently lost to follow-up and the remaining 54 patients were included in this cohort study  were excluded from this intervention but were followed up as per their initial consent. Patients with CD4 cell counts above 200/μL were followed without secondary prophylaxis.Pentamidine isethionate (Pentacarinat) secondary prophylactic treatment was started one month after the negative test of cure (completion of VL treatment). Every month, a dose of 4 mg/kg body weight of the salt reconstituted in 5 mL distilled water was re-diluted in 200 mL of 5% dextrose in saline or normal saline solution and infused over one hour with the patient in supine position. Patients were kept in the ward during administration and observed with frequent blood pressure monitoring for one hour before discharge. The blood glucose level was monitored prior to each infusion, and other metabolic panel tests  were run every 6 months.Patients were offered continued ART, co-trimoxazole prophylaxis and adherence counselling. Follow-up arrangements for HIV care were made at ART clinics. Blood samples for HIV viral load were sent to a regional reference laboratory every six months. Arrangements for ART regimen changes were made when indicated in communication with the respective ART clinics.Follow-up started on the date negative parasitology was achieved and ended 390 days (D390) after the initiation of the first VL treatment in the preceding trial. This means that those patients requiring more than one course of treatment to clear parasites during their VL treatment, and therefore achieving negative parasitology later, were followed up for less time than those who responded to one course of VL treatment . In partPatients eligible for pentamidine treatment were seen monthly for prophylactic treatment. There were two pre-specified follow-up time points for all patients, regardless of pentamidine prophylaxis; at 6 and 12 months after initiation of treatment for the current VL episode to check on long-term outcomes. They could present for unscheduled assessments as needed during the follow-up period ). All SAEs, drug related adverse events (AEs) and any event that could lead to pentamidine interruption were documented during the follow-up period but other non-serious AEs were not systematically captured.Relapse-free survival by D390 was specified in the protocol as the primary outcome of this cohort study. Time at risk was defined as the time from negative parasitology following VL treatment to the earliest of the following events: i) death, ii) relapse, iii) date of the D390 visit, and iv) date last seen (in case of loss to follow-up). Relapse-free survival was defined as reaching the end of the time at risk having neither died nor relapsed. Death (i) or relapse confirmed by positive parasitology (ii) correspond to a “failure” outcome. Conversely, alive and relapse-free at D390 (iii) or lost to follow up (iv) correspond to censoring.Relapse-free survival at one year was estimated using Kaplan-Meier methods to account for variable time at risk in all patients and within two sub-groups; i) CD4 ≥200 cells/μL not receiving pentamidine secondary prophylaxis and ii) CD4<200 cells/μL receiving pentamidine secondary prophylaxis. Although these groups were not defined a priori, risk factors were found to differ substantially between them. Graphical presentation of Kaplan-Meier analyses shows probability of failure rather than survival to illustrate timing of relapses and deaths in this cohort.Poisson regression was used to investigate univariable risk factors associated with relapse or death, again accounting for variable time at risk. This analysis was performed within the two sub-groups mentioned above. A multivariable model was built including those factors found to be associated with relapse or death in univariable analyses, i.e. with 95% confidence intervals (CIs) excluding the null effect of 1. In principle a single model could be fitted to both subgroups by including interactions between subgroup and the risk factors. In practice, however, this was not possible due to small cell frequencies.Compliance to pentamidine in those who were eligible was calculated as the percentage of patients who received all monthly treatments for which they were eligible, i.e. until relapse, death or D390 in surviving relapse-free patients.Safety analyses comprised of summarizing the proportion of patients experiencing SAEs, an SAE related to pentamidine, and an AE that required discontinuation of pentamidine treatment. AE is defined as any untoward medical occurrence  in temporal association with the use of the investigational treatment i.e. after the start of pentamidine. Causality relation to pentamidine is based on the known AEs listed in the Summary of Product Characteristics available from the manufacturer. Grading of the severity of the events was based on Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0 . For eveThe research protocol was approved by the Ethiopian regulatory authority , the National Research Ethics Review Committee (NRERC), the Institutional Review Board of the University of Gondar in Ethiopia, the Ethics Review Board of Médecins Sans Frontières, the London School of Hygiene and Tropical Medicine Research Ethics Committee, the Antwerp University Hospital Ethics Committee, and the Institute of Tropical Medicine, Antwerp Institutional Review Board. All patients were >18years old and were included into the study after written informed consent was given. Patients received all the required treatments free of charge including treatments for adverse events and intercurrent diseases. Food and transport support were provided.2. Most of the patients, 39 (72%), were already on antiretroviral treatment (ART) when VL was diagnosed with 27 (50%) being on ART for six months or more. About two-thirds had a high Leishmania parasite load (grade of +5 and +6) at VL diagnosis. Two-thirds of the patients had previously been treated with the Ambisome+miltefosine combination. Overall, 50% required more than one treatment course  were relapse VL cases, and 27 (50%) were malnourished with body mass index (BMI) < 18.5kg/mt course . More tht course .Of the 54 patients who were followed up, 22 had a CD4 count ≥200 cells/μL at the time of achieving negative parasitology. Of the 32 with CD4 count <200 cells/μL, 29 were started on pentamidine prophylaxis . The othLeishmania parasite load, BMI, ART status and duration and the VL treatment regimen used were comparable between those with a CD4 cell count <200/μL who started pentamidine, and those with a CD4 cell count ≥200/μL who did not receive pentamidine (The type of VL (primary or relapse), tamidine . Of thosThe percentage of patients who received all pentamidine treatments for which they were eligible was 76% .The Kaplan-Meier estimates of the percentage of all patients with relapse-free survival at one year was 50% (95%CI: 35–63%); 53% (95%CI: 30–71%) in the 22 patients with CD4 ≥200 cells/μL and 46% (95%CI: 26–63%) in the 29 patients with CD4<200 cells/μL who started pentamidine . The endPentamidine treatment was not interrupted or stopped due to any adverse drug reaction. One expected SAE was reported: renal failure, considered possibly related to pentamidine that led to death. This patient was also experiencing pyelonephritis, sepsis, VL relapse and multiple myeloma which might have contributed to the renal failure, either directly or by toxicity of the concomitant medications .Among patients with low CD4 counts (<200 cells/μL) at the time of VL cure and who started pentamidine prophylaxis, no statistically significant risk factors for relapse or death were identified .2, In univariable analyses of patients with higher CD4 counts (≥200 cells/μL) at the time of VL cure, higher rates of relapse or death were detected in relapse cases compared to primary cases, and in patients with normal BMI compared to low BMI  and BMI, only relapsed patients remained significantly associated with subsequent relapse or death (adjusted rate ratio (ARR) = 5.42, 95%CI: 1.1–25.8, The treatment of VL in HIV patients is complicated by a high rate of relapse in the first year after VL treatment ,15. RelaLong-term outcomes of VL in HIV patients are described in this prospective cohort study, with the use of pentamidine secondary prophylaxis for those with CD4 cell counts <200/μL. Parasitologically cured VL patients with HIV co-infection were followed up for one year. The study was conducted in Northwest Ethiopia which is one of the highest HIV-VL co-infection regions of the world. The population is largely young adult males, with relapsed VL status and malnutrition accounting for about half of the cases respectively and patients at different time period on ART. The results are likely to be generalizable to the rest of Ethiopia and East Africa. The management of patients in this cohort included offering ART to all and secondary prophylaxis to those with a CD4 count <200 cells/μL after achieving parasitological cure of VL. Three-quarters of the patients had 100% compliance for the monthly pentamidine infusions. Low CD4 count is a known risk factor for relapse of VL ,16. DespL. infantum transmission, using different drugs and different follow-up patterns.The probability of relapse-free survival at one year was 50% (95% CI: 35–63%) in all patients. One limitation of this study was that it was not adequately powered to test differences between the two sub-groups; CD4 ≥200 cells/μL without pentamidine versus CD4 <200 cells/μL and receiving pentamidine prophylaxis. Another limitation was that the allocation of pentamidine was dependent on CD4 count which complicates the interpretation of the results and does not allow for unbiased estimation of an overall effect of pentamidine prophylaxis, or an overall effect of CD4 count. Interestingly, comparable probabilities of relapse-free survival were seen in the two groups, 53% (95%CI 30–71%) and 46% (95%CI 26–63%) respectively. A direct comparison of the two groups (patients with CD4<200 given prophylaxis and patients with CD4>200 without prophylaxis) based on a hypothesis of a difference was not included in our study as we may not expect a difference between these two groups if pentamidine can reduce the risk of relapse or death due to low CD4, bringing the risk more in line with risk in those with higher CD4 and not on pentamidine, and we propose this as a hypothesis. According to previous reports, 60–70% of VL cases with HIV co-infection and on ART relapse within one year in the absence of secondary prophylaxis, the relapse rate being higher with a lower CD4 count ,15. The ≥200 cells/μL (n = 12), it was 82% (95%CI: 45%-95%), with up to 12 months of pentamidine prophylaxis. There was no apparent difference in relapse-free survival observed in the groups with CD4 <200 cells/μL between the two studies, based on the overlap between the 95% confidence intervals around relapse-free survival in each study.In a recently conducted interventional cohort at the same study sites in Ethiopia, primary, relapse and past VL cases were enrolled. Pentamidine was given to those who achieved negative parasitology for the VL episode if their CD4 cell count was <200 cells/μL, and to all relapse cases regardless of CD4 count. The relapse-free survival rate at one year in all patients was 71% . LookingThe potential benefit of pentamidine amongst patients in the current study can be hypothesized to result from pentamidine treatment reducing the rate of relapse amongst those more likely to otherwise do so, to a rate closer to that observed in patients with a higher CD4 count. Although the use of secondary prophylaxis reduced the risk of relapse, there was still a notably high number of patients who continued to relapse in both groups (CD4 <200 and CD4 ≥200 cells/μL). There are earlier reports showing relapse at higher CD4 count and also with secondary prophylaxis use ,11.Leishmania parasite load at diagnosis, the antileishmanial treatment regimen (Ambisome monotherapy vs Ambisome+miltefosine combination), the duration of VL treatment and BMI were evaluated. In the group of patients with CD4 cell count <200/μL and receiving pentamidine, no additional independent risk factors were found to be associated with an increased rate of relapse. There is a need for additional, adequately powered studies to assess the risk factors for persistent immunosuppression in these patients. VL disease while on ART and low CD4 count is a WHO AIDS-defining illness and an indicator of advanced HIV disease and possibly undiagnosed ART failure [Leishmania combination therapy and secondary prophylaxis seem insufficient to prevent relapse. The high relapse rate among these groups of patients indicates the need to explore other treatment modalities. These can be more frequent and/or higher doses of the prophylaxis or other interventions that can rapidly improve immunity [Multiple factors may play a role in the risk of VL relapse. In this study, CD4 count, duration on ART, VL relapse status (primary vs relapse), the  failure ,23. For immunity . Serum limmunity . Beyond immunity .In the group of patients who had a CD4 cell count above 200/μl at the end of VL treatment (stable on ART), past history of VL treatment (relapse) was an independent risk factor for subsequent relapse, as has previously been observed ,27. The Although the study was not adequately powered for a pre-specified magnitude of rate ratio, it is plausible to hypothesize a worse prognosis for patients whose VL episodes were hard-to-treat. Hard-to-treat patients are likely to include those who develop VL while on ART for more than 6 months, have a persistently low CD4 count (<200 cells/μL) after VL treatment (advanced HIV disease), those who require longer treatment to cure VL, those with treatment failure for a combination regimen and previous history of VL treatment . BecausePatients who require prolonged initial VL treatment (>1 month) might be those with advanced disease and high initial parasite load, or those receiving less effective initial treatment (e.g. Ambisome monotherapy). A highly effective initial VL treatment regimen (e.g. combination therapy) seems a more favorable approach, especially for hard-to-treat VL patients.Likewise, faster parasitological cure of VL and CD4 recovery ≥200 cells/μL (cure by D29) can be a sign of mild disease and relatively well-preserved immunity. Of the seven patients who responded to initial treatment (parasite clearance) by D29 and had CD4 count ≥200 cells/μL (thus without pentamidine prophylaxis), none relapsed or died. Although based on a very small number of patients, this could suggest that secondary prophylaxis may not be a priority for patients who respond to one course of treatment and achieve a CD4 level ≥200 cells/μL. This might be related to early diagnosis of VL in ART stable patients. On the other hand, the decision for secondary prophylaxis may need to be based on the CD4 level at the time of VL diagnosis rather than after VL treatment.In general, we have observed that the long-term outcome of VL in HIV patients is affected by multiple factors–importantly the level of immunity, history of VL treatment and the use of secondary prophylaxis. While a clear trend of benefit from the secondary prophylaxis is observed among those with low immunity, it should be noted that the treatment of these patients requires a multifactorial approach. Effective ART is a crucial component. Although the management of HIV included ART provision, clinical and CD4 cell monitoring; regular viral load determination was not possible due to the limited services available in the country during the study period and the delayed provision of results. The available data suggest that not all patients were on successful ART treatment and only a few had second line ART  Tables. L. donovani and L. infantum regions [In conclusion, the VL relapse rate in HIV co-infected patients is high irrespective of CD4 level. Secondary prophylaxis with pentamidine was found to be safe except in patients with risk factors for renal failure and could help prolong the disease-free survival of those with a CD4 count below 200cells/μL to a rate comparable to that for patients with CD4 count above 200 cells/μL and not receiving secondary prophylaxis. There are studies supporting its effectiveness and safety from both  regions ,16,30,31S1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Cryptococcosis is a global public health important infectious disease. HIV infection is the main risk factor estimated to account for 95% of cases in the middle- and low-income countries and 80% of the cases in high-income countries.Cryptococcal antigenemia (CrAg) among HIV/AIDS Patients on second-line ART Therapy at Ambo General Hospital and Nekemte Referral Hospital, Western Oromia, Ethiopia.The main aim of the study was to determine the prevalence and associated risk factors of Hospital-based cross-sectional study was employed from September 1, 2017, to October 30, 2017. Whole blood was tested for CrAg using Cryptococcal lateral flow assay  according to the manufacturer's instructions. The collected data were analyzed using SPSS version 20 software. Binary logistic regression models were applied to assess the association between predictors and outcome variables at 95% CI.nd line ART for an average of 2.5 years. The overall prevalence of Cryptococcal antigenemia in the study participants was 7.7% (14/183). Being male , poor adherence to ART , occupational exposures to contaminated soil , having non-separated house from chickens , CD4 T+ cell levels ≤ 100 cell/μL  and viral load >1000 copies/mL  were significant predictors of Cryptococcal antigenemia (P≤ 0.05).Among the study participants, 115(62.8%) were females and the median age of the participants was 35 (IQR: 14) years. Majority, 169(92.3%), have been living with HIV for ≥ 5.6 years and 124 (67.8%) stayed on 2Cryptococcal Antigenemia was significantly high in this study. Being male, occupational exposure to contaminated soil with avian droppings, CD4+ T cell levels <100 cell/μL and viral load >1000 copies/mL were significant predictors of Cryptococcal antigenemia. Therefore, public health measures, adherence to ART and early treatment are recommended.The prevalence of  Cryptococcus neoformans complex , occupational exposure to soil , non-separated house from domestic animals like hens , poor adherence to ART , CD4+ T cell counts ≤ 100 cell/μL  and higher viral load (HIV RNA >1000 copies/mL) . Therefore, male HIV/AIDS patients, those occupationally exposed to soil, having house non-separated from domestic animals, poor adherence, low CD4+ T cell count and with high viral load were 4.8, 6.8, 0.06, 0.12, 6.57 and 11.7 times more likely to harbor cryptococcal antigenemia than their counters, respectively.After adjusting for confounding factors, Cryptococcosis among HIV/AIDS patients on second-line ART attending two Western Oromia hospitals was determined. The prevalence in the current study (7.7%) is comparable to a study conducted in Addis Ababa (8.4%) Reviewers' comments:Reviewer's Responses to QuestionsComments 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: Yes**********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: Yes**********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 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: Authorship order is different on the manuscript than in the PLoS One system.Abstract Results: would prefer you to specify the median age (with interquartile range). % married is of no value and is distracting. Would remove that.% living with HIV and/or on ART -consider changing units to years instead of months. This is more intuitive/understandable for readersIntroduction:Line 59 -Define MLICsLine 61 -CrAg isn't antibodies, it's antigen. would remove the term anti-cryptococcal antibodies. replace with "antigen", same with line 63Methods:Line 90 -don't use contractions in formal writing. Same with line 132.Results:Line 162: don't capitalize "second line ART"Line 162 -saying 35% were in one age group is unhelpful. State the median age and interquartile range.Line 168 -change to yearsLine 170 -is this an error? the baseline CD4 was 163 +/- 182? That doesn't make sense.Line 173 -the prevalence is 7.7%. is this among peoplew ith CD4<100? On line 170 it says mean CD4 was 434. Very confused about the population you're describing.Line 221: Men tend to have more advanced HIV disease (ie. lower CD4 counts)Line 229: It's not clear from your results what the CD4 counts were of your patient population and those who were CrAg+Line 257: higher than what?In your figure, label your Y axis. Don't use decimal places unless it's useful. And put percentages behind your numbers.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  30 Sep 2019Response to the reviewer1. Authorship order is different on the manuscript than in the PLoS One system.Response: We have communicated with the journal office for another paper and confirmed the authorship order in the manuscript is the final acceptable order. The journal has similar rules which we thought will be applicable for this manuscript, too. It was difficult for the corresponding author to put his name last in the editorial manager.2. Abstract Results: would prefer you to specify the median age (with interquartile range). % married is of no value and is distracting. Would remove that.% living with HIV and/or on ART -consider changing units to years instead of months.Response: The age was described with median and IQR and we removed % marriage. And we changed unit of stay with HIV or ART in years instead of months.3. Introduction: Line 59 -Define MLICs Line 61 -CrAg isn't antibodies, it's antigen. would remove the term anti-cryptococcal antibodies? replace with "antigen", same with line 63 Response: We defined MLICs (Middle and low income countries) and HICs (High income countries). We removed the term “anti-cryptoccocal antibodies” and changed the “antibody” miswording to “antigen”.4. Methods:Line 90 -don't use contractions in formal writing. Same with line 132. Response: We fixed the abovementioned issue.5. Results: Line 162: don't capitalize "second line ART"Line 162 -saying 35% were in one age group is unhelpful. State the median age and interquartile range. Response: We modified the phrase “Second Line ART” into “second line ART” and the median age was used than mean age.6. Line 168 -change to years. Line 170 -is this an error? the baseline CD4 was 163 +/- 182? That doesn't make sense. Line 173 -the prevalence is 7.7%. is this among people with CD4<100? On line 170 it says mean CD4 was 434. Very confused about the population you're describing.Response: We changed months to years in line 168. Line 170…it was error. Baseline CD4+ T cell count was 163 +/- 82, not 163 +/- 182. In line 173….the prevalence of CrAg test positivity was 7.7% from all study participants. But 50% prevalence was among participants whose current mean CD4+ T cell count <100 cells/µL. plus the phrase saying “….CD4 was 434….” is about the current mean CD4 T cell count. Thus our study population are those with current mean CD4 T cell count of 434, but their baseline (before starting treatment) was 163+/-82.7. Line 221: Men tend to have more advanced HIV disease (ie. lower CD4 counts). Line 229: It's not clear from your results what the CD4 counts were of your patient population and those who were CrAg+Response: Line 221…it doesn’t necessarily mean men has advanced disease than women rather men usually have outdoor activities in Ethiopia which exposes them for Cryptococcosis infection which in turn resulted higher prevalence of CrAg test positivity among men. Line 229……We included the proportion of CrAg positive with respect to CD+ T cell count in results section. 8. Line 257: higher than what?Response: Line 260….higher. it is corrected into “….was significantly ‘high’ in the study area”.9. In your figure, label your Y axis. Don't use decimal places unless it's useful. And put percentages behind your numbers.Response: The figure was modified accordingly.AttachmentRebutal letter.docxSubmitted filename: Click here for additional data file. 12 Nov 2019Prevalence of Cryptococcal Antigenemia and Associated Factors Among HIV/AIDS Patients on Second-Line Antiretroviral Therapy at Two Hospitals in Western Oromia, EthiopiaPONE-D-19-20216R1Dear Dr. Mengist,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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 With kind regards,Kirsten Nielsen, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 22 Nov 2019PONE-D-19-20216R1 Prevalence of Cryptococcal Antigenemia and Associated Factors Among HIV/AIDS Patients on Second-Line Antiretroviral Therapy at Two Hospitals in Western Oromia, Ethiopia Dear Dr. Mengist:I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Kirsten Nielsen Academic EditorPLOS ONE


Isoniazid preventive therapy (IPT) is a key component of TB/HIV control, but few countries achieve high IPT coverage.Using a behavioural COM-B design approach, the intervention consisted of a training on IPT guidelines and tuberculin skin testing (TST), identification of the optimal IPT implementation strategy by the health care workers (HCWs) of 3 primary care clinics, and a 2-month mentoring period. Using routine register data, TST and IPT uptake was determined 3 months before and 5 months after the intervention. Records were reviewed to identify factors associated with IPT initiation and HCW fidelity to the guidelines. A survey among HCWs was conducted to determine barriers to IPT.3  were associated with IPT initiation. Time needed to perform a TST, motivating patients to return for TST reading, and low IPT patient awareness were the main barriers to IPT implementation.Two clinics implemented TST-guided IPT for all clients receiving HIV care, one clinic decided against use of TST. According to routine register data, the proportion of clients initiating IPT increased substantially at the clinic not opting for TST (6% vs 36%), but minimally (34% vs 37% and 0.7% vs 3%) in the two other clinics. TST uptake did not increase (0 vs 0% and 0.5%). In addition to poor IPT uptake, HCW fidelity to investigation for TB and timing of IPT initiation was poor, with only 68% of symptomatic patients investigated and IPT initiation delayed to a median of 374 days post-ART initiation. In multivariate analysis, pregnancy , recent HIV diagnosis , being on ART , and CD4 <500 cells/mmDespite using a behavioural intervention framework including training and participatory development of the clinic IPT strategy, HCW fidelity to the guidelines was poor, resulting in low TST coverage and low IPT uptake under primary care conditions. To achieve the benefits of IPT, health system level approaches including TST-free guidelines and sensitization are needed. Mycobacterium tuberculosis infection to TB disease in those at high risk [TB continues to be a major public health problem with 10 million new TB cases and 1.4 million deaths worldwide in 2015 [igh risk . The GloSouth Africa carries one of the world’s highest TB/HIV burdens, with close to one in every 100 South Africans developing active TB disease each year, and over 60% of all TB cases occurring in people living with HIV. The continued high TB notification rate highlights that the South African TB control strategy, which is mainly focused on passive case finding, fails to control the TB epidemic in South Africa , 6.Recent qualitative work assessing barriers to IPT implementation from the point of view of South African HIV care providers has suggested that the primary barrier to IPT use was lack of knowledge and experience . Other bImproving the implementation of an evidence-based practice such as IPT necessitates successful behaviour change at the patient, provider and health system levels. The COM-B framework identifies three main components of behavioural change  which can be targeted by interventions . We used2 with an estimated population of 138,329 [The study was performed in three primary care health facilities serving the Diepsloot community in Northern Johannesburg from August 2015 to November 2016. Diepsloot is a poor, densely populated, urban informal settlement of approximately 12 km 138,329 . AccordiWe used a before-after design to evaluate the change in uptake of IPT and TST following an intervention that consisted of (1) training, (2) participatory identification of the optimal IPT implementation strategy for each clinic, and (3) mentored implementation of the chosen strategy, followed by routine implementation.At each clinic, a 4-hour training session was organized for all clinic staff working with TB and HIV patients, with separate sessions tailored for clinicians, auxiliary nurses, counsellors and community health workers. The training was given by a study clinician in English, the language used by DOH for all staff training. The training covered the evidence on IPT effectiveness in people living with HIV and an in-depth overview of the 2014 South African DOH IPT guidelines. The theoretical knowledge covered was then applied to practical case studies to assure in-depth apprehension among all HCWs. During the last interactive session, all HCWs were encouraged to express concerns related to the IPT clinic flow and propose solutions. Clinicians were trained in TST placement using PPD RT/23 (2UT/0.1ml) and all relevant clinic staff were trained in TST reading. TST training was based on the Center for Disease Control and Prevention (CDC) TST training video and the TST training guide of the New Jersey Medical School Global Tuberculosis Institute . After tThe clinic flow and clinic environment were mapped and reviewed by the clinic manager and the clinician(s) in charge of TB and HIV care taking into account all concerns and solutions identified during the training. Using a participatory and iterative process, options for sustainable integration of IPT and its documentation were identified at each of the three clinics. Solutions to create a facilitating environment included a fast-track system for TST reading visits, full integration of IPT visits (except for TST reading visit) in the normal HIV care program, cooperation with local clinics for TST reading and documentation of IPT and TST results on hand-held patient cards. Options were discussed until a consensus on the optimal and feasible strategy at each of the three clinics was reached.To support IPT implementation and enhance treatment literacy, each clinic was given educational materials including patient TST cards, pamphlets and posters , and a poster with instructions for TST reading and placement (developed by CDC).To assure that the IPT strategy selected by the clinic was being implemented as intended, a study staff member was present to assist with IPT-related activities and monitor the relevant clinics’ processes and their documentation during the first 2 months of implementation. Study staff support was withdrawn after two months. Quarterly feedback on IPT uptake was given to the clinic managers.Data on IPT uptake and TST placement was collected using routine clinic registers in paper and/or electronic format. These registers collected information on IPT initiation in all people newly diagnosed with HIV (clinic 1 and 2) or in all clients receiving HIV care (clinic 3). The number of people initiated on IPT and number of TSTs placed was extracted from these registers for the three months prior to the intervention and 5 months after completion of the 2-month mentored IPT implementation.To assess the quality of the data collected in routine registers, data on IPT uptake was also collected through review of pharmacy reports of isoniazid mono-therapy prescriptions. This validation was only performed at one of the three clinics as detailed pharmacy records were not available at the other two clinics.a priori selected days distributed throughout the 5 months following the 2-month mentored IPT implementation. This record review was only performed at two clinics as medical files are kept by patients in the third clinic. We extracted information on age and gender, CD4 count, viral load, IPT initiation prior to the day of record review, presence of contra-indications for IPT , presence of symptoms suggestive of active TB , and results of sputum Xpert MTB/RIF, M. tuberculosis culture, and chest X-ray. We also documented whether TST was placed at that visit (and results thereof), whether IPT was initiated, and if so, its intended duration.To determine HCW fidelity to the guidelines and identify factors associated with IPT initiation, we reviewed the records  of all clients receiving HIV care at one of 8 To identify important barriers to the IPT program, a survey was done during the last two months of the observation period among all HCWs involved in IPT prescription using a structured questionnaire based on the COM-B model. The survey enquired about competency (‘Capability’), habitual attitudes (‘Motivation’), and hurdles related to the clinic flow (‘Opportunity’).To assess IPT and TST uptake before and after the intervention, the proportion of clients initiated on IPT were compared using either all clinic clients newly diagnosed with HIV or any clinic client presenting for HIV care as the denominator for the population of interest, depending on the data routinely collected by the clinic. Chi-square was used to compare proportions.To identify patient-level factors associated with initiation of IPT among those eligible, we performed a logistic regression analysis. Variables with a p-value ≤0.20 in the bivariate analysis were included and a backward selection method was employed to identify those factors independently associated with IPT initiation. The goodness-of-fit of the stepwise logistic regression model was assessed using the Hosmer-Lemeshow test.To assess implementation fidelity, a descriptive analysis was performed by comparing the decisions made by the HCWs to the ones expected when guidelines would have been followed accurately.To evaluate barriers to IPT implementation, frequencies and proportions were used to summarize the responses to closed survey questions.All analyses were performed in R.Ethical approval was obtained from the Health Research Ethics Committee of the University of the Witwatersrand . All identifying patient details were removed at time of data collection, recorded data were anonymous and securely stored. Waiver of consent was given by the ethics committee for review of routine clinic registers (clinic 1) and retrospective record review (clinic 2); all participants at clinic 3 gave written permission for use of routine data for research purposes.All three clinics opted to implement IPT for all clients receiving HIV care, but only two clinics opted to implement TST . Two difIPT uptake increased in all clinics, but the increase was only substantial in one and statistically significant in two clinics. The proportion of clients newly diagnosed with HIV initiated on IPT increased from 6% before to 36% after the intervention in clinic 2 (p<0.01), the proportion of clients living with HIV initiated on IPT increased from 0.7% to 3% (p<0.01) in clinic 3 , and theReview of the pharmacy records (in clinic 3) suggested potential underreporting of IPT in routine registers, with 5% of all 23,452 clients receiving HIV care being prescribed isoniazid monotherapy during 5 months post mentoring compared to 3% of these clients recorded in the registers to having been initiated on IPT during the same period (p<0.01).For patients receiving antenatal care, TSTs were–as intended- mostly (74%) done by 1 nurse. At the general adult HIV care and treatment clinic, all 22 clinicians were responsible for performing TST in their patients but only 12 placed one or more TSTs. The intervention failed to increase TST uptake, with no (0%) TST placement in the pre-intervention period and 0% (clinic 1) and 0.5% (clinic 3) of clients assessed by TST in the post-intervention period . Of the a priori selected 8 days, 1395 (92%) had a medical file available for record review , AFB sputum (n = 1), chest X-ray (n = 1), blood culture (n = 1), and urinary LAM (n = 4). IPT was started in 6 patients with presumptive TB, five of whom had not been evaluated for TB and one patient whose chest X-ray and abdominal ultrasound were not suggestive of TB.Among the remaining 1074 individuals, TB symptom screening was done in almost all (1056 or 98%) patients. Among the 1056 patients screened, 131 (12%) were classified to have presumptive TB. Overall, any investigation for active TB was performed in only 68% (89/131) of patients with presumptive TB, with large variability between clinics . The most frequent initial diagnostic used was sputum Xpert MTB/RIF . The remaining 9 patients with presumptive TB were assessed by other investigations including sputum culture (n = 1), blood culture (n = 2), urinary LAM (n = 3), fine needle aspiration of lymph nodes (n = 3), chest X-ray (n = 3) and abdominal US (n = 1). Of the 68 Xpert MTB/RIF negative patients, 37 (54%) were assessed by culture, all of which was negative for 3 (IQR 267–612) with a median time of 215 days (IQR 56–420) prior to the record review visit. Viral load suppression was present in half (52%) of the 736 clinic clients that had a viral load measurement available with a median time of 84 days (IQR 0–197) prior to the record review visit. A TST was performed at time of the record review visit in five of the 925 (0.5%) IPT eligible individuals. One TST was positive (14 mm), two were negative, and 2 patients did not return for TST reading. The TST result did not influence IPT eligibility as the two TST negative individuals were on ART. IPT was started in 76 of the 925 (8.2%) IPT eligible clinic clients, 63 of the 901 non-pregnant adults and 13 of the 24 pregnant women. Among non-pregnant adults, the median time from ART initiation to IPT initiation was 374 days (IQR 84–917) and the median CD4 count at IPT initiation was 374 cells/mm3. Among the 13 pregnant women started on IPT, two were not yet on ART at time of IPT initiation, six were on ART for more than one month, and five were on ART for less than 30 days. The intended IPT duration was specified in only 25 of 76 (33%) patients started on IPT but was according to the guidelines in all 25.Among the 925 IPT eligible clinic clients , median age was 39 years (IQR 32–46) and two thirds (627 or 68%) were female, of which 24 (3.8%) were pregnant . Most  of symptomatic patients investigated for TB, TST not implemented as intended (<1% coverage), low (8.2%) IPT initiation rate, and delayed initiation of IPT instead of as soon as “stable on ART” for non-pregnant adults or one month after ART initiation for pregnant women.3  were associated with IPT initiation in bivariate analysis. In multivariate analysis, pregnancy , recent HIV diagnosis , CD4 <500 cells/mm3 , and being on ART  remained independently associated with a higher odds of being initiated on IPT. Even among the patient groups where HCWs were more likely to initiate IPT, coverage of IPT was low: 54% of pregnant women, 16% of individuals with a recent HIV diagnosis, 11% of individuals with a CD4 count <500 cells/mm3, and 9% of people on ART.Age , pregnancy , recent (<90 days prior) HIV diagnosis , ART status  and CD4 <500 cells/mmThe main barriers to IPT implementation reported by HCWs were low patient awareness of IPT, time needed to counsel patients on IPT, burden to document IPT-related activities, and concerns regarding exclusion of active TB . Regardi3, and being on ART were independently associated with higher IPT initiation, but even among those groups, IPT initiation remained low. The main barriers experienced among HCWs were concerns regarding exclusion of active TB, burden to document IPT-related activities, and operational issues with TST.Despite comprehensive training, participation by health care workers in the identification of the most sustainable IPT strategy for each clinic, post-training mentoring and feedback on performance, we observed poor fidelity to the IPT guidelines under routine primary care conditions resulting in low IPT uptake and almost non-existent use of TST. Pregnancy, recent HIV diagnosis, CD4 <500 cells/mmThe low (8.2%) uptake of IPT was similar to the national IPT uptake estimates of approximately 10%  but far To date, few studies have assessed the operational feasibility, health systems aspects, HCW fidelity to IPT guidelines and resulting IPT coverage under real-life conditions. One study assessing IPT implementation in 49 South African health care facilities in 2010, when TST and ART status were not yet part of IPT eligibility assessment  found thRecent qualitative work suggested that lack of knowledge and experience were important barriers to IPT implementation . In our Our study had multiple strengths, including the use of a behavioural framework for intervention design and evaluation, the participation of health care workers in the design of the IPT implementation strategy at each clinic, the mixed methods approach, rigorous file review, large sample size, and evaluation of three primary care clinics under routine conditions. Our study also suffered from limitations. First, all clinics serve the same community, limiting generalizability to other urban and rural areas. Second, patient refusal can contribute to low IPT uptake, but we lacked sufficient data to assess this. Third, the study was conducted before South Africa implemented ART for all, limiting generalizability of our findings to the current situation.The global call for TB elimination underpins the urgent need to scale-up TB preventive therapy. IPT implementation under routine primary care conditions is challenging due to numerous barriers related to concerns regarding exclusion of active TB, logistical and human resource challenges. To assure sustainable high uptake of IPT, simplified guidelines, HCW and patient sensitization on the benefit of IPT, and political commitment are needed to scale-up of IPT. Reporting on accurate standardized indicators on cumulative IPT uptake will be required to evaluate the success of the IPT program. Continued research towards shorter, better-tolerated regimens might be needed to ultimately achieve the 90% IPT coverage target.


Increased second-line antiretroviral therapy (ART) failure rate narrows future options for HIV/AIDS treatment. It has critical implications in resource-limited settings; including sub-Saharan Africa (SSA) where the burden of HIV-infection is immense. Hence, pooled estimate for second-line HIV treatment failure is relevant to suggest valid recommendations that optimize ART outcomes in SSA.We retrieved literature systematically from PUBMED/MEDLINE, EMBASE, CINAHL, Google Scholar, and AJOL. The retrieved studies were screened and assessed for eligibility. We also assessed the eligible studies for their methodological quality using the Joanna Briggs Institute’s appraisal checklist. The pooled estimates for second-line HIV treatment failure and its associated factors were determined using STATA, version 15.0 and MEDCALC, version 18.11.3, respectively. We assessed publication bias using Comprehensive Meta-analysis software, version 3. Detailed study protocol for this review/meta-analysis is registered and found on PROSPERO (ID: CRD42018118959).A total of 33 studies with the overall 18,550 participants and 19,988.45 person-years (PYs) of follow-up were included in the review. The pooled second-line HIV treatment failure rate was 15.0 per 100 PYs (95% CI: 13.0–18.0). It was slightly higher at 12–18 months of follow-up , in children  and in southern SSA . Baseline values ; advanced clinical stage ; and low CD4 counts ) and suboptimal adherence to therapy  were the factors associated with increased failure rates.Second-line HIV treatment failure has become highly prevalent in SSA with alarming rates during the 12–18 month period of treatment start; in children; and southern SSA. Therefore, the second-line HIV treatment approach in SSA should critically consider excellent adherence to therapy, aggressive viral load suppression, and rapid immune recovery. In the past decade, rapid scale-up of antiretroviral therapy (ART) in sub-Saharan Africa (SSA) substantially reduced HIV/AIDS-related morbidity and mortality , 2. It hThe HIV-infected patients on ART are recalling earlier fears of death from the infection because of treatment failures . PatientThe HIV treatment failure involving second-line regimens has very narrow options for further switching, and this is a serious concern in resource-limited settings . The WorSuboptimal adherence  2015 statement recommendations . Identif18118959 .We performed a systematic literature search from PubMed, MEDLINE (Ovid), EMBASE (Ovid), CINAHL (EBSCOhost), Cochrane Library, Google Scholar, Health Technology Assessment, African Journals Online (AJOL) and ResearchGate. Websites of organizations and University repositories were also visited to retrieve any remaining relevant record including unpublished (gray) kinds of literature. In our search strategy, search terms we employed were “treatment failure”; “second-line”; “protease inhibitor”; “antiretroviral therapy”; and names of countries in the SSA. During the search, we accomplished a careful selection of keywords and indexing terms that did not limit the year of publication. In the search strategy, Boolean operators and truncations were also employed. The search was conducted from 15 December 2018 to 14 January 2019. Accordingly, all published and unpublished literature identified during the period of searching were retrieved.We set predefined inclusion and exclusion criteria for initial screening by titles or abstracts and evaluation of full texts for their eligibility assessment. We considered articles with at least an outcome of failure to second-line ART for their potential to be included. Next, we assessed the original articles reporting second-line HIV treatment failure after at least 6 month period of follow-up; reported in English language; and conducted in countries of SSA for their eligibility. In addition, we assessed the eligible original articles for quality using the Joanna Briggs Institute’s (JBI) critical appraisal checklist and articles with moderate (50–75%) to high (>75%) quality were considered as per the appraisers’ evaluation results. However, we excluded articles with outcomes not related to second-line therapy failure; with no separate failure data for SSA patients in case of mixed multi-center study settings involving SSA and other countries; and with no separate data of second-line therapy failure in studies involving first and second-line therapies during the screening and eligibility assessments.We identified and selected records retrieved through a search of the electronic databases and indexing services. Following this, we exported them to ENDNOTE reference software version 8.2 . Next, we identified, registered, and removed duplicates by the use of ENDNOTE. Accordingly, two authors, Dumessa Edessa (DE) and Mekonnen Sisay (MS), independently screened titles and abstracts of the retained records based on the predefined inclusion criteria. A third author, Fekede Asefa (FA), was consulted in case of disagreement between the two authors. With this, DE and FA individually collected and evaluated full texts of the retained articles for their quality and final eligibility assessment. In the end, we included articles that fulfilled the quality evaluation criteria.We accomplished quality assessment for the articles by employing the JBI’s critical appraisal checklist for cohort and analytical cross-sectional studies , 39. TwoTo extract relevant data, we employed a customized data abstraction format that has been prepared in a Microsoft Excel sheet. Two of the authors independently abstracted data pertaining to first author; year of publication; study design ; study region/country; study participants ; types of second-line ART (ritonavir-boosted protease inhibitor (PI)-based ART, PI-based ART with no ritonavir-boosting); sample size; median months of follow-up; person/patient-years (PYs) of follow-up; and event of interest (number of second-line therapy failure and factors associated with the failure).Proportion of second-line HIV treatment failure that includes clinical, immunological or virological failure, as defined by the WHO , 9 was t2) statistic). We also accomplished tests for factors associated with second-line therapy failure by using MEDCALC statistical software , version 18.11.3. Besides, we employed Comprehensive Meta-analysis software , version 3, for publication bias assessment. Similarly, we evaluated the presence of publication bias with the use of Egger's regression and Begg’s correlation tests. Lastly, we considered all statistical tests with p-values less than 0.05 (two-tailed) as significant.Proportion of second-line HIV treatment failure we pooled together was accomplished with the help of STATA software, version 15.0. Again, we performed sensitivity and subgroup analyses to minimize the risks of bias. With this, we used forest plots to graphically report the various meta-analysis results. We also applied the Mantel-Haenszel random-effects model to conduct meta-analyses at a 95% confidence level. Likewise, we assessed the heterogeneity status of the included studies and presented it with the use of Cochran’s Q test  and abstracts (n = 848). Of this 1,142 literature, 947 of them had unrelated outcomes of interest; 73 of them were discussion papers; 72 of them had outcomes of first-line therapy failure; 46 of them had mixed and/or other country studies, and 4 of them reported their outcomes with non-English languages. Accordingly, we conducted an eligibility evaluation of 98 full texts as per the predefined eligibility criteria for inclusion. Again, we excluded 65 studies with justifiable reasons . PRISMA flow chart depicting the selection, screening, and eligibility assessment process is shown in The 33 studies included for the systematic reviews and meta-analysis had a total of 18,550 participants, and 2,473 of them experienced treatment failures to their second-line HIV treatment. The study participants had a total of 19,988.45 PYs of follow-up. All the included studies were published during the year 2009 to 2018. Sample sizes for the included studies range from 40 patients enrolled by a study conducted in Uganda . The stu2 = 97.69%; P<0.001). The second-line treatment failures among the included studies range from 1.0/100 PYs (95% CI: 0.0–3.0 per 100 PYs) to 40.0/100 PYs (95% CI: 30.0–50.0)  .2 = 97.58%; P<0.001) at 12–18 month period of follow-up after second-line therapy initiation; 19.0/100PYs  among children; and 18.0/100 PYs  among patients in the southern SSA ; advanced clinical stage of HIV at baseline ; low peak CD4 cell counts at baseline (<100 cells/mm3) ; and suboptimal adherence to second-line therapy  were patient factors associated with the significantly increased occurrence of second-line ART failures .Different independent study reports also described depressive symptoms ; tubercuEgger’s regression test did not show any evidence for publication bias among the included studies . In addition, Begg’s correlation test did not also show any evidence of publication bias  .In this meta-analysis, the pooled estimate of proportion for second-line HIV treatment failure was 15.0 per 100 PYs of follow-up. This evidence is in line with reports from several studies that revealed a proportion of second-line HIV treatment failure as high as 25% , 65–69. The pooled treatment failure rates before 12 months and 12–18 months of follow-up after second-line therapy initiation were 17.0/100 PYs and 19.0/100 PYs, respectively. The failure rate was 9.0/100 PYs after 18 months of follow-up. This indicated that a relatively sustained virological response is expected after the 18 months of follow-up. Similarly, a multi-centered study in Asia and Africa found that the most frequent experience of second-line therapy failure  occurred during 6 to 11 months of follow-up compared to the 18 months and more duration of follow-up  [Subgroup analyses by regions revealed lower second-line HIV treatment failure rates in western (11.0/100 PYs) and eastern (13.0/100 PYs) regions of SSA compared to the rate in southern (18.0/100 PYs) region. These estimates are in line with the 19.6 million people living with HIV in southern and eastern regions compared to the 6.1 million people living with the infection in western and central Africa regions in 2017 . Since aHigh VL (≥ 5000 copies/ml) at second-line HIV treatment initiation increased the odds of treatment failure (odds ratio (OR) 5.67; 95% CI: 3.40–9.45; P<0.0001). Patients who experienced virologic failure with first-line therapy and switched to second-line therapy after 12 months were more likely to experience a further increase in VL as a potential indicator for second-line therapy failure . SeveralAn advanced clinical stage (stage III or IV) of HIV at the commencement of second-line therapy increased the odds of treatment failure . With this, baseline CD4 cell counts of < 100 cells/ml were linked to increased odds of treatment failure . Growing evidence relate the advanced HIV and lower peak CD4 cell counts at baseline to the increased rates of failure with second-line therapy , 87, 90.Some of the included studies also reported that patients with lengthy delays in initiating second-line therapy ; who werContrary to other studies, one of the included studies reports that obese or overweight patients had a reduced proportion of failure to second-line ART . Obese HAlthough the overall sample size was large enough, there are some limitations to note. First, the majority of data were derived from observational studies which resulted in a high degree of heterogeneity and a range of potential biases. As a result, we have used a random-effects model which is more appropriate in such anticipated heterogeneity. A series of subgroup analyses were also considered to reduce the degree of heterogeneity and presented them in percentages to indicate the extent of differences. Second, other additional potential explanations for second-line therapy failures including medication toxicities and drug-drug interactions might not have been adequately addressed. Third, we have included articles published only in the English language and this could under-or over-estimate the pooled proportion of second-line therapy failure in the SSA. Finally, the reporting of some variables pertaining to clinical and programmatic follow-ups were inconsistent, limiting the conclusiveness of the pooled factors associated with the second-line HIV treatment failure.The pooled proportion of second-line HIV treatment failure experienced by HIV-infected patients in SSA was found to be high. More common failure rates occurred at a 12–18 month period of follow-up after second-line therapy start, in children, and in the southern region of the SSA. Suboptimal adherence to second-line ART, higher HIV-RNA VL at baseline, lower peak values for CD4 cells, and advanced WHO clinical stages were among the key factors that have accelerated second-line HIV treatment failure in the setting. With this, prolonged delays in switching prior therapy, tuberculosis co-treatment, and other patient factors including younger age, depressive symptoms, underweight, and traditional medicine use were linked with the occurrence of second-line treatment failure. Therefore, optimal second-line HIV treatment approaches should critically consider immediate and aggressive VL suppression, rapid immune recovery, and excellent adherence to the therapy. Together with these approaches, more frequent clinical follow-ups and VL monitoring are recommended for the HIV-infected patients in SSA that help in rapid identification and intervention of failure cases. Finally, countries in the SSA should develop strategies and guidelines related to containment of second-line HIV treatment including intensive adherence support and intervention as routine clinical practice especially for patients with slow response to the therapies.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Second-line Antiretroviral Therapy (ART) regimens are used when patients develop treatment failure for first-line drug regimens. It is costly unaffordable and it is not widely available for patients in resource limiting setting, there is a need to maximizing the duration of stay on second-line regimen. This study was conducted to estimate the incidence rate of second-line treatment failure and to identify its predictors among adults living with HIV in the Amhara region.An institution based retrospective follow-up study was conducted from May to June 2017. A total of 1,011 adults on second-line ART who were enrolled between February 2008 and April 2016 were included for final analysis. Kaplan-Meier estimator curves were used to describe the survival function. Semi-parametric proportional hazard model was fitted to identify the predictors of treatment failure.The overall incidence of second-line treatment failure was 9.86 per 100 person-years. It was high during the first and the last year of follow-up. The rate of second-line treatment failure was higher for patients who didn’t change second-line regimens , who had poor ART adherence , and not taking INH  as compared to their counter group. The rate of treatment failure for patients who were under WHO clinical stage III at switch  was also lower as compared to clients who were under WHO clinical stage I. Furthermore, the rate of treatment failure was higher for clients who were under second-line regimen “TDF-3TC-LPV/r”  and “AZT-3TC-LPV/r”  as compared to patients under “ABC-ddI-LPV/r” regimens.A high incidence rate of second-line treatment failure was noticed in the study setting. The rate of second-line treatment failure was higher for patients who didn’t change drug regimens, who had poor ART adherence, and who were not taking INH. Therefore, addressing significant predictors to prevent treatment failure among ART patients is essential and sustainable monitoring to reduce the risk of treatment failure is also desirable. The Human Immunodeficiency Virus (HIV) has continued to be a major public health problem and claimed more than 39 million deaths since the beginning of the pandemic . Globall3 during the first years of ART follow-up and an average increase from 50 to 100 cells/mm3 per year at a steady state after a year [Optimal adherence to ART will minimize the development of drug resistance to Antiretroviral (ARV) medication, and it is key to suppress viral load, and decreasing the risk of mortality –9. Despir a year , 12.Second-line ART regimens are used when patients develop treatment failure for the first-line treatment regimens. It is estimated that 18.8% of people living with HIV were experienced second-line treatment failure in low-income countries , 14. In Second-line treatment is costly unaffordable, and it is not widely available for patients in resource limiting setting. There is a need to maximizing the duration of stay on the second-line regimen. Thus, this study was conducted to estimate the incidence rate of second-line treatment failure and to identify its predictors among adults living with HIV in the Amhara Region. The study has both public health and clinical importance. The suppression of viral load and reduction of the risk of treatment failure decreases HIV transmission, increases productivity, and enables to maximize the duration on second-line treatment.A multi-centered institution based retrospective follow-up study was conducted from May to June 2017. The Zonal hospitals of Amhara region namely, University of Gondar, Felege Hiwot referral, Debre Markos, Dessie referral, Debre Tabor, Woldiya, Finote Selam, and Debre Berhan hospitals were included in this study. All adults aged 15 and above and started second-line treatment between February 2008 and April 2016 were the study population. Accordingly, all adults aged 15 and above who were on second-line ART follow-up between February 2008 and April 2016 in the selected hospitals were included in the study. This study excludes HIV positive individuals with incomplete baseline information’s of CD4 cell count and WHO clinical stage.To get a representative image for the source population, the optimal sample size was calculated using sample size determination formula for survival analysis with assumptions of 95% level of confidence and 80% power, and it was 1098. However, the total numbers of patients on second-line treatment who fulfill the eligibility criteria in eight hospitals were 1,011, and all of these were included for the final analysis.3 following clinical failure or persistent CD4 levels below 100 cells/mm3 (at least two consecutive CD4 cell count in a row is less than100 cells/mm3) [The outcome variable of this study was time to second-line treatment failure. In this study, antiretroviral treatment failure is defined as a clinical failure, an immunological failure, or both. Clinical failure for adults is defined as a new or recurrent clinical event indicating severe immunodeficiency ) after 6 months of effective treatment. On the other hand, immunological failure is declared, when patients have a CD4 count at or below 250 cells/mmSocio-demographic variables , body mass index (BMI) at switch, taking Isoniazid (INH) preventive therapy, opportunistic infections (OI), CD4 cell count at switch, regimen modifications, WHO clinical stage (I-IV) at switch and ART adherence were analyzed in this study. ART adherence was measured by considering patients clinical compliance in scheduled visits. Patients adherence was classified as poor, fair, and good if there level of adherence was < 85, 85–95%, and > 95% respectively. Regimen modification was also considered when patients had regimen modification at least once in the follow-up period.The data collection checklist was organized to abstract data from the national ART follow-up form. This checklist was prepared for the collection of both baseline and follow-up socio-demographic, clinical, immunological, and treatment outcome related information that are important for the assessment of treatment failure. Consequently, the data were collected through chart review by trained nurses.All records of HIV patients who were switched to second-line ART between February 2008 and April 2016 were considered. Charts were retrieved using patient medical record numbers and ART registration numbers found in the database of health facilities. To assess the immunological and clinical response of the treatment, this study includes patients who had records of at least two CD4 cell count measurement and WHO-clinical stage. The quality of data was also assured by using a pretested checklist and trained data collectors. Data completeness and consistency were also checked by supervisors on daily bases. Moreover, the data clerks and case managers assisted data collectors by identifying patient records.The extracted data were checked for completeness, coded, entered, and cleaned into EPI-INFO version 7 and exported to R version 3.4 software for further analysis. An incidence rate was computed using person-time of observations. Person-time is the sum of the amount of time contributed by study participants in the follow-up period. Kaplan-Meier estimator curves were used to describe the survival function and to estimate the median survival time. Semi-parametric proportional hazard model was also performed to identify the predictors of second-line treatment failure. In semi-parametric model the baseline hazard function doesn’t need to be follow a particular statistical distribution which makes more robust than parametric approaches because it is not vulnerable to misspecification of the baseline hazard.The proportional hazard assumption was checked by Schoenfeld residuals Table . This as3.A total of 1,011 records of HIV positive patients on second-line treatment with complete information were analyzed, while 222 records of patients were excluded from the analysis because CD4 cell count and WHO-clinical stage didn’t measured for a minimum of two times. Nearly half (50.74%) of the subjects were male and about two-thirds (64.47%) were below the age of 35 years. About two-thirds (60.44%) of the ART clients started second-line ART at CD4 cell count level of below 100 cells/mm2 of BMI). Among ART clients who experienced second-line treatment failure, about 43.7, 46.46, and 83.74% of them were underweight, females, and had poor ART adherence respectively. Similarly, among clients who experienced treatment failure, nearly 56 and 45% of them were started second line ART at CD4 cell count level of below 100 cells/mm3 and under WHO clinical stage I respectively (Table Second-line regimens were modified for 576 (56.97%) patients. Nearly one-third 34.92%) of patients had poor ART adherence, 734 (72.60%) didn’t take INH preventive therapy, and 78 (7.72%) of patients had at least one opportunistic infection. Nearly half (46.3%), 8.8, 36.7, and 8.2% of patients were started second-line treatment under WHO clinical stage-I, II, III and IV respectively. The majority (84.67%) of clients were initiated second-line treatment on working functional status. About 39.07% of the HIV/AIDS patients were underweight  months. The overall incidence rate of second-line treatment failure was 9.86 per 100 person-years in the study setting. In this study, treatment failure was high during the first and the last year of follow-up period  was higher by 55% as compared to patients who modify the drug regimen at any time in the follow-up period. The rate of treatment failure was also higher by 40% for clients who had poor ART adherence  as compared to those who had good ART adherence. The rate of second-line treatment failure was lower by 32% for those patients who were under WHO clinical stage III at switch  as compared to patients who were under WHO clinical stage-I at switch. Furthermore, ART clients who were not taking INH preventive therapy  were 1.68 times more likely to experience treatment failure as compared to patients who were taking INH preventive therapy. Clients who were under second-line regimen “TDF-3TC-LPV/r”  were 1.55 times more likely to experience treatment failure than patients who were under second-line regimen “ABC-ddI-LPV/r”. Moreover, clients who were under second-line regimen “AZT-3TC-LPV/r”  were three times more likely to experience treatment failure than patients who were under second-line regimen “ABC-ddI-LPV/r”  months. This result is in agreement with previous studies conducted in African and Asian countries . HoweverIn the follow-up period, participants contributed a total of 2,575.63 person-years of observations, and the overall incidence rate of treatment failure was 9.86 (95%CI: 8.4–10.8) per 100 person years. The incidence rate of treatment failure for the current study was higher than previous studies conducted in northwest Ethiopia  and southeast Ethiopia  , 17. TheThe rate of treatment failure for clients who didn’t change the second-line drug regimens during resistance  was higher as compared to patients who modify the drug regimen. The possible reasons for this result might be the changed regimen is more effective than previous drugs in reducing side effect and drug-drug interaction. The other possible reason might be the modified drug regimens could reconstitute CD4 cell count. The rate of second-line treatment failure for clients who had poor ART adherence  was higher as compared to patients who had good ART adherence. It is reasonable that strict adherence to ART plays a crucial role in the success of therapy for peoples with HIV . The ratIn the present study, Tenofovir (TDF) and Zidovudine (AZT) based second-line drug regimens have been shown to be associated more with treatment failure than Abacavir (ABC) based drug regimens. A study conducted in Kenya showed that patients on AZT based regimens had better performance of physical and mental health summary score compared to those on TDF . On the Though we did our best to estimate the incidence rate of second-line treatment failure and to identify its predictors, it is not free from limitations. The retrospective nature of the study limited the inclusion of all possible factors that could affect the incidence rate of treatment failure. Variables such as hemoglobin level and side effects were some of the plausible factors that were not measured in this study. Unavailability of viral load testing which is the gold standard method for treatment failure is also the limitation of the study. Since treatment failure in this study was monitored by CD4 cell count and WHO clinical stage, the incidence rate of treatment failure might be underestimated.In this study, the incidence rate of second-line treatment failure among adults living with HIV was high. Changing the second-line drug regimen, ART adherence, WHO clinical stages, taking INH, and types of second-line drug regimen were a significant predictors of time to treatment failure. Therefore, addressing predictor variables to prevent treatment failure among ART clients is essential and close monitoring to reduce the risk of treatment failure is also desirable.