In addition, HIV-DNA species, immunological parameters, and clinical data of the patients were analyzed. We found that the possible causes for PV were divers, and both virologic and host parameters of this particular cohort were heterogeneous. We identified viruses with therapy-associated DRMs in six patients (30%); two of these were detected as minority variants. Five patients had sub-optimal drug levels (25%) and the baseline plasma viral loads were relatively high. Strikingly, we observed that >40% of the PV patients finally reaching HIV levels below the LOD later on showed up with episodes of low-level viremia (LLV). However, the amount of PBMC derived HIV-DNA species was not correlated with the likelihood of LLV after PV. According to our data, we conclude that drug-resistant viruses, sub-optimal drug level, and high baseline viral loads might be probable reasons for the prolonged RNA decline only in a sub-set of patients. In the absence of emerging DRMs and/or compliance issues, the clinical implications of PV remain unclear; however, PV appears to be a risk factor for episodes of LLV.After the start of antiretroviral therapy (ART), plasma HIV-RNA levels should fall below the limit of detection (LOD) within 24 weeks. Hence, the prolonged decline of HIV-RNA after ART initiation is defined as persistent viremia (PV). In this retrospective study, we analyzed factors associated with PV. Next-generation sequencing of viral RNA/DNA was performed to study viral evolution and the emergence of drug-resistance mutations in HIV-infected patients with PV ( Here, HIV-RNA and DNA were isolated using QIAamp Viral RNA Kit (Qiagen) and QIAamp DNA Blood Mini Kit (Qiagen), respectively, according to the manufacturer’s instructions. RNA was reverse transcribed using one-step RT-PCR kit (Qiagen), and PR and RT sequences were amplified using the Hot-StarTaq polymerase kit (Qiagen) according to a nested PCR protocol published previously . AccordiTotal and integrated proviral as well as two long terminal repeat (2-LTR) circle HIV-DNA were quantified using a pre-PCR followed by a real time PCR as described previously . BrieflyCmin) of each drug were correlated to the corresponding therapy application. The Cmin and Cmax values are obtained from the average measured values after intake in healthy volunteers as described in the corresponding patient information leaflet provided by the manufacturer. For darunavir, there are two Cmin and Cmax values depending on the mode of ingestion.Following a protein precipitation, drug levels were determined by an in house high-performance liquid chromatography–mass spectrometry (HPLC/MS/MS). Quantification was performed on the basis of a dilution series used on each run. Each controls for low and high are based on weighing of pure substance. For evaluation of the drug efficacy, the trough levels . Three patients did not reach pVL below the LOD and were lost to follow up. 16 of the 20 patients were treated with a PI-containing regimen combined with a NRTI-backbone, whereas only few of them received treatment regimens based on integrase inhibitors (INSTI) or NNRTI , while five of them were treatment experienced and restarted ART after treatment interruption. Before ART initiation, the median pVL was 302,000 copies/ml and dropped to 136 copies/ml during 237 days  with ART at the time of genotyping. The overall pVL decline after the ART initiation (if has been successful) required 459 days  to reach pVL <50 copies/ml  and sequenced plasma RNA as well as proviral DNA derived PCR amplicons. We identified viruses with therapy-associated DRMs in six patients (30%). As shown in Table n = 5; PV-02, PV-13, PV-16, PV-17, and PV-18), we found that the concentration of at least one drug of the current ART regime was below the therapeutic drug level Cmin. In one of these cases, also a new DRM was detected during PV (PV-16: V82A). Hence, sub-optimal drug levels can also be a reason for PV in a sub-set of patients.Since reduced drug absorption might be another cause for PV, we analyzed the drug-level concentrations in each patients plasma at the time of genotyping. In most cases, drug levels were found to be in the therapeutic range Tables , 3. HoweThe duration of PV has been correlated with pVL before ART initiation where patients with high BL pVL showed a longer decline period when compared to those with low BL pVL , 14. Furn = 11) underwent a therapy switch (Table n = 9), seven patients reached suppressed pVL within 17.2 (+/- 9.2) weeks (PV-10–PV-14 and PV-19–PV-20). Comparing the average time of decline for the groups with and without therapy switch, respectively, there was no significant difference . As shown in Table n = 6/14) of these patients showed sustained suppression of HIV-RNA. However, most interestingly, we found that the percentage of patients who showed up with episodes of LLV after reaching <50 pVL was particularly high. Here, 42.9% (n = 6) of patients with PV showed up with low-level viremia (LLV) during the follow-up time, which, in general, was more than threefold higher tendency compared to approx. 12% in non-PV patients  of the patients.We investigated whether PV might be associated with the occurrence of blips or of episodes of low-level viremia (LLV) during the follow-up time after the patients have reached pVL below the LOD. Here, a blip was defined as single virus detections that indicates transient viremia only, while, in contrast, LLV was defined as at least two consecutive pVL (>50 RNA copies/ml and <1000 RNA copies/ml). For this analysis, we only included patients who received a first-line therapy and who reached pVL <50 RNA copies/ml (n = 17) into two smaller groups. We compared the outcome of the group of patients having low drug levels (LD) or drug-resistant viruses (n = 8) with the group of patients without these features (n = 9) but did not found different frequencies of LLV experience after PV. Interestingly, we did not observe blips in the group without LD and DRMs, while the blip frequency in the other group was comparable to studies published previously [Based on this finding, we sub-divided the group of patients finally reaching LOD (<50) regardless whether they received the first-line or multi-line therapy  show a low probability of achieving viral suppression within 72 weeks . When an+ cells and by antiretroviral drugs [In former studies, a significant higher risk for the development of drug resistance was detected in patients whose viral load decreased with a relatively slow rate . In our al drugs , 28. Heral drugs . Thus, tThe importance of the viral reservoir, additional host factors, or compliance issues as the underlying reason for PV is further emphasized by the fact that patients with PV in this study were especially prone to show up with low-level viremia episodes during the follow-up time. We found that the rebound probability and risk to show up with LLV after a period of PV were more than threefold higher (~43%) when compared to the above-mentioned studies by Moore et al. and Sklar et al. (~12%) , 26. HerCmin values of each drug and therapy application form to determine whether drug levels were available in sufficient quantities. The drug levels were measured only once at the time of genotyping, so that only a strong generalized representation is possible. Although our sample size was small, this work clearly showed the heterogeneity of the underlying factors of PV and the urgent need of identifying markers for classifying PV according to its clinical outcome. The major finding of this study, however, was the observation that PV appears to be a risk factor for undergo episodes of LLV.Since the overall number of patients in our study was low and multiple factors seemed to independently influence the occurrence of PV, this study has certain limitations. In addition, the patients studied were treated with different ART regimens, so the influence of drugs on PV could not be adequately evaluated. In a comparative analysis of treatment-naïve patients receiving NRTI and INSTI versus NNRTI and boosted PI, for instance, the benefit of INSTI, in particular raltegravir, on RNA plasma decline comes obvious . Since t


Emphasis on adolescent HIV has increased worldwide as antiretroviral treatment has greatly extended life expectancies of HIV-positive children. Few evidence-based guidelines exist on the optimal time to disclose to an adolescent living with HIV (ALHIV); little is known about the medical effects of disclosure. This study looked to determine whether disclosure is associated with improved medical outcomes in ALHIV. Prior work has tended to be qualitative, cross-sectional, and with an emphasis on psychosocial outcomes. This paper addresses the adolescent cohort retrospectively , building upon what is already known about disclosure.Retrospective, longitudinal clinical record reviews of ALHIV seen at Kericho District Hospital between April 2004 and November 2012 were performed. Patient demographics and clinical outcomes were systematically extracted. The student’s t-test was used to calculate changes in mean CD4 count, antiretroviral therapy (ART), and cotrimoxazole adherence pre- vs. post-disclosure. Linear regression modelling assessed for trends in those clinical outcomes associated with age of disclosure.Ninety-six ALHIV  were included; most (73%) entered care through the outpatient department. Nearly half were cared for by parents, and 20% experienced a change in their primary caregiver. The mean time in the study was 2.47 years; mean number of visits 10.97 per patient over the mean time in the study. Mean disclosure age was 12.34 years. An increase in mean ART adherence percentage was found with disclosure . Younger disclosure age was associated with significantly higher mean CD4 counts over the course of the study (p = 0.001), and a nonsignificant trend toward a higher mean ART adherence percentage (p = 0.055).ART adherence and improved immunologic status are both associated with disclosure of HIV infection to adolescent patients. Disclosure of an HIV diagnosis to an adolescent is an important means to improve HIV care. Adolescents and young adults account for 40% of the world’s new HIV infections, and remain a socially and economically vulnerable population. In 2012Despite these risk factors and for HIV transmission and acquisition, challenges to effective adolescent antiretroviral therapy (ART) are encountered in low- and middle-income countries. Such challenges include psychosocial issues such as peer pressure, the stigma of HIV infection, and school-related concerns, as well as poor medication adherence–9. Adherdisclosure–has been looked at somewhat extensively vis-à-vis the effect it has on access to and retention in care, on how and when the disclosure process should be performed, and on the psychosocial benefits of disclosure to the pediatric or adolescent patient[Opportunities exist to affect adolescents’ and emerging young adults’ prognosis, through their own understanding and ownership of a disease process. Depending on an individual adolescent’s resiliency, support network and resources, knowledge of his or her own HIV infection can either be remarkably valuable or potentially devastating. Specific knowledge of one’s own HIV status–t patient,11–19. Ht patient,20–24; hTo date, most studies have been primarily qualitative psychosocial assessments, and few have focused on medical outcomes. Quantitative studies and outcomes data are far less prevalent; one study done in Romania showed that a patient’s knowledge of his or her HIV infection was related to delayed disease progression, while aDeveloping countries and regions may still be hampered by infrastructure and resource deficits, which could serve to amplify difficulties with adolescent ART adherence. Donor nation supply channels and recent donor nation austerity measures may impact ART availability. Although non-availability of first-line antiretroviral (ARV) medications is not common in public (e.g. KDH) or established private  treatment facilities in Kenya, stockouts have been reported in 34% of free-standing clinics and 43% of NGO-led facilities. “Youth-To that end, this study aims to determine whether disclosure is associated with improved medical outcomes in ALHIV.The United States Army Medical Research Unit—Kenya/Walter Reed Project (USAMRU—K/WRP) collaborated with the Kenya Minstry of Health (MOH) and the Kenya Medical Research Institute (KEMRI) and initiated an HIV care and treatment program in the South Rift Valley Province of Kenya in 2004. This HIV care and treatment program’s objective was to support and improve existing health care systems to provide both medical and psychosocial care to HIV-infected patients, including children and adolescents. Currently the program supports eleven district-level HIV treatment hospitals in the South Rift Valley Province, including Kericho District Hospital (KDH), a public hospital which was the site of the study. KDH serves as a referral center for smaller health facilities in the region.The study population included all HIV-infected persons who were enrolled in the KDH clinics, who were between the ages of 9 and 19 years during the retrospective study period, and who were enrolled for at least one year during that age range. Other inclusion criteria were documented patient care in the KDH pediatric HIV clinics between 1 April 2004 and 1 November 2012, and at least one predisclosure and one postdisclosure visit. Patients who did not have a documented disclosure date were excluded.This retrospective, coded clinical chart review aimed to describe the disclosure status, characteristics and outcomes of adolescents who were enrolled in the KDH HIV clinics. Individual medical records were reviewed, and study variables consolidated on a data collection form, which was de-identified by design. The KDH information technology chief, and KDH data clerks, who were not part of the study, were the only personnel with access to the protected key linking data collection forms to medical records. No study personnel had access to this link. All databases were password protected. Analyses proceeded on a coded and protected study database that was formed by extraction of data from individual outpatient medical records kept at the outpatient HIV clinic in Kericho. To ensure data was veritable, ten percent of data extractions were reviewed with the patient record for accuracy.th percentile for sex and age; 11) absolute CD4 count; 12) viral load; 13) hemoglobin ratio, calculated as the patient hemoglobin relative to the 50th percentile for sex and age; 14) ART adherence; and 15) presence of additional diagnoses, with description when available. Measurement of ART adherence involved caretaker or patient self-report through clinical interviews, standardized with the Morisky Medication Adherence Scale[Variables extracted and studied included: 1) study ID; 2) clinic enrollment date; 3) sex; 4) date of birth; 5) date of death, when applicable; 6) entry point to care; 7) primary caregiver; 8) HIV diagnosis disclosure status; 9) date of disclosure, when applicable; 10) body mass index (BMI) ratio, calculated as the patient BMI relative to the 50nce Scale, and an A systematic process of full disclosure is incorporated into the management of the adolescent patients at KDH: 1) the age of the adolescent guides when and how disclosure is done; 2) baseline and updated information is gathered as to what the adolescent knows about his or her clinical management; 3) the healthcare team ascertains the relationship of the caregiver to the adolescent patient, which helps to support the adolescent if disclosure is done during the visit; 4) the healthcare team supports the caregiver if disclosing the HIV status during the clinic visit; 5) the patient is educated on ailments that are managed in the hospital; 6) HIV/AIDS and modes of transmission are discussed with the adolescent; 7) during disclosure, the adolescent is linked to support groups that are held after the scheduled clinic visit. There, he or she is involved in activities that are geared toward patient education and coping with long-term treatment.Data analysis on the coded study database was done using Stata 13 . Demographic data was captured and reviewed for patterns. Disclosure age was calculated to be the age of the patient at the first visit marked “disclosed.” Adherence to cotrimoxazole and ART was calculated as the percentage of visits the patient was deemed to be adherent per the standardized clinical adherence tools. A t-test was used to calculate differences between patient mean cotrimoxazole adherence percentage, mean ART adherence percentage, mean CD4 count, and mean viral load, before and after disclosure. Finally, linear regression was used to look at relationships between disclosure age and mean ART adherence percentage and mean CD4 count, while controlling for demographic variables.The study was conducted according to the Declaration of Helsinki, local regulatory requirements, Protection of Human Subjects (45 CFR 46), and DoD regulations on the protection of human subjects (32 CFR 219). The protocol was reviewed and approved by the institutional review boards (IRB) or independent ethics committees of the Kenya Medical Research Institute, which includes the Center for Clinical Research Scientific Committee, the Kenya Medical Research Institute (KEMRI) Scientific Steering Committee, and the KEMRI National Ethics Review Committee; and the Walter Reed Army Institute for Research (WRAIR) IRB. To ensure confidentiality, only a password-protected and de-identified electronic database was used for data analysis, with the protected patient-study ID key never available to study personnel, or anyone outside of the data clerks and KDH IT chief. As no human subjects were contacted during the retrospective study, with the strict procedures in place to maintain a de-identified database, since consent would necessarily compromise nondisclosure status to those patients unaware of their HIV infection, and since the study presented no more than minimal risk of harm to subjects, KEMRI and WRAIR justified a waiver of informed consent to the study.Ninety-six patients met inclusion criteria and were considered in the analysis . Among t2 = 0.2756) while controlling for sex, entry point to care, main caregiver, caregiver change, and total number of visits. No significant difference was found in mean ART adherence percentage with earlier ages (in years) of disclosure  while controlling for the same variables. None of the control variables were found to be significantly correlated to either of the outcome variables.A correlation was found between disclosure age and mean CD4 . Our findings are consistent with previous qualitative and cross-sectional studies that have described improved adherence with disclosure,26,29,30prevent clinical decline through proactively involving an adolescent in his or her clinical care at an earlier age. Our finding of improved mean ART adherence percentage with disclosure to adolescent patients is consistent with the improved mean CD4 count seen with earlier disclosure.Translated into clinical terms, and based on our regression model, each year of disclosure delay is associated with a substantially lower mean CD4 count . This relationship between older age and lower mean CD4 count may be related to one of the reported rationales for disclosure of HIV infection to an adolescent—waiting for HIV disease progression and related illness before disclosing—that has been described previously. This coThe KDH disclosure process involves linking adolescents with HIV support groups, where peers with similar challenges and concerns may seek assistance. Although disclosure is generally a logical requisite to participate in ALHIV support groups, it is impossible to state which aspect or aspects of disclosure  are responsible for improved adherence. There was no data in the medical record that recorded or described support group involvement or the extent of that involvement. ART adherence and health effects of adolescent peer group participation are interesting areas for future and continued research.Our study has some limitations. Disclosure is difficult to define, and is a process more so than an absolute, yet our measurement was binary out of necessity. Although disclosure was consistently and systematically assessed at each clinic visit over the ten-year study period, there may have been inter-rater variability over time. It is also quite possible, particularly in the older adolescents, that patients were aware, or had a suspicion, of their HIV diagnosis even before they were formally told by their clinical teams and caretakers. It is difficult to know the length of such a transition period, or to know the effects of delayed disclosure on a suspecting adolescent.As with many similar studies in low and middle income countries, lack of resources leads to gaps in data. This study was conducted before the World Health Organization’s 90-90-90 targets and push for viral load tracking as the desired clinical outcome, and we were unable to assess for an association between disclosure and viral load given the paucity of viral load assays completed during the study period. As sites similar to Kericho are able to consistently track viral loads in addition to adherence and CD4, future prospective studies are needed to look for links between disclosure and clinical outcomes in ALHIV.In conclusion, our results support disclosure of an HIV diagnosis to an ALHIV in order to improve ART adherence and CD4 counts. Our findings may further indicate that disclosure should occur proactively, at younger ages and in developmentally appropriate ways, before CD4 decline; this is an area of interest for future research. This study adds to the body of growing literature that supports the importance of earlier disclosure to adolescents, and helps to inform the continued discussion on disclosure of an HIV diagnosis to adolescents.S1 File(CSV)Click here for additional data file.


Treatment options among Human Immunodeficiency Virus (HIV)-infected children are limited as only a few Highly Active Antiretroviral Therapy (HAART) are approved worldwide for paediatric use. Among children, frequent changes in HAART regimen can rapidly exhaust treatment options, and information addressing this issue is scarce.The aim of the study was to determine factors associated with the modification of initial HAART regimen modification among HIV-infected children.A retrospective study was performed among HIV-infected children aged 18 and below, that received HAART for at least six months in a tertiary hospital in Malaysia. Factors associated with modification of initial HAART regimen were investigated.Out of 99 patients, 71.1% (n=71) required initial HAART regime modification. The most common reason for HAART modification was treatment failure . Other reasons included drug toxicity , change to fixed-dose products , product discontinuation  and intolerable taste . The overall mean time retention on initial HAART before regimen modification was 3.32 year ± 2.24 years . Patient's adherence was the only factor associated with initial regimen modification in this study. Participants with poor adherence showed a five-fold risk of having their initial HAART regimen modified compared to those with good adherence .Poor adherence was significantly associated with initial regimen modification, intervention to improve patient's adherence is necessary to prevent multiple regimen modification among HIV-infected children. ThisThere is a lack of data addressing the reasons and factors associated with initial HAART regimen modification among paediatric HIV patients in the Malaysian clinical setting. It is crucial to understand the factors that lead to the initial HAART regimen modification. This is especially true in the paediatric population in order to assist in better decision making of first HAART choice. Optimization of initial HAART in younger patients could lead to better interventions, as well as reduced risk of abrupt HAART interruption. The aim of this study was thus, to determine the factors associated with initial HAART regimen modification among paediatric HIV patients who receive HAART treatment in Malaysia.22.1This was a retrospective study, performed in a Malaysian paediatric infectious disease referral centre in Hospital Kuala Lumpur, Kuala Lumpur, Malaysia. HIV-infected children, aged 18 and below, that had received HAART  for at least 6 months from January 2008 to December 2015, were included. The HAART regimen included in the study were two Nucleoside Reverse Transcriptase Inhibitors (NRTI) - zidovudine and lamivudine, and a Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI) or Protease Inhibitor (PI).2.2Patient's information was collected by using a standardized patient data collection sheet. The information included patient's age on HAART initiation, gender, race, main carer during HAART treatment, World Health Organization (WHO) clinical disease staging at HAART initiation, baseline immunodeficiency staging using CD4 count at HAART initiation, baseline viral load, initial HAART regimen used, any regimen modification, reason for modification if occurred, the new HAART regimen used, follow up viral load and immunodeficiency staging using CD4 count at the time of regimen modification, history of previous antiretroviral exposure before HAART initiation and patients’ adherence level to HAART treatment.2.31) [In this study, HAART was defined as: a regimen that consists of at least three antiretroviral drugs from at least two drug classes that were approved for HIV infection based on international and national guidelines , 12. Reg1) . Treatme1) . Previou1) .2.4Data was analysed using SPSS  version 23. Descriptive statistics were used to determine overall frequency of initial HAART regimen modification and to identify the most common reason for regimen modification. Univariate and multivariate logistic regression analysis was conducted to determine factors associated with initial HAART regimen modification. Factors tested in the univariate analysis were gender, ethnicity, age on HAART initiation, main carer, adherence, antiretroviral exposure before HAART, initial HAART regimen, baseline immunodeficiency at HAART initiation and WHO clinical stage at HAART initiation. Crude and adjusted odd ratios (OR), 95% confidence interval (CI) and p-value were presented for regression. The mean duration of retention on initial HAART before regimen modification was analysed using ANOVA or T-test. A p-value ≤ 0.05 was considered significant.33.11. A total of 110 paediatric HIV infected patient records were screened from January 1, 2008 to December 31, 2015. A total of eleven patients were excluded as HAART was initiated for less than six months. Patient's demographic profiles and clinical characteristic are shown in Table 1. All patients were diagnosed as mother-to-child transmission patients. Among the 99 patients, 53.5% (n = 53) were male. The ethnicity of more than half of the participants were Malay , followed by Chinese , Indian , indigenous  and others . Other ethnicities were patients mainly from Myanmar and Thailand.Demographic characteristics of the patients  are shown in Table 3.2The overall percentage of HIV-infected children that required initial HAART regimen modification in this study was 71.1% (n = 71). Modification of two or more agents (treatment failure and drug toxicity) occurred in 53 (52.5%) patients. Treatment failure defined as virological or clinical or immunological failure was the most common reason for initial HAART regimen modification within the studied population , whilst drug toxicity occurred in 14 (19.7%) patients. Modification of one agent occurred in 18 (17.8%) patients and accounted for the remaining cases that required initial HAART regimen modification: a change to fixed-dose products , product discontinuation  and intolerable taste .The median duration of retention time on initial HAART before a regimen modification among the study population was 3 years (IQR 2). The time taken for modification of two or more agents was a median of 3.3 years (IQR 2), whilst it took a median of 2.5 years (IQR 0.2) for modification of one agent (p >0.05). Patients with good adherence showed a significantly longer mean retention time on initial HAART  compared to patients with poor adherence  (p = 0.013). No other significant findings were observed.3.32). It was demonstrated that the factors affecting initial HAART regimen modification were previous antiretroviral exposure , adherence , and WHO clinical stage at HAART initiation . Patients with a previous exposure to antiretroviral were 8.5-fold more likely to modify initial HAART regimen compared to those that were not exposed. Poor adherence increased the risk of modification of initial HAART by 5.8-fold compared to good adherence.In order to assess factors affecting modification in initial HAART regimens, a univariable analysis was performed (Table 2). Results showed that only patients' adherence level remained associated with initial HAART regimen modification in the studied population in a logistic multivariate analysis. Patients with poor adherence demonstrated were 5.2-fold more likely to have their initial HAART regimen modified compared to those with good adherence .Multivariable logistic regression analysis was conducted to determine the factors associated with initial HAART regimen modification when adjusting for potential confounders (Table 4As in most parts of the world, the younger population in Malaysia remains vulnerable to HIV. In the current work, the ethnic balance of HIV paediatric patients observed, clearly represents the Malaysian population . Free HAHAART is vital for maximal clinical benefit in HIV-infected paediatric patients . DespiteVarious factors have led to initial HAART modification . Among fReasons for poor adherence to HAART among HIV infected children is often multifactorial , 28, 29.As with all studies, there were a few limitations in the current work. Firstly, being a retrospective study, the accuracy of the data could not be confirmed with the patients or clinicians. Furthermore, there were only three participants who fell within the age category of 10-19 years old, and a larger, multi-centred study could reduce these limitations in future work. More importantly, it should be noted that this study intended to serve as a baseline data of paediatric HAART modification in the local setting, looking into general changes of HAART. Thus, the broader term of HAART modification was used in the current work, taking into account major modifications involving two or more agents such as drug toxicity, and minor modifications involving one agent such as product discontinuation. However, findings suggest that drug toxicity and product discontinuation may not be related to differences in adherence. Further work could be performed by separating these two outcomes for a more specific results. In addition to this, other variables such as calendar year effects of drug modification may also affect clinical outcome of HAART. Hence, generalizability of the study should be done with caution.In conclusion, the overall percentage of children infected with HIV that required initial HAART regimen modification was high and the most common reason for initial HAART modification was treatment failure. Poor adherence was found to be an independent predictor of initial HAART regimen modification within the local population. It is important to note that although HIV clinics tend to stress on the importance of adherence to all patients, other factors may also affect medication adherence. Furthermore, findings of the study suggest that more work should be performed to determine factors that may affect HAART modification as adherence of HAART medication may not apply to modifications involving toxicity and product discontinuation.


Cryptococcus from a clinical specimen, and detection of cryptococcal antigen. Without appropriate treatment, cryptococcosis is fatal. Early diagnosis and treatment is the key to treatment success. Treatment of cryptococcosis consists of three main aspects: antifungal therapy, intracranial pressure management for cryptococcal meningitis, and restoration of immune function with antiretroviral therapy (ART). Optimal integration of these three aspects is crucial to achieving successful treatment and reducing the mortality. Antifungal therapy consists of three phases: induction, consolidation, and maintenance. A combination of two drugs, i.e. amphotericin B plus flucytosine or fluconazole, is preferred in the induction phase. Fluconazole monotherapy is recommended during consolidation and maintenance phases. In cryptococcal meningitis, intracranial pressure rises along with CSF fungal burden and is associated with morbidity and mortality. Aggressive control of intracranial pressure should be done. Management options include therapeutic lumbar puncture, lumbar drain insertion, ventriculostomy, or ventriculoperitoneal shunt. Medical treatment such as corticosteroids, mannitol, and acetazolamide are ineffective and should not be used. ART has proven to have a great impact on survival rates among HIV-infected patients with cryptococcosis. The time to start ART in HIV-infected patients with cryptococcosis has to be deferred until 5 weeks after the start of antifungal therapy. In general, any effective ART regimen is acceptable. Potential drug interactions between antiretroviral agents and amphotericin B, flucytosine, and fluconazole are minimal. Of most potential clinical relevance is the concomitant use of fluconazole and nevirapine. Concomitant use of these two drugs should be cautious, and patients should be monitored closely for nevirapine-associated adverse events, including hepatotoxicity. Overlapping toxicities of antifungal and antiretroviral drugs and immune reconstitution inflammatory syndrome are not uncommon. Early recognition and appropriate management of these consequences can reinforce the successful integrated therapy in HIV-infected patients with cryptococcosis.Cryptococcosis has been one of the most common opportunistic infections and causes of mortality among HIV-infected patients, especially in resource-limited countries. Cryptococcal meningitis is the most common form of cryptococcosis. Laboratory diagnosis of cryptococcosis includes direct microscopic examination, isolation of  Cryptococcosis is an important opportunistic infection among HIV-infected patients particularly in sub-Saharan Africa and in South and Southeast Asia. It is estimated that more than 600,000 deaths each year globally are from cryptococcosis . The mosART has proven to have a great impact on survival rates among HIV-infected patients with cryptococcosis , 4. The Cryptococcus is believed to occur mainly after inhalation of desiccated yeast cells or basidiospores into the alveoli. Other proposed portals of entry include gastrointestinal tract, direct inoculation from trauma, and transplantation of an infected organ . Thi. Thi2]. Cryptococcus are rarely required [Pulmonary cryptococcosis in HIV-infected patients is frequently a manifestation of disseminated disease. Hence, these patients should be evaluated for CNS involvement and disease dissemination, even if they are asymptomatic. A CSF examination, culture, as well as testing for cryptococcal antigen in the CSF and serum should be performed. Disseminated disease should be treated with the same regimen as that of cryptococcal meningitis , 9. For required .Similar to pulmonary cryptococcosis, cryptococcal infection of other organs is often a manifestation of disseminated disease, especially in HIV-infected patients. These patients should be evaluated for CNS involvement and disease dissemination as well , 9. For Positive serum cryptococcal antigen has been shown to be associated with subsequent cryptococcal disease –110. ScrPregnant women with cryptococcosis represent a special population. Amphotericin B can be used safely without risk of teratogenicity in humans (FDA pregnancy category B) , 9, 115.2O or 50% of initial opening pressure are recommended [In cryptococcal meningitis, intracranial pressure rises along with CSF fungal burden and is associated with morbidity and mortality –12. Incrommended . Brain iommended . Interesommended . This fiommended . PatientMedical treatment such as corticosteroids, mannitol, and acetazolamide are ineffective and should not be used , 9. A reAfter appropriate treatment, clinical improvement should be observed, usually within 2 weeks. In patients with cryptococcal meningitis, increased intracranial pressure usually resolves. Follow-up CSF cultures gradually turn negative. It is recommended by several experts that a CSF culture at 2 weeks should be sent to determine CSF sterility after induction phase . A persiManagement of patients who fail to achieve CSF sterility at 2 weeks awaits more data, but experts suggest induction therapy for another 2 weeks and a follow-up CSF culture obtained . NonethePersistence is arbitrarily defined as lack of clinical improvement and continued positive cultures after 2–4 weeks of appropriate therapy . In suchCryptococcus spp. and other potential pathogens are warranted. Disease relapse may be more likely if the patient has not yet received ART or has virologic failure. Patients who received inadequate induction treatment and/or had poor adherence to consolidation/maintenance therapy are also at high risk for disease relapse. Relapse was more frequent in cohorts using induction therapy with only fluconazole monotherapy [Relapse is defined as a recurrence of symptoms, after an initial resolution, with a positive CSF culture after ≥4 weeks of treatment , 9. It rotherapy , 88. Amootherapy . Anotherotherapy . Yet anootherapy . After aotherapy . Of noteotherapy . Newer aotherapy . Like neART constitutes an essential component in the management of AIDS-related cryptococcosis. Most patients are antiretroviral-naïve at the time of diagnosis of cryptococcosis. Delayed ART initiation places the patients at risk for other opportunistic infections. However, too early ART initiation increases the risk of developing IRIS. Consequently, the time as to when ART should be commenced is crucial. Delayed initiation of ART until at least after completion of induction therapy and possibly until after completion of consolidation therapy, has been recommended . A recenIn general, any effective ART regimen is acceptable. There is no data regarding the benefit of one certain ART regimen over another. Amphotericin B does not affect the CYP450 enzyme system; therefore, pharmacokinetic interactions between amphotericin B and most antiretroviral agents are not expected . HoweverAs a relatively modest inhibitor of CYP3A4, fluconazole has the potential to increase concentrations of NNRTIs. No significant interactions have been observed with efavirenz . A cliniAmphotericin B has multiple adverse effects, e.g. nephrotoxicity, anemia, electrolyte abnormalities, and infusion reactions . TherefoRegarding overlapping toxicity with antiretroviral drugs, both amphotericin B and tenofovir can cause nephrotoxicity. Concomitant use of these agents should be considered with caution. In a cohort study of 222 HIV-infected patients, prior exposure to amphotericin B was found to be a risk factor for nephrotoxicity . In contAnemia represents a common toxicity with amphotericin B therapy in HIV-infected patients with cryptococcal meningitis. A recent study has demonstrated that amphotericin B-induced anemia was mostly transient and did not impact mortality . AmphoteFluconazole may cause prolongation of the QT interval either directly or by inhibiting the hepatic metabolism of other QT-prolonging agents. A clinical trial to assess QT intervals in 141 HIV-infected patients with cryptococcal meningitis has demonstrated that there were no differences of QTc interval prolongation between those that received and did not receive fluconazole 800 mg/day in addition to amphotericin B in the induction phase of treatment . HoweverImmune reconstitution inflammatory syndrome (IRIS) is characterized by clinical deterioration with symptoms and signs of inflammation, resulting from exaggerated host immune responses against pathogens or antigens during immune reconstitution. IRIS can occur after ART initiation, re-initiation, or switching to a more active regimen following virological failure. It has a wide range of clinical manifestations as well as severity. IRIS symptoms often mimic those of infections, and represent a diagnostic challenge for clinicians.IRIS can be classified into paradoxical IRIS and unmasking IRIS. The former describes a worsening, after an initial clinical improvement of a pre-existing infectious process following ART initiation, while the latter describes a subclinical disease that becomes clinically apparent after ART introduction. Consensus definitions of cryptococcal IRIS have been proposed by the International Network for the Study of HIV-associated IRIS (INSHI) .Paradoxical cryptococcal IRIS has been reported in 8–49% of patients with known cryptococcosis at ART initiation . The repPathogenesis of IRIS remains to be further elucidated and is currently an active area of research –155. A nCryptococcal paradoxical IRIS is a diagnosis of exclusion. Patients with clinical deterioration should be initially managed similarly to those with suspected relapse disease, since other possible etiologies of clinical deterioration require distinct management as mentioned above. Shelburne et al. showed that patients with paradoxical IRIS returned later, were more likely to receive ART, had more CD4 count increases and more viral load declines in response to ART, and had higher CSF opening pressure than those with relapse disease . Higher Management of paradoxical cryptococcal IRIS is based on data from case reports and expert opinions. ART and antifungal agent(s) continuation are generally recommended , 9. For In the unmasking IRIS, the symptoms arise after ART initiation , 168. GiThe mortality in HIV-infected patients with cryptococcosis is high during the first few months of treatment. Managing the two diseases more effectively during this critical period is essential to improve the patients’ survival and quality of life. The time to start ART in HIV-infected patients with cryptococcosis has to be deferred until 5 weeks after the start of antifungal therapy. Integrated therapy of HIV and cryptococcosis including antifungal therapy, intracranial pressure management for cryptococcal meningitis, and ART to restore immune function is the key to success. Integration of care for both cryptococcosis and HIV using a single facility and a single health care provider is a model to deliver integrated therapy for both diseases and manage overlapping toxicities of antifungal and antiretroviral drugs and IRIS more effectively.


Assessment of virological outcomes among HIV-infected patients receiving protease (PR) inhibitor-based second-line regimen are uncommon in Cambodia. The objective of this study is to assess the virological effectiveness of this regimen as well as impact of adherence boosting for patients experiencing virological failure.The 2PICAM study  is a cross-sectional study of HIV-infected adults on PR inhibitor-based second-line regimen since at least 6 months, conducted in 13 representative sites, comprising more than 90% of the target population. Adults with HIV RNA above 250 copies/mL (threshold of the assay) at inclusion received boosted adherence counseling during 3 months followed by HIV RNA control. For confirmed virological failure, genotype resistance test was performed and expert committee used results for therapeutic decision.3  and >350/mm3 , duration of PI-based regimen >2 years , ATV-containing regimen  and high level of adherence . After adherence counseling, 63 (46.7%) patients were rescued while 72 (53.3%) were not. For the 54 patients with genotype resistance tests available, high or intermediate levels of resistance to lopinavir, atazanavir, and darunavir were reported for 13 (24%), 12 (22.2%), and 2 (3.7%) patients, respectively. Change to an alternative PR inhibitor-based regimen was recommended for 17 patients and to third-line regimen, including integrase inhibitors for 12.Among the 1,317 adults enrolled, the median duration of second-line regimen was 5 years. At inclusion, 1,182 (89.7%) patients achieved virological success (<250 copies/mL) and 135 (10.3%) experienced a virological failure (>250 copies/mL). In multivariable analysis, factors associated with virological success were: CD4 cell count between 201 and 350/mmThis study reports high rate of virological suppression of second-line regimen and importance of adherence boosting prior to deciding any change of ART regimen. Genotype resistance tests appear necessary to guide decisions. Such information was of great importance for National HIV Program to adapt guidelines and program needs for third-line regimen. Global ART coverage among estimated number of individuals living with HIV had reached approximately 41%—or 15 million people—by March 2015 –5 and chIn 2010, a multi-cohort analysis of 27 ART programs in resource-limited settings [only four with routine viral load (VL) monitoring] including three countries of Southeast Asia  reported treatment failure, defined as the first diagnosis of clinical, immunological or virological failure, for 19% of patients receiving PI-based second-line therapy for more than 6 months . More reThe present study reports the results of a nationwide cross-sectional study aimed to assess the virological effectiveness of PI-based second-line regimen in Cambodia as well as the impact of adherence boosting in patients experiencing virological failure. HIV drug resistance mutations in patients experiencing confirmed virological failure despite adherence boosting are also described and options for alternative second-line or third-line regimen are discussed.At the time of the study in 2013, according to WHO recommendations, the first-line ART regimen in Cambodia consisted of two nucleoside reverse-transcriptase inhibitors (NRTIs) plus a non-nucleoside reverse-transcriptase inhibitor (NNRTI) and TDF + 3TC + EFV as a fixed-dose combination was recommended as the preferred option  is a cross-sectional assessment conducted in 13 representative ART sites (6 in Phnom Penh and 7 in province), selected because they displayed more than 30 adult patients on PI-based regimen. Overall, second-line regimen patients from all these sites comprised more than 90% of the total number of adult patients on PI-based regimens in Cambodia. At each site, patients were exhaustively enrolled if they were HIV-infected adults (age > 18 years old), on PI-based second-line regimen since at least 6 months and if they accepted to participate to the study.Patient recruitment began on February 2013 and ended on April 2014. Data about clinical events and previous antiretroviral regimen were collected retrospectively and reported on the Case Reporting Form the day of inclusion. Samples for CD4-cell count and HIV RNA VL testing were collected.http://www.hivfrenchresistance.org).HIV RNA VL was done at NCHADS laboratory by using the G2 Generic HIV-1 VL ANRS kit . HIV RNA was extracted from plasma using an Arrow extractor (Nordiag). The real-time PCR Detection was conducted on the ABIprism 7000 (Applied Biosystems). By using 0.2 mL of plasma and Qiagen extraction, the threshold of this assay is 250 copies/mL. Bulk sequencing of reverse transcriptase (RT), protease (PR), and integrase (IN) genes were initially performed at the Institute Pasteur of Cambodia and later at NCHADS’s laboratory. Nested-PCR amplified fragments were sent to the Macrogen Company  for sequencing. Chromatograms, sent back electronically (around 3 days later) were verified and analyzed using Ceq2000 (Beckman Coulter) software. Nucleotides and amino acid sequences were compared with the HXB2 reference sequence using Mega 4 software. Resistance-associated mutations (RAMs) and drug resistance analysis were performed using GREG2010 software according to ANRS algorithms (Adults with detectable VL at inclusion (>250 copies/mL) received boosted adherence counseling every month during 3 months according to national guidelines and returned for VL control at month 4 (VLM4). For those patients, the aim of the three interviews with the psychosocial counselor will be to correct and/or strengthen their adherence to treatment. Virological failure was defined as a VLM4 still above 250 copies/mL after adherence boosting. In case of confirmed virological failure, genotype analysis including RAMs and drug resistance analysis were sent to a Clinical and Virological Expert Committee (CVEC) in charge of providing recommendations to clinicians about the most appropriate ART regimen to propose to each patient.Adherence to ART was measured using a validated scale comprising 14 items. In this scale, patients provided information about the doses taken during the 4 days preceding the survey, about whether they respected the dose schedule during the previous 4 days and 4 weeks, and whether treatment interruption had occurred for at least two consecutive days within the previous 4 weeks. The algorithm proposed by Carrieri et al.  of Pasteur Institute (Paris).The primary outcome was the prevalence of virological success at inclusion, defined as an HIV VL < 250 copies/mL. The secondary outcomes were 1/the proportion of patients in virological success (<250 copies/mL) after 3 months of boosted-adherence counseling among those in virological failure at inclusion 2/the prevalence of drug resistance defined by the presence of RAMs inducing resistance according to the ANRS algorithm to one or more drugs in the standard second-line regimen(s) used in Cambodia.Potential explanatory variables included age, sex, CD4 cell-count, duration, and type of PI-based regimen and adherence level.P value of <0.2. Age and sex were kept in the final model as they were a priori specified confounders. Stata software version 14.2  was used for all statistical analysis.First, a univariable logistics regression analysis was conducted to examine the relationship between virological success at inclusion and each potential explanatory variable. Then, a multivariable logistic regression model was conducted by including those factors with evidence of an association in the univariable analysis and a Among the 1,348 PI-based regimen patients recruited for the study, 31 were excluded because of non-eligibility criteria and 1,317 patients were enrolled for evaluation. Results of the VL assessment at inclusion revealed that 1,182 patients (89.7%) had VL below 250 copies/mL and 135 (10.3%) had a VL above 250 copies/mL , and 29.9% of these patients were in an advanced stage of immunosuppression with CD4 below 200 cells/mm3. The duration on PI-based regimen was 3.9 years (IQR: 1.6–6.2) at the time of inclusion, and 97 patients (71.9%) were receiving ATV/r-based regimen while 35 (25.9%) were receiving LPV/r-based regimen and 3 (2.2%) an association ATV/LPV/r.The median age of patients with a VL above 250 copies/mL was 42 year-old (IQR: 35–48), and 87 (64.4%) were males  and >350/mm3  compared to CD4 cell-count <200/mm3, duration of PI-based regimen >2 years  compared to duration <2 years, ATV-containing regimen  compared to LPV-containing regimen and high leve of adherence  compared to low level of adherence.Factors associated with PI-based second-line regimen success at time of inclusion are reported in Table After 3 months of boosted adherence counseling, a VL control (VLM4) was performed for all patients with detectable baseline VL Figure . Among tGenotypic resistance test (GRT) was performed in all 72 patients with confirmed virological failure after 3 months of adherence boosting. The description of observed RT and PR drug resistance mutations is reported in Figure Reverse transcriptase sequences were available in 65 samples because of 7 PCR amplification failures. According to ANRS resistance algorithm, high or intermediate level of resistance to AZT and TDF was reported in 35 (53.8%) and 20 (30.8%) of them, respectively.Protease sequences were available in 54 samples because of 18 PCR amplification failures. High or intermediate level of resistance to LPV/r, ATV/r, and DRV/r was reported in 13 (24.0%), 12 (22.2%), and 2 (3.7%) patients, respectively. Globally, 20 (37%) patients had resistance to PI , and 34 (63%) did not present resistance to PI .For the 54 patients with GRT available including PR mutations, the recommendation of the CVEC was to continue current regimen for 25 (46.3%) patients (absence of resistance), change to an alternative PI-based regimen using drugs available in Cambodia  for 17 (31.5%) and change to a needed third-line regimen including IN inhibitors for 12 (22.2%) patients.For the six patients without GRT available for either RT or PR, no change of ART regimen was recommended.For the 12 patients with available GRT for RT but not for PR, the recommendation of the CVEC were to not change for seven patients (absence of resistance), change with available drugs for three patients, and change with an association including a PI/IN inhibitors for two patients because of resistance to all NRTI.The present study reports a high rate of virological suppression for HIV-infected adults on PI-based second-line regimen in Cambodia with 90% of patients in virological success at time of inclusion. In addition, almost half of those with a detectable VL could be rescued following 3-months of boosted adherence counseling giving a final 94.5% success rate under PI-based regimen. Higher success rates at baseline were observed in patients with high CD4 cell count, longer duration of PI-based regimen, ATV-containing regimen and high level of adherence. Overall, 5.5% of patients on PI-based regimen had confirmed virological failure in Cambodia and 12 of them were already in urgent need of third-line regimen. Such information was of great importance for the National HIV Program to adapt HIV management guidelines and program the needs for alternative second- and third-line regimen.Similar positive virological outcomes have been previously reported in Cambodia on a small cohort of 70 patients under lopinavir-based regimen  and in TThe control of adherence is a critical factor of ART success and was significantly associated with virological success in our study. WHO recommends adherence support intervention prior to deciding any change of ART regimen. Indeed, it was found that a significant number of patients could be rescued after initial identification of treatment failure but the effectiveness of such a strategy still remains unclear. We report such adherence boosting strategy is effective among PI-based second-line patients with 46.7% patients with detectable VL being rescued having an undetectable control VL after 3 months. Following this result, HIV national program has revised in 2017 the national guidance to enhance ART adherence. Similar results of adherence boosting strategy were reported in India where 34% of PI-based regimen patients with virological failure had a suppressed VL after 6 months  and more3), being on ATV/r containing regimen, having a high adherence level and a longer duration of PI-based regimen were independently associated with success. The longer delay of PI-based regimen associated with success might be surprising but could suggest that higher risk of failures and death occurred early after PI-based regimen initiation, as it was reported in India  for patients failing second-line regimen if VL remains above 1,000 copies/mL after 3 months of adherence boosting. In our study, among the 72 patients with confirmed VL failure after adherence boosting, 52 had a VL above 1,000 copies/mL and should have been switched to a third-line regimen. However, according to the results of GRT, only 12 were really in need of third-line regimen, which should include an IN inhibitor. The majority of the remaining patients can continue their current regimen or could benefit from adapted alternative second-line regimen. Because of the high cost of third-line drugs, the use of HIV GRT before switching to third-line regimen could be a cost-effective intervention, which needs further investigation. WHO recently recommend expansion of HIV drug resistance test and encourage innovative research in this topic to have the greatest public health impact . The useIntegrase inhibitors are essential components of third-line regimen but both raltegravir (RAL) and dolutegravir (DTG) are not currently available in Cambodia. The choice of one of these drugs will be important for National program awaiting further recommendations. RAL have shown favorable outcomes in association with LPV/r , 30 but Our study had some limitations. First, this is a cross-sectional study and we are not able to provide data on long-term follow-up, especially for patients with confirmed virological failure. Second, this study was limited to adults HIV patients and data about the effectiveness of second-line regimen in children and adolescents are still pending. Third, the findings related to the type of PI used could be due to bias by indication.In conclusion, this study reports the high rate of virological suppression of PI-based second-line regimen in Cambodia nation wide and the importance of boosted adherence counseling prior to deciding any change of ART regimen. Such a very high virological success rate of PI-based regimen after a median duration of 5 years reveals the strength of such regimen and is very encouraging concerning the duration of available PI-based second-line regimen.HIV genotyping appears necessary to guide decisions for alternative second-line or third-line regimen. IN inhibitors are key components of potential third-line regimen, and DTG could represent an interesting option. Results of this study were of great importance to adjust HIV national guidelines and to pave the way to a further assessment of a third-line regimen option in Cambodia.Patients were enrolled if they have signed a consent form after receiving appropriate study information. The study was approved by the National Ethics Committee for Health Research in Cambodia (NECHR) and the “Pole Intégré de Recherches Cliniques” (PIRC) of Pasteur Institute (Paris).Design of the study: EN, SAN, BS, VK, LF, MCV, PL, and VS. Collection of data: EN, SAN, VK, BS, and SS. Virological analysis: EN, CM, SON, and CC. Data interpretation: all authors. Data analysis: OS, EN, LF, BS, and MM. Writing committee: OS, EN, BS, LF, CC, and VS. Revising the work: all authors. Final approval of the version: all authors. Agreement to be accountable for all aspects of the work: all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Abbott Real-Time and Cobas AmpliPrep/TaqMan (v2.0), and virological failure defined as ≥ 1000 HIV-1 RNA copies/ml. Plasma from patients experiencing virological failure were processed for sequencing of HIV-1 protease-reverse transcriptase using the ANRS-AC.11 resistance testing protocol; drug resistant mutations were interpreted using the ANRS-AC11 algorithm; and phylogenetic analysis was performed using MEGA.v.6.The national antiretroviral therapy in the Republic of Chad provides free of charge antiretroviral regimens and therapeutic monitoring for patients receiving antiretroviral therapy nationwide. For a successful programmatic uptake, these efforts merit to be supported by thorough assessments of antiretroviral therapy response and HIV-1 drug resistance surveillance, especially with risks of cross-resistance due to the gradual stavudine phasing out in such national settings. We therefore evaluated the virological response to antiretroviral therapy, HIV-1 drug resistance emergence and circulating HIV-1 clades in a Chad context. A cross-sectional and prospective study was conducted among 116 patients  receiving first-line antiretroviral therapy for ≥ 6 months in Ndjamena, Chad, in 2011–2012, enrolled consecutively. To ensure accuracy, plasma viral load was concomitantly measured using Majority of patients was receiving zidovudine plus lamivudine plus nevirapine (46%), stavudine plus lamivudine plus nevirapine (41%) and tenofovir plus emtricitabine plus efavirenz (11%), for a median time-on-treatment of 5 [IQR 4–7] years. The rate of virological failure was 43% (50/116), with 86% (43/50) sequencing performance. Overall, 32% (37/116) patients presented ≥ one major drug resistant mutation(s), with 29% (34/116) to nucleos(t)ide reverse transcriptase inhibitors ; 86% (37/43) to non-nulceos(t)ide reverse transcriptase inhibitors ; and 2% (1/43) to protease inhibitors . Six HIV-1 subtypes were found: 30% circulating recombinant form (CRF02_AG), 30% J, 16% G, 9% A, 9% D, 5% F.In Chad, almost half of patients are failing first-line antiretroviral therapy after 5 years, with considerable drug resistant mutations at failure. Absence of K65R supports the use of tenofovir-containing regimens as preferred first-line and as suitable drug for second-line combinations, in this setting with significant HIV-1 genetic diversity.The online version of this article (10.1186/s13104-017-2893-1) contains supplementary material, which is available to authorized users. HIV/AIDS remains a major cause of death worldwide, and especially in sub-Saharan Africa (SSA) where over 71% of the global AIDS epidemic is concentrated in only 12% of the world population. In spite of a reduction in HIV-associated morbidity and mortality in SSA, about half of people living with HIV (PLHIV) still ignore their status, suggesting a potential growing burden of HIV in this region of the world .Located in central Africa, Chad is a country with 3.3% of HIV prevalence in the sexually active population i.e. 15–45 years) for a national population of 11.4 million inhabitants 5 years f. InteresART management and laboratory monitoring are effective and free-of-charge in the national AIDS program in Chad since 2007, with first-line regimens consisting of two nucleoside reverse transcriptase inhibitor (NRTI) and one non-NRTI (NNRTI). As per the World Health Organization (WHO) recommended guidelines, preferred first-line regimens since 2012 consist of “tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV)” or “zidovudine (AZT), lamivudine (3TC) and nevirapine (NVP)”; following phasing-out of Triomune due to lipodistrophy/lipoatrophy and peripheral neuropathy significantly associated with “stavudine” (d4T)-containing regimens , 8, 14. As first-line regimens used in Chad mainly consist of drugs with low-genetic barriers to resistance, risks of HIV-1 drug resistance (HIVDR) emergence are concerning. Of note, over 60% of ART failure was previously reported , 16, supIn this study, we sought to ascertain the rate of virological failure (VF), the level of drug resistance mutations and HIV-1 genetic diversity among people living with HIV (PLHIV) receiving first-line ART as per the Chadian AIDS program in N’Djamena. in Chad, between 2011 and 2012. This hospital was selected as sentinel site based on its role as the national reference center in Chad, its long-term experience on ART and its technical capacity in providing reference laboratory monitoring for ARV management. Participants were eligible if: (a) receiving first-line ART for ≥ 6 months, (b) self-reported adherent to prescribed ARV medications, (c) registered and followed-up on ART at the study clinic, and (d) providing consent as study participants.A prospective and cross-sectional study was conducted in PLHIV receiving first-line ART at the National Reference General Hospital of N’djamena A non-probability sampling was used, by which patients were conveniently enrolled based on accessibility throughout the study period.Following informed consent, participants were interviewed and assessed for eligibility criteria, then enrolled as study participants if eligible.g for 10 min. Plasma aliquots of 1 ml were prepared and stored at − 80 °C.Whole blood was collected in two EDTA tubes of 4 ml each, through venipuncture, and plasma was collected following centrifugation at 2000To evaluate the stage of disease progression, CD4 T lymphocytes were measured for all patients at baseline and at 6 [± 2] months of ART, based on the fluorescent activated cell sorting approach, using the commercially available FACS Count as per the manufacturer’s instructions .Laboratoire de Référence Sida du CHU) in Liege, Belgium, using two different approaches purposely to ensure accuracy on these non-B viral populations: (1) COBAS® AmpliPrep/CobasTaqman® HIV-1 version 2.0 (v2.0) which is based on in vitro amplification of HIV-1 RNA from plasma with detection thresholds ranging from 20 (lower) to 10,000,000 (upper) RNA copies/ml designed specifically for HIV-1 groups M and O; (2) the Abbott RealTime HIV-1 test Ref 2G3190 which is based on in vitro amplification by RT-PCR for the quantification of HIV-1 in plasma. Both PVL tests were performed as per manufacturers’ instructions.Plasma viral load (PVL) was performed at the AIDS Reference Laboratory of the University Health Center , as per the manufacturer’s instructions. Briefly, 140 µl plasma samples stored at − 80 °C was extracted using lysis and wash buffers, followed by elution of 60 µl RNA.RNA extraction was performed on plasma using the QIAamp mini kit (The ANRS AC11 protocol was used for amplification and sequencing of the protease (PR) and reverse transcriptase (RT) regions of HIV-1, using GeneAmp PCR System 9700 thermal cycler . Briefly® 96 PCR (Macherey–Nagel).Revelation of PCR products was done using 4% ethidium bromide agarose gel electrophoresis, with an expected size of 731 pb for RT and 507 for PR, including positive and negative controls alongside a molecular ladder (Tacklt ™ ΦX174 RF DNA/Hae III Fragment). Amplicons were purified PCR using NucleoFastAs per the ANRS AC11 protocol, HIV-1 PR-RT was sequenced using overlapping primers by deoxyterminators . Sequenchttp://www.hivfrenchresistance.org/). Viruses with a mutant or a mixture of wild type and mutant, at an amino acid’s position, were considered to have the resistant variant. Patients were considered as harbouring wild type viruses if their viral load was < 1000 RNA copies/ml  or with a non-amplifiable sample.Following the sequencing protocol used , HIV-1 Dhttp://www.hiv.lanl.gov/).Sequence alignment was done using CLUSTAL W version 1.7, then sequences were trimmed and gaps closed . PhylogeP < 0.05 considered statistically significant. Spearman correlation was used for quantitative data on PVL results obtained from the two different instruments, with R ≥ 0.8 considered as a strong positive correlation.Data were processed using EPI INFO version 3.3.2. Chi square test was used for analysing categorical data on VF according to ART regimens, including 95% CI, with a 3, min–max: 11–684 cells/mm3.Overall, 116 PLHIV treated at the National Reference General Hospital of N’djamena were enrolled in the study, divided into 59% (68) female versus 41% (48) male. Mean age of these participants was 41 (± 6.87) years, min–max: 17–84 years  were receiving nevirapine (NVP) plus lamivudine (3TC) plus zidovudine (AZT) or stavudine (d4T), followed by 11% on a tenofovir-containing regimen , and 109 samples yield comparable levels of HIV-1 RNA copies between both assays. A strong positive correlation (R2 = 0.96016) was observed between both PVL assays  viremia with Cobas AmpliPrep/TaqMan v2.0. Overall, both PVL assays accurately detect all patients with VF (≥ 1000 HIV-1 RNA copies/ml). Further characteristics of the two assays are provided in Additional file All one hundred and sixteen study participants were tested for PVL using both described quantitation assays  experienced VF. Between the two most prescribed regimens, VF was higher with d4T-containing (49% 24/49]) compared to other regimens (39% [26/67]), odd ratio (OR): 1.514  K103N/S/E, 26% [11/43] Y181C/V/F/L, 2% [1/43] L100I, 2% [1/43] F227L and 2% [1/43] P225H, followed by other DRMs observed at lower rates. Of note, Y181C/F and K103N were observed concomitantly in three (7%) cases. Thus, in the entire study population, NNRTI DRMs exhibit 28% (32/116) and 21% (24/116) high-level resistance to nevirapine and efavirenz respectively  patients, classified as VF, were reported without any DRMs, suggesting possible poor adherence in spite of the self-reported adherence registered prior to enrolment. The list of genotypic scores associated to each RTI is provided in Additional file Only one patient (< 1%) was reported with major DRMs to protease inhibitors (PI/r), among which M46I, I54V, and V82S, indicating either an event of transmitted PI-associated DRMs or unknown past-exposure to PIs. Minor mutations found were mainly polymorphisms: K20I/M (21), L10I/V (15), L90W (1), L76S (1), N88D (1), V11I (1), V32L (1) and G48R (1).The 43 protease-RT sequences generated clustered within six clades (five pure subtypes and one recombinant). Of note, the two equally most prevailing were subtype J (30%) and CRF02_AG (30%), followed by subtypes G (16%), A (9%), D (9%) and F (5%), as shown in Fig. The success of combination ART has remarkably changed the paradigm in the AIDS epidemics globally. However, such achievements could rapidly be hampered in settings where ART is mainly based on drugs with low genetic barrier to resistance , 22, 23.Our findings indicated that, about 5 years after ART initiation, almost half of PLHIV on first-line NRTI/NNRTI regimens in Chad would be experiencing VF, suggesting close virological monitoring is needed in the country ART program , 24–27. Entre-aide 92 team from Paris is assessing the feasibility of using GeneXpert for point-of-care VL in the cities of Moundou and Am Timan, in order to scale-up access to VL in Chad [The high rate of VF in our cohort could be attributed to infrequent PVL measurement in 2011–2012 due to the centralized system, thus resulting to delayed testing and suboptimal monitoring. At the moment of the study, VL was possible only at the National Reference General Hospital of N’djamena, the only facility nationwide whereby VL is routinely offered since 2005 to date. Presently, the  in Chad . TherefoAmongst those experiencing VF, relatively lower rates of NRTI and NNRTI mutations were found, possibly due to suboptimal adherence, in the frame of poor ART monitoring. This calls for an improved adherence support to enhance and sustain viral suppression in the country .For an accurate evaluation of virological response, a strong positive correlation in PVL was reported for samples tested concomitantly with two different platforms, which in turns confirms assay reliability in settings where non-B HIV-1 prevails . Of noteAs levels of acquired HIVDR are higher to NNRTIs 32%) compared to NRTIs 29%), conferring high-level resistance to NVP and EFV (mainly due to K103N and Y181C), NRTIs are more prone for use in second-line combinations in such ART program –32. Of n% compare%, conferA higher prevalence of CRF02_AG was found in Chad as compared to previous findings , 12, posA study limitation would be the “self-reported” adherence, making it difficult to verify the reliability of recalls. Phasing out of D4T may suggest not representativeness of the data on current ART regimens. However, D4T is the same drug class with AZT , and several patients have been exposed to these drugs. Our findings are therefore useful for all patients with past-exposure to the drug class of TAMs , 43.Complementary studies are needed to ascertain response after switch from first- to second-line , 44, 45,Our findings address issues that could be translated into policies. Of note, in addition to the need for closed viral load monitoring for timely detection of treatment failures, the high failure rate of ART in Chad also indicates a rapid switch of patients from first- to second-line ART regimens. Rapid switch to second-line ART lead to increased costs of ARVs provision for the national HIV program in Chad, thus representing a major programmatic challenge for such RLS. Interestingly, the unusually low rate of DRMs, developing after significant periods of treatment failure, suggests suboptimal adherence, thereby underscores the usefulness to closely monitor ART adherence and the need for confirming VF  before deciding on ART switch. Such measure would help clinicians in distinguishing elevated viremia due to non-adherence, thereby limiting unnecessary switch to second-line regimens while saving related-costs in the national ART program of Chad.About half of Chadian PLHIV experienced VF by medium-term after ART initiation, suggesting closed virological monitoring using commonly available commercial assays. For successful phasing-out of d4T, DRMs supports using current WHO-recommended TDF plus TFC (or 3TC) plus EFV as preferred first-line regimen in Chad, while LPV/r is potentially active as second-line backbone, alongside “TDF” or “ABC”, in association with NRTIs. The predominant rate of recombinant AG is a quest for further investigation within the sub-region.Additional file 1. PCR and sequencing Primers (ANRS AC 11). The primer sequences are designed for amplification and sequencing reactions of protease and reverse transcriptase regions.Additional file 2. Study participants by age range. The table details the study population by range age from 17 to over 60 years old, divided by male and female.Additional file 3. Characteristics of assays used for plasma viral load. The table reports the minimal and maximal values, as well as mean and median of viral loads, obtained on one hand with Cobas and on the other hand with Abbott.Additional file 4. Level of resistance to reverse transcriptase inhibitors. The table presents the proportion of patients with levels of genotypic susceptibility score following the Stanford algorithm.


Using the cryptococcal antigen (CrAg) lateral flow assay (LFA), screening is possible at the point of care (POC). However, critical shortages of health staff may limit adoption. This study investigates the feasibility of lay counsellors conducting CrAg LFA screening in rural primary care clinics in Lesotho.Cryptococcal meningitis is one of the leading causes of death among people with HIV in Africa, primarily due to delayed presentation, poor availability and high cost of treatment. Routine cryptococcal antigen (CrAg) screening of patients with a CD4 count less than 100 cells/mm3 were screened with CrAg LFA. All tests were performed by lay counsellors. CrAg-positive asymptomatic patients received fluconazole, while symptomatic patients were referred to hospital. Lay counsellors were trained and supervised by a laboratory technician and counsellor activity supervisor. Additionally, nurses and doctors were trained on CrAg screening and appropriate treatment.From May 2014 to June 2015, individuals who tested positive for HIV were tested for CD4 count and those with CD4 <100 cells/mm3. Of these, 128 (99%) were screened with CrAg LFA and 14/128 (11%) tested positive. Twelve of the 14 (86%) were asymptomatic, and received outpatient fluconazole. All commenced ART with a median time to initiation of 15.5 days [IQR: 14–22]. Of the asymptomatic patients, nine (75%) remained asymptomatic after a median time of 5 months  of follow up. One (8%) became co-infected with tuberculosis and died and two were transferred out. The two patients with symptomatic cryptococcal meningitis (CM) were referred to hospital, where they later died.During the study period, 1,388 people were newly diagnosed with HIV, of whom 129 (9%) presented with a CD4 count <100 cells/mmCrAg LFA screening by lay counsellors followed by pre-emptive fluconazole treatment for asymptomatic cases, or referral to hospital for symptomatic cases, proved feasible. However, regular follow-up to ensure proper management of cryptococcal disease was needed. These early results support the wider use of CrAg LFA screening in remote primary care settings where upper cadres of healthcare staff may be in short supply. CM is3 ,4.Many deaths from cryptococcal disease are attributed to delays in seeking healthcare, resulting in diagnosis when disease has already progressed to an advanced stage, at which point available treatments are less effective. Limited access to rapid diagnostic tests and materials for lumbar punctures (LPs) add to these delays . Access Early initiation of ART in CM patients can result in potentially life-threatening immune reconstitution syndrome (IRIS), and for that reason it is recommended that ART is delayed by 4–6 weeks following effective antifungal therapy. However, the presence of CrAg in the blood is highly predictive of future development of CM , and manIn countries where CD4 testing is predominantly laboratory based, several strategies to increase CrAg screening among at-risk patients are possible, such as laboratory-based “reflex” testing. This strategy entails automatic testing for CrAg among patients with a CD4 count <100 cells/mm3, using remnant serum from the CD4 tests. South Africa is currently doing this, in line with the National Strategic Plan for HIV, STIs and TB 2012–2016 [Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free and Deliverable to those who need it [Point-of-care (POC) testing using CrAg LFA offers an alternative strategy, allowing for immediate intervention. The CrAg LFA test has demonstrated high sensitivity (100%) and specificity (ranging from 94–100%) when used with whole blood, serum or cerebrospinal fluid (CSF) –15. AddiLesotho, with a population of 2.1 million, is a small highland country with the third highest HIV prevalence in the world, at 23.2% among adults aged 15–49 . ART wasLesotho’s population is largely rural, with 70% of people living outside of cities . LaboratThe prevalence of CrAg in patients entering ART programs in Lesotho has not been reported to date. Screening has not been implemented despite recommendation by the WHO and the existence of national guidelines that provide for this, which means that cases often go unidentified and untreated. Slow adoption may be attributable to human resource constraints, in particular the severe shortage of qualified healthcare workers.In the Roma- Semonkong area, most patients on ART are managed at the clinic level and in some clinics CD4 testing for ascertaining ART eligibility is done at the point of care by lay counsellors using PIMA machines. Given the widespread use of PIMA in Roma-Semonkong sub-district and the familiarity of lay counsellors with POC technology, the district is an ideal setting for the introduction of POC CrAg LFA screening.Since May 2014, MSF has supported routine screening using CrAg LFA followed by pre-emptive treatment of positive patients with outpatient fluconazole in 3 rural health facilities in the Roma-Semongkong sub district of Maseru district, Lesotho. Among our catchment population all clinics had access to PIMA, whereas nationwide PIMA access is variable and difficult to quantify due to the absence of national level data. This study describes the feasibility of implementing a screen-and-treat strategy for cryptococcal disease using lay counsellors and nurses in rural primary care clinics.3 with the POC CrAg LFA test  was implemented in three primary care rural clinics in the Roma- Semonkong sub-district , where the PIMA POC CD4 analyser was readily available.Screening of all HIV-infected patients with a CD4 count <100 cells/mmIn order to reduce nurse workload and improve patient flow, lay counsellors were trained to perform CrAg-LFA testing on capillary whole blood.A laboratory technologist trained several lay counsellors to collect finger-prick specimens, to read and record CrAg LFA results and to dispose of materials in a safe, bio-hazardous waste bin. Training materials included visual support aids, package inserts and standard operating procedures (SOPs) developed jointly with the Ministry of Health (MoH) Quality Assurance (QA) Office.A CrAg register was provided to each clinic for result recording. As part of on-going monitoring and supervision, a laboratory technologist and a counselor supervisor visited the clinics monthly to ensure adherence to SOPs and good clinical practice. To ensure that CrAg-positive patients received proper clinical management at the time of presentation, nurses were trained to appropriately treat asymptomatic disease and to refer symptomatic patients. Likewise, doctors at the referral hospital were provided with training in diagnosing and treating CM .Lay counsellors collected finger-prick specimens and performed CrAg-LFA testing in designated HIV counseling and testing (HCT) rooms. CrAg LFA testing was performed according to the manufacturer’s instructions.In order to ensure appropriate performance of all test strips included in each box, CrAg-positive and negative controls were performed for each box of tests. If both positive and negative control samples were correctly identified by the LFA, then all test strips included in that box were deemed to be working and ready for use.3 [In line with WHO’s Rapid Advice on diagnosis, prevention and management of cryptococcal disease in HIV-infected adults, adolescents and children, CrAg screening was carried out for ART-naïve HIV positive adults (>18 years old) with a CD4 count <100 cell/mm3 ,19. CrAg3 .Nurses were trained on the signs and symptoms of CM, including fever, headache, altered mental status, seizure, and neck stiffness . CrAg-poThis pilot project was carried out after screening for cryptococcal disease had been incorporated into Lesotho national guidelines. We applied for ethical approval for this work to the Lesotho National Heath research ethics committee. The committee exempted the proposal from research ethics review on the basis that it constituted the implementation of national guidelines. Because we were implementing something that was already part of national guidelines, participants were not asked to provide informed consent to participate, and this was not asked for by the ERB. The only patient samples used were finger-prick blood specimens for routine care, collected by Lesotho lay counsellors for CrAg LFA testing. No samples were stored or collected for any other reason.Demographic and clinical data for the 128 CrAg-LFA-screened people are presented in 3 compared with 53 cells/mm3 in females (p = 0.6).The median age was 35, . There was no difference in the mean age and mean CD4 count between patients testing positive or negative for CrAg. However, there was a statistically significant association with gender, with all but one of those testing positive being male (13/14). Median CD4 count at presentation for males was 50 cells/mm3. Of these, 128 (99%) were screened with CrAg-LFA, and 14 (11%) tested positive  had a CD4 count <100 cells/mmpositive .Twelve CrAg-LFA-positive persons were asymptomatic, and received outpatient fluconazole . All werThe remaining two CrAg-positive patients were diagnosed with CM and were referred to hospital where they subsequently died.One patient died during the follow up period, among those that tested CrAG positive but without showing symptoms of meningitis.3. This is the first CrAg prevalence data from ART clinic attendees in Lesotho, and although considerably higher than the prevalence observed in neighboring South Africa [We describe the successful implementation of point-of-care testing by lay counsellors and pre-emptive treatment of asymptomatic CrAg positive patients by nurses at primary care clinics in rural Lesotho. CrAg was detectable in 11% of patients with CD4 counts <100 cells/mmh Africa , these fh Africa ,9,20–22.Given the high CrAg prevalence observed and the high likelihood of mortality without pre-symptomatic or early disease identification and treatment, we consider routine screening and targeted pre-emptive antifungal treatment for those testing CrAg-positive to be a worthwhile intervention, particularly in settings like Lesotho, where the prognosis is poor once meningitis develops.The cost-effectiveness of this intervention has been supported through recent sub-Saharan and Southeast Asian data, which should provide an impetus for policy makers to consider this intervention, even in resource-limited settings ,23. PrevTo our knowledge, this is the first study showing how CrAg prevalence differs by sex ,7,9,20. The need for close supervision provided a particular challenge, especially during the initial phase of implementation. Fluconazole treatment involves 3 phases that are conducted over a fairly lengthy amount of time, and adherence to dosing protocols required constant reinforcement. This was complicated by the fact that we were working in remote areas, where access to secondary level care is limited. Furthermore, the relatively low number of CrAg-positive patients restricted the opportunities to supervise and discuss the cases with lay counsellors and clinicians during the consultation time. Achieving a high training coverage of staff was limited by high turnover and workload. Consequently, frequent refresher trainings were needed.We did not perform LPs on all asymptomatic patients because they were not available in nurse-operated, rural clinics. Hospital referral was difficult, and even at the hospital level it is difficult to ensure that a LP is carried out. Of our 2 referred patients, we have evidence of only 1 receiving a LP. Both, however, received treatment with amphotericin B, and fluconazole at the correct dosages. While more research is needed to investigate optimal indications for LPs, offering systematic LPs to all patients with a positive CrAg would not be feasible at the primary care level in resource-limited settings.Other challenges included poor documentation by clinicians, which compromised any potential retrospective review of cases. This was a problem particularly in respect of the recording of fluconazole treatment , outcomes  and referrals.The lack of adequate monitoring could contribute to the deaths among CM cases, as the lack of management of amphotericin-related toxicities remains a challenge in settings such as Lesotho. However, misdiagnosing early invasive cryptococcal disease as cryptococcal antigenaemia, or unmasking OIs, could also contribute to deaths in both arms (CrAg positive and negative). As it was not feasible to carry out a LP on all those with a CD4 <100 to identify early invasive disease, we sought to establish a safety net by closely following up all patients in the community, so that appropriate management could be instituted should symptoms develop.The two deaths in confirmed CM cases occurred during the amphotericin course, and could be related to its toxicities, although this was not confirmed.The main objective of the short-course (5–7 days of amphotericin B deoxycholate administration) strategy recommended by WHO is to reduce the risk of complications related to amphotericin B administration in places where laboratory monitoring is scarce or absent, like rural Lesotho . HoweverCryptococcus in the meninges, and developed meningitis after commencing fluconazole as an outpatient, although the patient also had TB and it was not possible to ascertain the actual cause of death. Since CrAg-positivity is associated with increased risk of death from TB [One death was observed among the asymptomatic CrAg-positive group. This patient may have already had  from TB , both diA secondary consequence of this intervention was that additional cases of CM that would have previously gone undetected were now identified, exposing deficiencies in the clinical management of CM.Our short follow up period (median 5 months) limited monitoring for CM development in asymptomatic CrAg-positive patients, despite pre-emptive treatment. However, our observation period covered the highest mortality risk period reported in similar resource-limited settings . AdditioCryptococcus sp. in blood or CSF) was a limitation. Although, through the use of the CrAg-LFA positive quality control, we were able to ensure proper performance of each test batch.The lack of external quality control for an ideal comparison between lay counsellor’s performance and gold standard test , proved feasible as a modest addition to overall clinic workload. However, close follow-up was needed to ensure proper management of cryptococcal disease and other OIs.Further operational research in this area should address improved strategies for quality control, as well as patient referral and follow up. Given that TB is the principal cause of death among patients with advanced HIV disease, future work could also address how CrAg screening can be better integrated within a more comprehensive approach for the screening and management of people with advanced HIV disease.The results of effective implementation of CrAg-LFA screening and monitoring of disease-free follow-up among those who received pre-emptive treatment suggest a positive impact of this intervention, particularly in a setting where the disease is largely undiagnosed and untreated. These early results support the wider use of CrAg-LFA POC screening in remote primary care settings.In summary, this is as far as we are aware the only study that demonstrates the efficiency of lay counsellors performing CrAg-LFA screening, and documenting patient clinical outcomes. This is relevant within health systems where nurses have little capacity for additional activities, and where financial constraints limit the likelihood of more highly paid staff being employed to carry out this activity.