Kenya’s guidelines for prevention of mother-to-child transmission of HIV (PMTCT) recommend routine viral load (VL) monitoring for pregnant and breastfeeding women.We assessed PMTCT VL monitoring and clinical action occurring between last menstrual period (LMP) and 6 months postpartum at 4 Kenyan government hospitals. Pregnant women enrolled in the HIV Infant Tracking System from May 2016-March 2018 were included. We computed proportions who received VL testing within recommended timeframes and who received clinical action after unsuppressed VL result.Of 424 participants, any VL testing was documented for 305 (72%) women and repeat VL testing was documented for 79 (19%). Only 115 women (27%) received a guideline-adherent baseline VL test and 27 (6%) received a guideline-adherent baseline and repeat VL test sequence. Return of baseline and repeat VL test results to the facility was high (average 96%), but patient notification of VL results was low (36% baseline and 49% repeat). Clinical action for unsuppressed VL results was even lower: 11 of 38 (29%) unsuppressed baseline results and 2 of 14 (14%) unsuppressed repeat results triggered clinical action.Guideline-adherent VL testing and clinical intervention during PMTCT must be prioritized to improve maternal care and reduce the risk of HIV transmission to infants. Prevention of mother-to-child transmission (PMTCT) services for pregnant women and mothers living with HIV throughout the antenatal, delivery, and postpartum phases are critical to reducing perinatal transmission and preserving the health of the mother and child. Despite significant uptake of antiretroviral therapy (ART) among pregnant women with HIV, adoption of recommended regimens has been gradual, with only 38% of pregnant women at selected Kenyan facilities receiving guideline adherent treatment between 2013 and 2016. , 8 HowevRoutine maternal VL level provides a mechanism to identify elevated maternal viral load early so that corrective action can be taken to suppress maternal viral load  and/or reduce the risk of transmission (intensified infant prophylaxis). , 17 As sThe objective of the present study was to evaluate implementation of Kenya’s PMTCT VL testing guidelines at four facilities, including documented baseline and repeat tests, return of test results, and clinical action taken in response to ART treatment failure, highlighting gaps and opportunities for strategic intervention.At the time of the study, Kenya’s PMTCT guidelines recommend early antenatal attendance, ART initiation <14 weeks gestation, and routine VL monitoring of pregnant and breastfeeding women. –25 GuideKenya has over 9,000 health facilities, across six levels of care. Level 1–3 facilities  focus on primary care and community health. Facility levels 4 and 5  provide more specialized services, including HIV care and maternity services, while level 6 facilities  provide highly specialized services, in addition to HIV care and maternity services. Since 2013, maternity services  have been provided free of charge in government facilities.  In the eWe conducted a retrospective record review to evaluate guideline-adherent VL testing during PMTCT at four Kenyan government hospitals. The four government hospitals included in the study were in geographically diverse cities and ranged from level 4 to level 6, to capture the range of facilities that offer both HIV and maternity services. Details of the study hospitals are shown in At all facilities, patient viral load samples were collected at the facility’s internal laboratory. Samples were then shipped to the facility’s designated central laboratory for processing. Each hospital’s PMTCT program was supported by the HITSystem 2.0, a web-based intervention that tracks clinical engagements during PMTCT and Early Infant Diagnosis (EID), sends providers alerts for missed or late services, and sends clients automated text messages reinforcing ART adherence and timely appointment attendance.  This verPMTCT client data collected by routine entry in the HITSystem 2.0 were supplemented with data captured through retrospective review of paper-based facility records for VL testing history. Collected data included dates of ART initiation, ANC enrollment, last menstrual period (LMP), estimated date of delivery (EDD), VL sample collection, return of VL results, and clinical action in response to an unsuppressed VL result . For thThis study was a retrospective chart review. All patient records/information was anonymized and de-identified prior to analysis. As such, retrospective review study participants were not required to provide informed consent for their clinical records to be used in this study. The Institutional Review Boards at the University of Kansas Medical Center and Kenya Medical Research Institute approved this study.”VL tests occurring between the LMP date and 6 months postpartum were included in analysis, and each patient’s first VL test within this period was designated as the baseline. We assessed the proportion receiving guideline-adherent VL testing at baseline and repeat intervals. For patients newly initiated on ART, guideline adherent VL baseline testing was defined as (1) ART initiation by 14 weeks gestation and (2) baseline VL testing within 6 months of ART initiation. For patients who were previously initiated on ART, guideline-adherence baseline testing was defined as (1) occurring after LMP or at confirmation of pregnancy and (2) within the targeted timeframe of early antenatal engagement (≤14 weeks gestation). If baseline VL tests results were suppressed, repeat VL tests were indicated within 6 months of the date the baseline sample was obtained. Among those with unsuppressed baseline or repeat VL results, guidelines recommended a repeat test within 3 months. All VL samples (baseline and/or repeat) collected ≤30 days after recommended schedule were designated as guideline-adherent.Descriptive statistics were used to report demographic characteristics of study participants, uptake of baseline and repeat VL tests, and engagement of participants with test result notification and clinical action. Chi-square tests were performed to compare proportions of patients with guideline adherent baseline and repeat VL testing among health care facilities and proportions of the previously versus newly ART-initiated patients.Patient enrollment by year and study site are described in 2 = 20.91, p < .0001). There was also a significant difference between sites for receipt of any VL testing by 6 months postpartum, with proportions ranging from 53.7% to 86.2%, χ2 = 20.05, p < .0001, as well as receipt of both baseline and repeat testing by 6 months postpartum, ranging from 9.9% to 29.1%, χ2 = 18.18, p = < .0001.Among the 424 enrolled PMTCT patients who were included in analyses, 119 women (28%) did not receive any documented VL test by 6 months postpartum. Among the 305 women (72%) who received a baseline VL test, 213 (70%) received it antenatally at a mean gestational age of 19.4 weeks and 92 (30%) received it postpartum at a mean of 12.5 weeks post-delivery. Baseline VL tests were followed by a repeat VL test within 6 months of delivery for 79 (19%) women. Twenty-six (33%) repeat VL tests were administered in the antenatal stage, 1 (1%) was administered at delivery, and the remainder were administered postpartum. Only 8% of those newly initiated on treatment received a baseline and repeat test, and only 2% of those previously initiated on treatment received a baseline and two repeat tests by 6 months postpartum . Those w2 = 13.61, p < .0001). Across the four study sites, the proportions of patients who received guideline-adherent baseline VL tests varied significantly, ranging from 14.9% to 40.0% .One hundred fifteen clients (27%) received a baseline VL test adherent to Kenya’s 2016 guidelines per their personal ART history . A signi2 = 1.11, p = .293). Receipt of the guideline-adherent baseline and repeat test sequence did not vary significantly across study sites, ranging from 4.8% to 10.9% of patients .Twenty seven of the patients with guideline-adherent baseline tests subsequently received guideline-adherent repeat VL tests, while 27 others received delayed repeat testing . Thus, 62 = 0.08, p = 0.774). Of the 38 patients with unsuppressed VL results, 11 (29%) were followed up with documented clinical action: 9 patients were given enhanced adherence counseling and 2 others were prescribed a change in ART regimen. Additionally, 7 (18%) patients with unsuppressed baseline VL received repeat testing in accord with the guideline-recommended accelerated 3-month turnaround.Of the 305 baseline test samples submitted to centralized laboratories, 294 (96%) VL results were reported back to the clinic. The mean turnaround time for VL results was 2.3 weeks. Patient notification was documented for only 106 (36%) of the baseline test results returned to the clinic. Thirty-eight (13%) baseline test results indicated viremia above the threshold for unsuppressed VL (≥1000 copies/ml); with no significant differences among newly ART-initiated and previously ART-initiated patients  VL results were returned to the clinic (mean turnaround time of 2.0 weeks), and patient notification was documented for 38 (49%) patients. Fourteen (18%) of the repeat test results were unsuppressed: 2 (14%) were followed up with documented clinical action (enhanced adherence counseling) and an additional 2 (14%) had a second documented repeat test within 3 months in accordance with the guidelines.Very limited prior data on VL testing uptake in Sub-Saharan African PMTCT programs suggest that only 35–50% of pregnant women on lifelong ART regimens receive any VL testing. , 10, 22 A well-functioning clinic-laboratory interface is critical to ensure HIV diagnostic tests lead to quality, actionable results for patients. , 31 VL mScience relating to PMTCT is a rapidly evolving field of study, prompting frequent guideline changes to reflect most up-to-date evidence.  This stuSeveral emerging adaptations to traditional viral load testing processes, which may help address gaps in PMTCT VL monitoring. eHealth interventions that support clinical decision making and patient outreach have proven successful at improving completion of guideline-adherent care and increasing patient notification for EID results,  and may Limitations of the study include the fact that all data come from patients who enrolled programmatically in the HITSystem. Although this version of the intervention provided no direct support for VL testing adherence, it surpasses the local standard of care and may bias the results toward greater ART and appointment adherence. If so, the true proportions of women receiving VL tests and VL-driven clinical action are likely even lower. Also, the population enrolled in this study, at four hospitals, is a small sample of PMTCT clients and facilities nationwide. Despite that constraint, the chosen study sites represent diverse settings from the standpoint of geographic region and facility size, so documented engagement in services there are likely to reflect the situation in many Kenyan localities. In addition, the timing and duration of antenatal PMTCT engagement varied at the individual level, thus patients who received late baseline testing (after delivery) may not have been eligible for retesting by 6 months postpartum. Furthermore, this study is limited to retrospective review of facility records, and thus, does not include exploration of site-specific factors that may have influenced uptake and compliance with guidelines. Nonetheless, these data accurately describe the proportion of women receiving guideline adherent antenatal enrollment and VL monitoring between the targeted intervention window of pregnancy through 6 months postpartum.Unique contributions of this study include that it followed patients longitudinally and thus captured maternal VL testing uptake both antenatally and postpartum, including baseline and repeat testing. By collecting the documented dates of VL testing and VL-driven clinical action–in conjunction with dates of ANC enrollment, ART initiation, and delivery–we were able to measure for each patient the adherence to Kenya’s national PMTCT guidelines. An important feature of this study was the analysis of clinical actions taken in response to high VL test results, providing insight into the fulfillment of VL testing’s intended purposes.The investment in VL monitoring during pregnancy and postpartum can avert perinatal HIV transmission and protect women’s health. Data collected from PMTCT programs at these sites suggest the need for enhanced communication channels between providers, central laboratories, and patients in order to reinforce guideline-adherent test orders, patient notification, and rapid clinical action. Meeting these goals is critical to realizing a maximum return on current investment and optimizing PMTCT outcomes.S1 Dataset(XLS)Click here for additional data file.


CYP2B6, CYP3A5, ABCB1, UGT2B7, and SLCO1B1 was done. CD4 cells-count was measured at baseline, 12th, 24th, and 48th weeks of cART. Among HIV-only cohort, plasma efavirenz concentration and median CD4 cell count were significantly higher in Tanzanians than Ethiopians, and both CYP2B6 genotype and population-variation were significant predictors of efavirenz plasma concentration. Within-population analyses indicated a pronounced efavirenz autoinduction in Tanzanians as reflected by a significant decrease of plasma efavirenz concentration over time (p = 0.0001), but not in Ethiopians. Among TB-HIV cohort, there were no significant between-population differences in plasma efavirenz concentrations or CD4 cell-recovery, and CYP2B6 genotype but not population-variation was a significant predictor of efavirenz plasma exposure. In Tanzanian patients, short-term anti-TB co-treatment significantly reduced the mean plasma efavirenz concentration in CYP2B6*1/*1 genotype at week-4 (p = 0.005), but not at week-16 of cART. In Ethiopian patients, anti-TB cotreatment increased the mean plasma efavirenz concentration among CYP2B6*6 carriers at week-4 (p = 0.003) and week-16 (p = 0.035) of cART. In general, long-term anti-TB co-treatment increased plasma efavirenz concentration at week 16 of cART in both Ethiopians and Tanzanians being higher in CYP2B6*6/*6 > *1/*6 > *1/*1 genotypes. In TB-HIV patients, baseline body mass index (BMI), viral load, and WHO clinical-stage but not genotype, population-variation, or efavirenz concentration were significant predictors of immunologic outcome at week-48. In summary efavirenz auto-induction, pharmacokinetics, and the immunologic outcome are influenced by population-variation, anti-TB co-medication, and CYP2B6 genotype. CYP2B6 genotype is a significant predictor of efavirenz plasma exposure regardless of population-variation or antituberculosis co-treatment, but population-variation is insignificant during antituberculosis treatment. CYP2B6 genotype, population, and geographic differences need to be considered for efavirenz dosage-optimization.Efavirenz-based combination antiretroviral-therapy (cART) is the recommended regimen during tuberculosis (TB) therapy. In a multi-national parallel prospective-cohort study, we investigated the impact of population and pharmacogenetic variations for efavirenz pharmacokinetics, auto-induction, and immunologic outcome during antituberculosis treatment. A total of 921 treatment-naïve HIV patients with (196 Ethiopians and 231 Tanzanians) or without TB co-infection (285 Ethiopians and 209 Tanzanians) were enrolled and treated with efavirenz-based cART. TB-HIV patients started rifampicin-based anti-TB therapy 4 weeks before cART. Efavirenz plasma concentrations were measured on the 4th and 16th weeks of cART. Genotyping for  Tuberculosis (TB) continues to be the most common cause of mortality among patients infected with human immunodeficiency virus (HIV). The burden of this dual global epidemic of HIV and TB is extremely high in sub-Saharan Africa with the World Health Organization (WHO) estimating 86% of the deaths related to HIV associated TB coming from this region . EffectiCYP2B6*6 variant allele, which is more common among African populations as compared to Caucasians, tend to experience higher levels of efavirenz when it is co-administered with rifampicin   and efavirenz and the patients were followed up to 1 year to monitor the immunological outcome. A parallel cohort of ART naïve HIV-only patients (209 Tanzanian and 285 Ethiopian) were enrolled as a control group and initiated efavirenz-based cART only and followed up for the same duration of time  titled “Optimization of tuberculosis and HIV co-treatment in Africa: Pharmacokinetic and pharmacogenetic aspects on drug-drug interactions between rifampicin and efavirenz.” The study design, population eligibility criterions were described previously . In brie of time .The study received ethical approval from Institutional Review Board (IRB) of the Muhimbili University of Health and Allied Sciences in Dar es Salaam, Tanzania, by the IRB of Faculty of Medicine, Addis Ababa University, and Ethiopian National Ethics Review Committee and by the IRB of Karolinska Institutet in Stockholm, Sweden. Prior written informed consent was obtained from all study participants.4 cell count, HIV RNA determination, hepatitis B surface antigen, anti-hepatitis C antibody, serum albumin, renal function tests, and liver enzymes including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and direct and total bilirubin levels. Change in CD4 count from baseline was monitored at 12th, 24th, and 48th weeks of ART in both TB-HIV and HIV only cohorts.At enrollment baseline, clinical and laboratory parameters were assessed. Pretreatment laboratory tests included complete and differential blood counts, hemoglobin, platelet count, CD13C6-efavirenz as internal standards where the lower limits of quantification were 0.01 µg/ml. The efavirenz calibration range was 0.01–10 µg/ml. Linear regression with 1/x weighing resulted in correlation coefficients of r2 0.99. Accuracy and precision (within-batch and batch-to-batch) of the assay fulfilled all recommendations of FDA guidelines.At 4 and 16 weeks of initiating efavirenz-based cART, a 16-h post-efavirenz dose blood samples were collected in vacutainer Cell Preparation Tubes (CPT) tubes . Blood samples were centrifuged  to prepare plasma and stored at −80°C. Plasma efavirenz concentrations were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS) as described previously . EfavireABCB1 c.3435C > T rs1045642, C:11711730_20 for CYP2B6*6 c.516G > T rs3745274, C:30720663_20 for UGT2B7 g.-327G > A rs7662029 (UGT2B7*2), C:26201809_30 for CYP3A5*3 c.6986A > G rs776746, C:30203950_10 for CYP3A5*6 14690G > A g.14690G > A, C:32287188_10 for CYP3A5*7 g.27131_27132insT rs241303343, C:_1901697_20 for SLCO1B1 c.388A > G rs2306283 (*1b), and C:30633906_10 for SLCO1B1 c.521T > C rs4149056 (*5) on ABI 7500 FAST . The final volume for each reaction was 10 μl, consisting of 2 X TaqMan Universal PCR Master Mix (Applied Biosystems), 20 X drug metabolizing genotype assay mix, and 10 ng genomic DNA. The PCR profile consisted of an initial step at 50°C for 2 min and 50 cycles with 95°C for 10 min and 92°C for 15 s. Genotyping for SLCO1B1 c.388A > G (rs2306283) and c.521T > C (rs4149056) in Tanzania was done using Light Cycler H based method . Genotyping for the common functional variant alleles in five relevant genes for efavirenz disposition were done using allelic discrimination TaqMan genotyping assays  with the following ID number for each single nucleotide polymorphisms (SNP): .Baseline socio-demographic and laboratory parameters were described as means and standard deviations (SD) or medians and interquartile range (IQR) for continuous variables and as percentages for categorical variables. The normality of kinetic data was assured by transforming the data to log 10 values before statistical analysis. Independent t-tests were used to compare log-transformed plasma efavirenz concentration at 4 and 16 weeks after cART initiation between Tanzanian and Ethiopian patients. Predictors of efavirenz plasma concentrations were assessed using univariate and multivariate regression analysis. Linear regression models were used to estimate predictors of log efavirenz concentrations at 4 and 16 weeks, separately. Similarly, the predictors of immunological outcomes at week 48 were assessed. Log-binomial regression analysis was used to estimate the relative risk of efavirenz concentrations <1 µg/ml at 4 and 16 weeks, separately. To take into account the repeated measure design and examine predictors of efavirenz longitudinally, we used generalized estimating equations (GEEs) with an exchangeable working covariance and identify the link to produce population-averaged mean differences in efavirenz concentrations while the log link for the population-averaged relative risk of efavirenz <1 µg/ml. Variables in univariate analysis with a A total of 921 treatment naïve HIV patients with or without TB-HIV coinfection were enrolled in a multi-national parallel prospective cohort study: 427 TB-HIV co-infected patients (196 in Ethiopia and 231 in Tanzania) and 494 HIV patients without TB coinfection (285 in Ethiopia and 209 in Tanzania). The HIV only cohort treated with efavirenz-based cART only were used as a control group to examine the impact of population differences on efavirenz-rifampicin drug interaction. The sociodemographic, clinical, and laboratory, pharmacogenetic, and pharmacokinetic data of the HIV-only cohort is described previously .2 compared to the Tanzanians (49.5 vs. 39.3% respectively) (p = 0.03). The mean (±SD) hemoglobin was 10.6 ± 2.2 with Tanzanians (19%) having significantly more patients with a hemoglobin of <8.5g/dl compared to the Ethiopians (10%) (p = 0.02). The patients were severely immunocompromised with a mean CD4 cell count of 97.7 cells/µl with over half having a CD4 cell count of <100 cells/µl in both settings.The sociodemographic, baseline clinical, and laboratory characteristics of the TB-HIV cohort are presented in CYP2B6, CYP3A5, UGT2B7, ABCB1, and SLCO1B1 genotype and variant allele frequency distribution between Ethiopians and Tanzanian TB-HIV patients is presented in CYP2B6*6 and CYP3A5*6, the distribution of all other variant alleles genotypes for were significantly different between the two study populations. Though not significant, Tanzanians had a higher allele frequency of the defective CYP2B6*6 variant allele than Ethiopians. Proportionately more Tanzanians had the *6/*6 allele compared to the Ethiopians (58.8% compared to 41.2%). The defective variant allele CYP3A5*7 was absent in Ethiopians but occurred at a frequency of 10% in Tanzanians. Due to the high frequency (70%) of the defective CYP3A5*3 variant allele, the frequency of functional CYP3A5*1 allele in Ethiopians was significantly lower in Ethiopians (22.3%) than Tanzanians (50.4%). Likewise, the defective SLCO1B1*5 variant allele occurs at a much higher frequency in Ethiopians than Tanzanians.Three hundred and twenty-eight TB-HIV coinfected patients had their genotype determined (145 Tanzanians and 183 Ethiopians). Comparison of versus TB-HIV patients co-treated with efavirenz (EFV) based cART and rifampicin based anti-TB therapy at the 4th and 16th week of ART is presented in th and 16th weeks of cART (Comparison of plasma efavirenz concentration between Ethiopian and Tanzanian HIV patients receiving efavirenz-based cART only (control group)  of cART Figure 1CYP2B6*6 genotype group at week 4 and 16 of cART is presented in CYP2B6*1/*1 genotype group (p = 0.005), but no significant effect was observed in Tanzanian CYP2B6*6 carriers. On the other hand, though not significant, there was a trend of having higher mean efavirenz plasma concentration among those co-treated with rifampicin than those without rifampicin in Tanzanian CYP2B6*6 carriers (p = 0.18) at week 16 of cART but not in CYP2B6*1/*1 (p = 0.68).Comparison of plasma efavirenz concentration while on (EFV+RIF) and off (EFV-only) rifampicin co-treatment among Ethiopians and Tanzanians stratified by CYP2B6*6 allele both at week 4 (p = 0.003) and at week 16 (p = 0.035). Though not significant, a similar trend was observed in CYP2B6*1/*1 genotype at week 4 (p = 0.18) and at week 16 (p = 0.36). Overall long-term rifampicin co-treatment increased plasma efavirenz concentration in Ethiopians being higher in CYP2B6*6/*6 > *1/*6 > *1/*1  genotypes were significant predictors of efavirenz plasma concentrations.Predictors of efavirenz plasma concentration in HIV patients without TB coinfection is presented previously . In briep = 0.07), but results did not reach statistical significance. CYP2B6 genotype was a significant predictor of efavirenz concentrations at both 4 and 16 weeks. In the longitudinal model, patients with CYP2B6*6/*6 and *1/*6 alleles had 1.38 (95% CI: 0.98–1.78) and 0.52 (95% CI: 0.28–0.77) greater log efavirenz plasma concentrations than patients who had CYP2B6 *1/*1 genotype. Predictors for efavirenz plasma concentrations below the therapeutic window threshold (<1 µg/ml) were also assessed. Statistically significant differences were seen in the CYP2B6 genotype distribution and efavirenz levels where 71% of the patients with <1 µg/ml were CYP2B6 *1/*1 and 48% of patients with supra-therapeutic efavirenz concentrations (> 4 µg/ml) were *6/*6 (p < 0.001). Multivariate analysis showed that patients with CYP2B6 *1/*6 and *6*6 genotype had 0.50 (95% CI: 0.29–0.87) and 0.23 (95% CI: 0.05–1.03) times the risk of having efavirenz concentrations <1 µg/ml as compared to those with *1/*1 genotype respectively  times higher risk of having efavirenz concentrations <1 µg/ml compared to those who did not have both alleles.Predictors of plasma efavirenz concentrations in TB-HIV patients co-treated with rifampicin based anti-TB therapy is presented in ectively Table 4.4 cell count at baseline, 12th, 24th, and 48th week of cART between Ethiopians and Tanzanians stratified by treatment group are presented in 4 cell count was significantly higher in Tanzanians than Ethiopians at week 24 (p = 0.01) and week 48 (p = 0.001) of cART (p < 0.001). Likewise, and the percent change of CD4 cell count from baseline by week-24 (p = 0.03) and 48 (p = 0.02) were higher in Tanzanian than Ethiopians.Comparison of the median and interquartile range (IQR) of CD4 cell count at week 24 (p = 0.82), week 48 (p = 0.19), or percent change in CD4 cell count from baseline by week 24 or 48 of cART was observed among TB-HIV patients co-treated with rifampicin based anti-tuberculosis. Though not significant the CD4 cell-count remained consistently higher in Tanzanians than Ethiopians (4 cell count from 98 (±63) cells/µl at baseline, to 231 cells/µl at 24 weeks, and 271 cells/µl at 48 weeks after cART initiation. Tanzanian patients had an overall higher median CD4 cell count compared to Ethiopians at week 24 (207 vs. 200 cells/µl) (p = 0.38) and at week 48 post cART (271 vs. 241 cells/µl) (p = 0.057). At the 48 weeks of cART, twice as many Ethiopians compared to Tanzanians had CD4 cell counts below 200 cells/µl (66% vs. 34% respectively).In contrast, no significant between-country differences in median CDhiopians Figure 44 cell count by week 48 is presented in Predictors of CDCYP2B6 genotype for efavirenz plasma exposure was prominent regardless of population variation or rifampicin co-medication, the importance of population variation was prominent only when efavirenz-based cART was given alone. This may indicate that findings during rifampicin based anti-TB co-treatment could possibly be extrapolated between black African populations, but not when efavirenz-based cART is given alone. To the best of our knowledge, this is the first parallel prospective study to explore the importance of between population variation for antiretroviral and antituberculosis drug interaction and its impact on efavirenz pharmacokinetics, auto-induction, and immunologic outcome in two genetically different sub-Saharan Africa population.In the present study we performed a parallel comparative multicenter prospective observational study to assess the importance of population, geographic, and pharmacogenetic variations for efavirenz pharmacokinetics and immunological outcome during antituberculosis cotreatment in two genetically different east African populations, Ethiopians, and Tanzanians. HIV patients without tuberculosis co-infection were enrolled as a control group to investigate the impact of population and geographic differences in the absence of anti-TB co-medication. Our result indicates different efavirenz plasma exposure and immunologic outcome profiles between Ethiopians and Tanzanian HIV patients depending on the presence or absence of antituberculosis co-medication. Tanzanian HIV patients treated with efavirenz-based cART only had significantly higher plasma efavirenz concentration and immunologic recovery than Ethiopian counterparts. However, this difference became insignificant during rifampicin based anti-TB co-medication. While the impact of via CYP2B6 and CYP3A4/5 enzymes  as suggested previously  that influence efavirenz pharmacokinetics  is absent in Ethiopians  of the Muhimbili University of Health and Allied Sciences in Dar es Salaam, Tanzania, by the IRB of the Faculty of Medicine, Addis Ababa University and the Ethiopian National Ethics Review Committee and by the IRB of Karolinska Institutet in Stockholm, Sweden. The patients/participants provided their written informed consent to participate in this study.EA, OM, EM, and JB contributed to the conception and design of the study. SM, WA, AH, EM, GY, EA, OM, and EM conducted the clinical study and data collection. EA, AH, EN, and JB conducted plasma drug quantification and genotyping. SM, EA, and CS conducted data analysis and interpretation. EA and SM wrote the draft manuscript. All authors contributed and approved the final article.This study was supported by grants from the European and Developing Countries Clinical Trial Partnership (grant number CT.2005.32030.001) Swedish Research Council (Vetenskapsrådet) grant number: 2015-03295. The research analyzed and reported in this publication was supported by the Fogarty International Center of the National Institutes of Health under Award Number D43 TW009775. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.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.


The World Health Organization (WHO) published a guideline for advanced HIV disease in 2017 and a guideline relevant to resource-limited settings for HIV-associated CM in 2018.4 Cryptococcal antigen (CrAg) screening and pre-emptive treatment reduced all-cause mortality among ambulatory participants in a randomised clinical trial in Zambia and Uganda.5 Following an evaluation of reflex versus provider-initiated screening, national reflex laboratory CrAg screening was implemented in South Africa in 2016.7 Recently completed clinical trials conducted in resource-limited settings have provided evidence for the best first-line antifungal regimens for CM and the role of corticosteroids in CM.9 Finally, international and local advocacy efforts have resulted in increasing, yet still limited, access to flucytosine and a reduced cost of liposomal amphotericin B for the treatment of CM.10Six years after the Southern African HIV Clinicians Society cryptococcal disease guideline was published in 2013, cryptococcal meningitis (CM) remains an important cause of mortality among antiretroviral treatment (ART)-naïve and ART-experienced HIV-seropositive adults in South Africa.+ T-lymphocyte (CD4) count < 200 cells/µL who are initiating ART for the first time, switching after ART failure or re-entering into care after prior disengagement.We now recommend CrAg screening for all adults or adolescents with a CD4Reflex laboratory CrAg screening is recommended as the preferred approach in South Africa, although alternative approaches may be more suitable for other countries in Southern Africa.To diagnose CM, lumbar puncture (LP) is recommended for all patients with a new positive CrAg screening test result.The recommended induction regimen for CM is 1 week of amphotericin B deoxycholate (1 mg/kg/day) and flucytosine (100 mg/kg/day in four divided doses), followed by 1 week of fluconazole .We have recommended alternative induction regimens for CM among patients with renal dysfunction, including liposomal amphotericin B-based options.A higher dose of fluconazole is now recommended during the 8-week consolidation phase for CM .Maintenance treatment for CM is recommended to be continued for at least 12 months and until a single CD4 count is > 200 cells/µL and the HIV viral load is suppressed.We have included new recommendations for the prevention, monitoring and management of flucytosine toxicities.Although a manometer is the most accurate way to measure raised intracranial pressure, we have suggested alternative options for assessment of elevated pressure when it is unavailable.Patients with a positive blood CrAg test result in whom CM is ruled out by LP  should be given oral fluconazole alone as induction therapy (adults: 1200 mg for 2 weeks). Antiretroviral treatment may be commenced immediately.We recommend immediate referral for LP in all patients with a new positive blood CrAg test who initiated ART within the 4-week period prior to the CrAg test. Among those with a negative CSF CrAg test (i.e. in whom CM is excluded), ART is recommended to be continued and fluconazole pre-emptive therapy should be initiated. Among those with a new diagnosis of CM during the first 4 weeks of ART, the guideline panel thought that there was clinical equipoise in terms of a decision to continue or interrupt ART.Antifungal susceptibility testing is now recommended if a patient has a single relapse episode of CM and other causes have been excluded.12 Screening for subclinical cryptococcal disease has a proven mortality benefit among HIV-seropositive patients with low CD4 counts. In the Reduction of Early Mortality among HIV-infected Subjects sTarting AntiRetroviral Therapy (REMSTART) trial, HIV-seropositive outpatients with a CD4 count < 200 cells/µL, who were randomised to receive CrAg screening and community-based ART adherence support, had a 28% reduced all-cause mortality rate compared to those receiving standard of care (intervention: 134/1001 [13%] vs. standard: 180/998 [18%]).5 While the previous Southern African guideline recommended CrAg screening for patients with CD4 < 100 cells/µL, this approach will fail to detect a substantial proportion of cases that occur in patients with CD4 counts between 100 cells/µL and 200 cells/µL. A meta-analysis estimated that the pooled global prevalence of cryptococcal antigenaemia was 6.5% among those with CD4 count < 100 cells/µL and 2% among those with a CD4 count between 101 cells/µL and 200 cells/µL.13 A post hoc analysis of REMSTART trial data revealed an important mortality benefit of CrAg screening and pre-emptive treatment in subgroups of patients with a CD4 count < 100 cells/µL and with CD4 count of 101 cells/µL – 200 cells/µL.4 Thus, we now recommend that 200 cells/µL should be used as the CD4 threshold below which patients should be screened for cryptococcal disease, where resources permit  in whom the incidence of CM is much lower.15 Even though data for adolescents are limited, the WHO currently recommends screening for adolescents. South African data indicate an increasing incidence of CM from the age of 10 years onwards.15HIV-seropositive adults and adolescents (≥ 10 years) with a CD4 count < 200 cells/µL are recommended to be screened for cryptococcal antigenaemia. If screening is initiated by a clinician  and not performed reflexively in the laboratory, the expert panel recommends that screening must be restricted to adults and adolescents initiating ART for the first time, switching after treatment failure or re-initiating ART (after an episode of interruption that was > 3 months) with a CD4 count < 200 cells/µL and no prior CM. Although adults or adolescents with a CD4 count < 200 cells/µL who are virally suppressed on ART may also be at risk of cryptococcal disease,16 A South African modelling study comparing the two strategies and using a CD4 threshold of < 100 cells/µL demonstrated that reflex screening was more cost-effective and saved more lives.7 Although the CrAg latex agglutination (LA) test format has been more extensively evaluated for the diagnosis of cryptococcal disease, a rapid CrAg lateral flow assay is simpler to perform on blood samples and more accurate.17 The National Health Laboratory Service (NHLS) expanded its reflex laboratory CrAg screening service across South Africa in October 2016.6 Between October 2016 and March 2019, more than 600 000 persons were screened, with a CrAg+ prevalence of 5.7% . However, the real-world effectiveness of this screen-and-treat strategy in terms of impact on mortality has yet to be determined. The laboratory turnaround time is very short for both CD4 count and reflex CrAg screening test results; however, initiation of ART should not be unnecessarily delayed while awaiting CrAg test results in patients with advanced HIV disease who have no clinical features of meningitis.With reflexive laboratory CrAg screening, remnant blood samples (submitted to the laboratory for CD4 testing) are tested automatically for CrAg below a specified CD4 threshold. This approach is superior to clinician-initiated screening  in terms of screening coverage.18 CrAg-positive patients who are subsequently identified as having CM should be managed as per Recommendation 3. In the latter group, ART should be commenced 4–6 weeks after the introduction of antifungal therapy. Patients in whom CM is ruled out by LP (a negative CSF CrAg test is the most rapid method to establish this) should be given oral fluconazole alone as induction therapy (adults: 1200 mg for 2 weeks). This is followed by standard consolidation and maintenance treatment regimens as per Recommendation 3. In these blood CrAg-positive/CSF CrAg-negative patients, ART may be commenced immediately, with the caveat that there is no published evidence for this recommendation.19 Adolescents and children should receive fluconazole 12 mg/kg/day (to a maximum of 800 mg per day) for 2 weeks followed by standard consolidation and maintenance treatment. The timing of ART initiation should be the same as adults.All patients who screen CrAg-positive for the first time should have an LP performed. The absence of symptoms of meningitis does not exclude CM: approximately one in three patients with asymptomatic cryptococcal antigenaemia has concurrent CM.18 Patients with headache, nausea, vomiting or other signs of CM should be treated as such (as per Recommendation 3), whereas those without symptoms can be treated with fluconazole alone, as for patients in whom CM has been excluded. However, this approach will result in some cases of asymptomatic or subclinical CM being missed. If a screening blood CrAg titre is available , patients with CrAg titres > 160 may be considered at high risk of CM or cryptococcal disease-related death.21 Such patients should be carefully monitored for CM signs/symptoms or considered for empirical CM treatment. Community adherence support is recommended for all patients who screen CrAg-positive and are followed up as outpatients, particularly among those who decline an LP.5Patients who decline an LP can be stratified according to the presence or absence of meningitis symptoms , althoug22 the panel recommends immediate referral for LP and CSF analysis in all patients with a positive blood CrAg test who initiated ART in the 4 weeks prior to the CrAg test. Among patients with a negative CSF CrAg test (i.e. in whom CM is excluded), ART can be continued and fluconazole pre-emptive therapy should be initiated. Among patients on a new ART regimen with a positive CSF CrAg test (i.e. a new diagnosis of CM), data suggest that mortality may be increased if CM is diagnosed in the first 4 weeks of ART, possibly mediated through a mechanism of unmasking immune reconstitution inflammatory syndrome (IRIS).23 Not all studies have demonstrated this and we do not know if ART interruption would reduce this mortality risk. Potential harms of ART interruption include HIV resistance (more likely with NNRTI-based regimens but less likely with dolutegravir-based regimens) and the risk of HIV disease progression and other opportunistic infections. The panel thought that there was clinical equipoise for the decision whether to continue or stop ART in this group, and we therefore do not, at this stage, recommend ART interruption. This remains a research question.Current same-day HIV test-and-treat strategies mean that some patients may start ART before a positive reflex blood CrAg result is received. Given the increased risk of mortality associated with early ART in CM,Patients with a history of CM do not need to be routinely screened. Cerebrospinal fluid and blood specimens may remain CrAg-positive for months to years after a CM diagnosis and successful treatment of cryptococcal disease and therefore if these tests are positive in the absence of symptoms and signs, this is not an indication of relapse. However, if a patient with prior CM is screened, found to be CrAg-positive and has new symptoms or signs of meningitis, a full evaluation should be undertaken for relapse disease (refer to Recommendations 2 and 7). If the patient does not have new symptoms or signs of meningitis, the clinician should ensure that the patient receives adequate fluconazole maintenance therapy (refer to Recommendation 3).25 In a pregnant CrAg-positive patient without laboratory-confirmed CM or an asymptomatic woman who declines an LP, the patient should be counselled regarding the risks and benefits of fluconazole treatment. Fluconazole has rarely been associated with human teratogenicity when administered in the first trimester, particularly at higher doses (≥ 400 mg/day). However, patients with a positive blood CrAg test treated with ART and no fluconazole have a substantial risk of progression to meningitis, which has a high mortality. The panel therefore recommends that pregnant women should be counselled that the benefits of fluconazole outweigh the risks in this situation. An option that could be considered in women who are in the first trimester of pregnancy is to treat with fluconazole 200 mg daily until the second trimester. All women exposed to fluconazole in the first trimester should be referred for a high-resolution ultrasound scan before 20 weeks of gestation to detect congenital abnormalities. For mothers who are breastfeeding, consultation with an experienced medical practitioner is also recommended as fluconazole is present at concentrations similar to maternal plasma concentrations in breast milk and can be transmitted in large amounts through breast milk to the infant.26Cryptococcal antigen-positive patients who are pregnant should be offered an LP before a decision is made regarding management. If the patient has laboratory evidence of CM, she should be treated for CM with standard treatment.27 Consultation with a medical practitioner experienced in the care of HIV-seropositive patients is recommended.Patients with a history of liver disease or with evidence of clinical liver disease deserve careful monitoring of serum liver enzyme and bilirubin levels  because fluconazole may cause liver injury.Cryptococcus neoformans is the most commonly detected pathogen causing meningitis in South Africa.28 All HIV-seropositive adults and adolescents with clinically suspected meningitis or a positive blood CrAg test should be investigated for CM. HIV-seropositive children aged < 5 years are considered to have advanced HIV and, if symptomatic, should also be investigated for CM.3 Patients with CM usually seek care with symptoms and signs related to inflamed meninges (including neck stiffness), raised intracranial pressure  and encephalitis (including memory loss and new-onset psychiatric symptoms).29 Cutaneous lesions and pulmonary involvement  may also occur. Symptomatic relapses are common and are most often because of inadequate or premature cessation of maintenance fluconazole treatment.30 The incidence of CM is much lower among children15; children with CM may present with an acute onset of illness and focal neurological signs may be less common  has been most widely studied in pre-clinical and clinical evaluations,32 and the accuracy of other assays is still unclear. Cryptococcus neoformans can be cultured from CSF within 72 h among patients with a first episode of CM. Cryptococcus gattii is occasionally confirmed on culture  and these infections should be managed as for C. neoformans among HIV-seropositive patients.33 There is no need to routinely order a baseline CSF CrAg titre because most patients are diagnosed when the CSF fungal burden is already high and the antifungal regimens for a first episode are standardised and not influenced by the CrAg titre (refer to Recommendation 3). A point-of-care CrAg LFA may be performed on CSF at the bedside if laboratory facilities are not immediately available.34 Antifungal susceptibility testing should not be routinely requested for a first episode because the vast majority of antifungal minimum inhibitory concentrations (MICs) are low at first diagnosis and, even if elevated, the relevance is difficult to interpret in this setting.35 If opening pressure was not measured at the time of diagnostic LP, an LP should be repeated to measure the pressure once a diagnosis of CM is confirmed (refer to Recommendation 6). Cerebrospinal fluid obtained from therapeutic LPs during the course of a hospital admission should not be routinely submitted for laboratory analysis because this is a waste of resources and does not impact on treatment management.An LP is required to confirm the diagnosis and establish the aetiology of suspected meningitis. Lumbar puncture may also alleviate symptoms that are a direct result of raised intracranial pressure. For a suspected first episode of CM, CSF should be submitted to the laboratory for a CrAg test and fungal culture. India ink test is not recommended as the only rapid test because of its lower sensitivity (78% compared to CSF CrAg test).and symptoms and signs of meningitis are very likely to have CM and should be started empirically on antifungal treatment (refer to Recommendation 3). Patients without focal neurological signs should then be referred to a centre where LP can be performed, while patients with focal neurological signs need to have a CT brain scan performed first followed by an LP (if this is not contraindicated by CT brain findings). Although the expert panel is aware that it may be difficult to access a CT brain scan in rural settings, the panel recommends that there should be urgent referral for a CT scan before LP in blood CrAg-positive patients with focal neurologic signs, whenever possible.Focal neurological signs are relatively uncommon in CM, except for a sixth cranial nerve palsy. Where focal neurological signs are present, a computed tomography (CT) brain scan should be performed before LP to exclude the presence of space-occupying lesions. If a CT brain scan cannot be performed immediately in the case of focal neurological signs or if an LP is not immediately available to make a diagnosis of meningitis, serum/plasma/whole blood may be tested for CrAg to determine if the patient has disseminated cryptococcal disease. Patients with a positive blood CrAg test A careful history should be taken including dates of previous episodes of CM and the patient should be assessed clinically for signs and symptoms of meningitis. An LP is indicated if the patient has signs and symptoms of meningitis. Cerebrospinal fluid should be submitted for fungal culture, with plates incubated for at least 14 days to detect slow fungal growth. Rapid tests are not useful for the diagnosis of subsequent episodes because both CSF India ink and CSF/blood CrAg tests may remain positive for months to years even if treatment has been successful. Antifungal susceptibility testing may be considered for a first relapse episode (see below and refer to Recommendation 7).37Resolution of symptoms and signs should be used to monitor the response to treatment. An LP should not be routinely performed after 7 (or 14) days of induction antifungal treatment to document conversion of CSF from culture-positive to culture-negative because the expert panel advises routinely changing from induction to consolidation phase treatment at 14 days. Given that a fungal culture result may take up to 14 days to become available, the culture result will not affect the timing of this change. If symptoms persist or recur during induction or at day 7 or 14, an LP should be repeated to re-measure the opening pressure, which may increase despite successful CSF sterilisation. There is no evidence to support extending the duration of induction treatment. The patient should be investigated for other causes of a poor clinical response, the most common being raised intracranial pressure. Patients with raised intracranial pressure should be managed according to Recommendation 6. Daily therapeutic lumbar puncture, and a CT brain scan if possible, is advised. If the cause of a poor clinical response cannot be found, consider referral to a secondary or tertiary centre for review unless the patient’s prognosis is already deemed to be very poor. Cerebrospinal fluid CrAg tests may remain positive for months to years and CrAg titres are not recommended to be routinely measured to monitor the response to treatment. Blood CrAg titres are also not useful to monitor the response to treatment for both CM and asymptomatic antigenaemia.38 Because there are no established clinical breakpoints for C. neoformans and fluconazole, it is useful to test isolates from the initial and subsequent episodes in parallel in an academic or reference laboratory and document a fourfold (double dilution) change in MIC that may suggest resistance.25 This requires that the initial isolate is stored, which may not always be possible at a diagnostic laboratory. Minimum inhibitory concentrations should be interpreted by an experienced clinical microbiologist in conjunction with the clinical history. In the absence of paired isolates, epidemiologic cut-off values can be applied to distinguish wild-type and mutant strains.39 Refer to Recommendation 7 for the management of patients with fluconazole-resistant isolates. Non-susceptibility to amphotericin B is very unusual and susceptibility testing for this antifungal agent should not be performed. Flucytosine resistance develops with monotherapy; hence, combination treatment is always recommended.40 Baseline flucytosine MIC testing is not routinely recommended at present.Antifungal susceptibility testing should be considered if the patient has a relapse episode and the causes listed in The antifungal treatment of HIV-associated CM is divided into three phases: induction, consolidation and maintenance .4 Flucytosine is not currently registered in South Africa or any other African country.41 A flucytosine product manufactured in Poland was acquired by Mylan in 2016. Despite previously lapsed registration in South Africa and approval by the WHO Pre-Qualification Program, the South African Health Products Regulatory Authority (SAHPRA) requires a full dossier to be submitted. The panel and the Southern African HIV Clinicians Society support advocacy efforts to provide greater access to flucytosine through fast-track SAHPRA registration and development of additional generic products, particularly in light of the findings of the recently published Advancing Cryptococcal Treatment in Africa (ACTA) trial.42 This trial demonstrated a substantial mortality reduction among participants randomised to flucytosine-based combination therapies  because the 2-week regimen was associated with lower 2- and 10-week mortality rates than 1 week of this drug combination in the ACTA trial . Maintenance fluconazole should only be stopped when the CD4 count is documented to be > 200 cells/µL (on one occasion is sufficient) and the most recent HIV viral load is suppressed.During the induction phase, amphotericin B should be prescribed at a daily dose of 1 mg/kg/day and flucytosine as a (divided) daily dose of 100 mg/kg/day . FluconaIf there is renal impairment at the time of diagnosis of CM with a calculated creatinine clearance of 30 mL/min – 50 mL/min, the recommended induction regimens are the following:If liposomal amphotericin B is available, switch to 1 week of liposomal amphotericin B (3 mg/kg/day – 4 mg/kg/day) and flucytosine, followed by 1 week of fluconazole (both fluconazole and flucytosine dose adjusted according to creatinine clearance) .One week of amphotericin B deoxycholate (1 mg/kg/day) and flucytosine (dose adjusted according to creatinine clearance), followed by 1 week of fluconazole (dose adjusted according to creatinine clearance) .If there is renal impairment at baseline with a creatinine clearance of < 30 mL/min or a deterioration in renal function, the recommended induction regimens are the following:If liposomal amphotericin B is available, 1 week of liposomal amphotericin B (3 mg/kg/day – 4 mg/kg/day) and flucytosine, followed by 1 week of fluconazole (both fluconazole and flucytosine dose adjusted according to creatinine clearance).Two weeks of fluconazole (dose adjusted according to creatinine clearance) and flucytosine (dose adjusted according to creatinine clearance).If neither flucytosine nor liposomal amphotericin B is available, administer a single dose of amphotericin B deoxycholate (0.7 mg/kg) and daily fluconazole (dose adjusted according to creatinine clearance) and monitor creatinine clearance daily. Administer amphotericin B deoxycholate (0.7 mg/kg) on alternate days if the creatinine clearance remains static and give as many doses as possible over the 2-week induction period.Clinicians should be very clear when prescribing liposomal amphotericin B versus amphotericin B deoxycholate. The daily doses are different.Intravenous fluids should be administered to all patients. Tenofovir and amphotericin B deoxycholate can be used together provided the patient’s renal function is normal and serum creatinine does not double on treatment. Among patients with renal impairment, ART regimens containing tenofovir should be adjusted, switching to an alternative antiretroviral agent such as abacavir. If the patient’s initial reason for a reduced creatinine clearance was dehydration and this has been corrected with fluid administration, the patient can be switched back to amphotericin B deoxycholate at normal doses. Flucytosine has a short plasma half-life and is cleared unchanged via the kidneys. Flucytosine requires dose adjustment if there is renal impairment; where patients are dialysed, it should be given after dialysis . Flucona25Cryptococcal fungaemia (i.e. a positive blood culture) should be managed as per CM. The treatment of cryptococcomas, pulmonary cryptococcosis and other forms of culture-confirmed disseminated cryptococcal disease is beyond the scope of this guideline. Readers are advised to consult the 2010 Infectious Diseases Society of America guideline.47The panel does not recommend a fluconazole dose increase among patients receiving rifampicin because the induction of fluconazole metabolism by rifampicin causes only moderate reductions in fluconazole exposure and because of the high doses of fluconazole that are now being recommended for induction and consolidation treatment.9 Refer to Recommendation 7 for the use of corticosteroids among patients with IRIS.The expert panel advises against adjunctive corticosteroid therapy in the initial management of CM.If patients with priorly treated CM develop immunological failure on ART and their CD4 count drops below 200 cells/µL after secondary prophylaxis has been stopped, the panel advises restarting fluconazole at 200 mg daily. This may be considered for patients with priorly treated cryptococcal antigenaemia too. Refer to the Maintenance phase (secondary prophylaxis) for duration of treatment.Among patients who stop taking fluconazole maintenance prematurely and then return for care but are asymptomatic, the panel advises simply restarting fluconazole 200 mg daily and monitoring closely for recurrence of meningitis. Symptomatic patients should be fully investigated for CM. Community adherence support for ART and fluconazole should be arranged. Refer to the Maintenance phase (secondary prophylaxis) for duration of treatment.Therapeutic LPs are the best form of ‘analgesia’ for headaches associated with raised intracranial pressure. Paracetamol can be used but not non-steroidal anti-inflammatory drugs (NSAIDs) because of the nephrotoxicity concerns with amphotericin B deoxycholate. Morphine may also be appropriate and is not contraindicated in the presence of raised intracranial pressure.48 Acute kidney injury and electrolyte abnormalities may be prevented by pre-hydration, by avoiding concurrent use of other nephrotoxins (e.g. NSAIDs and aminoglycosides) and by routine administration of potassium and magnesium supplements. Phlebitis is very common among patients receiving amphotericin B and increases the risk of localised cellulitis as well as sepsis. Anaemia commonly occurs among patients receiving amphotericin B and can be clinically significant particularly among those with a low baseline haemoglobin level. Decreases in haemoglobin of greater than 2 g/dL occurred in 50% – 71% of patients over 2 weeks’ treatment in an individual-level analysis of data from several trials.49 It is important to also exclude other treatable causes of anaemia and consider transfusion among symptomatic patients. It should be noted that both the nephrotoxic effect of amphotericin B deoxycholate and the decrease in haemoglobin are less commonly observed when using the preferred regimen of 1-week amphotericin B combined with flucytosine compared to regimens using 2 weeks of amphotericin B (8Major adverse effects of amphotericin B deoxycholate include acute kidney injury (AKI)  usually in the second week of therapy, hypokalaemia, hypomagnesaemia, anaemia, febrile reactions and chemical phlebitis.ericin B .852 The reported prevalence of toxicity from recent trials, using lower doses for shorter periods, has been much lower. In the ACTA trial, the preferred regimen of 1-week amphotericin B combined with flucytosine 100 mg/kg/day was not associated with an increase in severe neutropenia compared to flucytosine-free regimens.8 The bioavailability of oral flucytosine was reduced among Thai patients with advanced HIV disease compared to the intravenous formulation, minimising toxicity but with no demonstrated difference in early fungicidal activity.53 For these reasons, therapeutic monitoring of serum levels is not recommended in the setting of HIV-related CM. Flucytosine plasma clearance is closely related to creatinine clearance and flucytosine thus accumulates with impaired renal function; this may lead to increased risk of toxicity. Close monitoring of renal function and appropriate dose adjustment according to estimated creatinine clearance are therefore essential.54Flucytosine is associated with bone marrow toxicity and resultant anaemia, neutropenia and thrombocytopenia. This was observed particularly in early studies when flucytosine was used at high doses for prolonged periods of time.Amphotericin B deoxycholate: Amphotericin B deoxycholate powder  should be refrigerated between 2 °C and 8 °C and protected from light.55 The total daily dose of amphotericin B is calculated based on a dose of 1 mg/kg/day; amphotericin B deoxycholate powder from each 50 mg vial should be aseptically reconstituted in 10 mL of sterile water. The calculated volume of the concentrate (i.e. reconstituted drug in sterile water) should be injected into a 1 L bag of 5% dextrose water and shaken to mix well. Amphotericin B deoxycholate should never be mixed with normal saline or half-normal saline as it will precipitate. Once mixed, the solution, containing ≤ 0.1 mg of amphotericin B per 1 mL of 5% dextrose water for infusion through a peripheral intravenous line, must be infused within 24 h of preparation, or else it should be discarded.55 A test dose is not recommended and protection of the solution from light with a brown bag is unnecessary.55 The line that is used for amphotericin B infusion should not be used to simultaneously administer other medications. The solution should be infused over 4 h or more (infusion < 4 h can result in cardiac complications). Once the infusion is complete, the line should be flushed immediately with normal saline.Flucytosine: Flucytosine (500 mg tablets) should be stored below 25 °C in a cool, dry area. With normal renal function, the total daily dose is 100 mg/kg/day given in four divided doses (i.e. every 6 h) per os  per os . Nausea + per 10 mL ampoule) infused over 2 h before administration of amphotericin B deoxycholate. This reduces renal toxicity and hypokalaemia. Patients should be given 1200 mg potassium chloride orally twice daily  and up to 1500 mg magnesium chloride orally daily  for the duration of treatment with amphotericin B deoxycholate.26 Routine pre-emptive potassium supplementation should not be given to patients with pre-existing renal impairment or hyperkalaemia. To minimise the risk of phlebitis, lines should be flushed with normal saline immediately after amphotericin B infusion is complete. The empty bag should not be left attached to the intravenous line. The intravenous line should be removed if the patient develops a fever after the infusion or at the first sign of redness or discomfort at the insertion site. Febrile reactions may occur; in order to prevent recurrence, the infusion should be administered at a slow rate over the first half hour while observing the patient closely and treatment such as paracetamol may be required.Patients should be pre-hydrated with 1 L of normal saline containing 1 ampoule of potassium chloride  was documented among 12% of participants enrolled into the Cryptococcal Optimal ART Timing and Adjunctive Sertraline for the Treatment of HIV-Associated Cryptococcal Meningitis clinical trials where they received amphotericin B and fluconazole .57 Patients with baseline grade 1 neutropenia did not have a higher 30-day mortality compared to those without neutropenia. A baseline full blood/differential count should be done where available and the risk–benefit ratio of flucytosine therapy should be considered in the setting of baseline neutropenia. However, we strongly recommend the use of flucytosine even when there is baseline neutropenia because of the substantial mortality benefit over alternative regimens. Both amphotericin B and flucytosine are associated with anaemia and thus serum haemoglobin should be monitored. A full blood count should be done at least weekly. A baseline differential count should be done if available and be repeated with subsequent full blood counts. Creatinine clearance needs to be monitored in order to adjust flucytosine dose. This is particularly important where baseline renal function impairment exists or where flucytosine is administered with amphotericin B deoxycholate  levels if symptoms of hepatitis or jaundice develop while patients are on fluconazole, but routine ALT monitoring is not indicated.+ <3.3 mmol/L), additional intravenous replacement is required: up to 2 ampoules of potassium chloride (20 mmol K+ per 10 mL ampoule) in 1 L of normal saline 8 hourly. Among those who develop hypokalaemia, serum potassium should be monitored daily until it is resolved. If hypokalaemia remains uncorrected, serum magnesium should be checked (if this test is available) and/or oral magnesium supplementation should be doubled. Intravenous magnesium sulphate may be considered for persistent hypokalaemia and hypomagnesaemia. If serum creatinine doubles from baseline, one dose of amphotericin B deoxycholate may be omitted and/or pre-hydration may be increased to 1 L of normal saline 8 hourly; serum creatinine should then be monitored daily. If serum creatinine improves, amphotericin B may be restarted at a dose of 0.7 mg/kg/day and/or alternate-day treatment could be considered. If creatinine remains elevated or repeatedly rises, liposomal amphotericin B should be substituted if available or amphotericin B deoxycholate should be stopped and an alternative regimen should be used (refer to Recommendation 3). If febrile reactions occur with amphotericin B, paracetamol 1 g may be given 30 min before infusion or, for severe reactions, hydrocortisone 25 mg IV can be administered before subsequent infusions.22For significant hypokalaemia (serum K3) develops, then we recommend to reduce the flucytosine dose and repeat a neutrophil count as soon as possible. If the neutropenia is confirmed, then withhold flucytosine until counts recover. If the patient was on the preferred 1-week amphotericin B and flucytosine regimen, fluconazole may be added to amphotericin B, and extension of amphotericin B treatment to a second week should be considered.Bone marrow suppression is uncommon at the recommended induction flucytosine dose for 1 week only, especially if renal function is normal . Other causes of a low neutrophil count should also be considered. However, if grade 4 neutropenia  before commencing tenofovir. The panel advises that, among patients who present with relapse of CM or a first CM episode after interrupting ART, ART should also be restarted after 4–6 weeks. One situation where ART may be delayed further is if a patient is still symptomatic with headaches at the visit when ART is about to be started. In such a situation, a LP should be repeated to measure pressure and for fungal culture to exclude persistent culture positivity. Antiretroviral treatment should be deferred and such patients may require further LPs or amphotericin B to ensure control of symptoms before starting ART . Asymptomatic CrAg-positive patients who decline consent for LP or for whom LP is contraindicated should start ART after at least 2 weeks of antifungal treatment .62 Raised pressure may be present at the diagnosis of CM or develop while the patient is on treatment and may result in severe headaches, vomiting, confusion or depressed level of consciousness, ophthalmoplegia  and visual disturbance/loss. Clinicians need to consider raised intracranial pressure and manage appropriately if a patient exhibits these symptoms or signs at any stage of the management of CM. To alleviate raised pressure, therapeutic LPs are indicated. New-onset hypertension may be a sign of increased intracranial pressure (i.e. part of Cushing’s triad) and should prompt an LP to measure opening pressure instead of anti-hypertensive medications , remove 10 mL – 30 mL CSFIf opening pressure is > 25 cm HRepeat LP whenever there are symptoms or signs of raised intracranial pressure Daily therapeutic LPs may be requiredCM, cryptococcal meningitis; CSF, cerebrospinal fluid; LP, lumbar puncture.The panel agreed that it is a good practice to measure the CSF opening pressure whenever a diagnostic LP is performed. However, in practice, the opening pressure may not have been measured at the initial diagnostic LP. Thus, once the diagnosis of CM is made, an LP should be repeated to measure CSF opening pressure, particularly if the patient still has a headache . The pressure should be measured using a manometer with the patient lying down and without excessive spinal flexion. Approximately 15% of patients with initially normal intracranial pressure will develop raised intracranial pressure during treatment; thus, all patients should be monitored daily for headache or signs of raised intracranial pressure that should prompt an LP.Manometers are not readily available in all centres or settings. In the absence of a manometer, the CSF pressure can be crudely estimated in various ways:63Drop counting: obtaining ≥ 40 drops of free-flowing CSF in 60 s using a 22-gauge spinal needle suggests a high CSF pressure.An ‘eyeball test’: a powerful squirt of CSF from the LP needle suggests a high CSF pressure.2O although these sets consistently under-estimate the opening pressure.64Makeshift manometers from intravenous line sets can be used to estimate opening pressure in cm HThe panel cautions that the above methods may be prone to under-estimating the actual CSF pressure based on a manometer reading.2O), then 10 mL – 30 mL CSF should be drained . Then the need for pressure relief should be dictated by recurrence of symptoms of raised intracranial pressure. Patients may require daily LPs. Where a manometer is not available and there are clinical symptoms or signs of raised intracranial pressure, we advise performing an LP and removing 20 mL – 30 mL of CSF. A symptomatic improvement after the therapeutic LP would support the symptoms having been due to raised intracranial pressure . The patient should then be reviewed on subsequent days for ongoing symptoms and signs of raised intracranial pressure, which would indicate the need for further therapeutic LPs.If the opening pressure is raised . There are situations in which there is a recurrence of symptoms but CSF fungal cultures are negative. The causes are summarised in When a patient seeks care for a recurrent episode of meningitis, it is not always possible to immediately be sure what the aetiology is. The initial assessment should include:An evaluation of the patient’s adherence to fluconazole consolidation and maintenance phase treatment (using self-reported and pharmacy refill data).An enquiry as to whether the patient has recently started ART to support a possible IRIS diagnosis.An LP to measure opening pressure, assess CSF inflammation and for a prolonged fungal culture (request 14 days of incubation in the laboratory) on at least 3 mL – 5 mL of CSF. There is no role for India ink staining or CSF/blood CrAg assays in establishing the cause of recurrence as these tests may remain positive for months to years even in patients after successful treatment – refer to Recommendation 2.If the CSF is culture-positive and non-adherence does not appear to be the cause, then fluconazole susceptibility testing should be considered. Susceptibility testing should be performed in an academic/reference laboratory. The panel recommends this when there has been at least one documented relapse despite reported good adherence (Recommendation 2).If the cause of the recurrence is attributed to non-adherence, then the patient should be treated as for the first episode (Recommendation 3). The reasons for non-adherence should be explored and the patient should receive additional adherence counselling, preferably together with a treatment supporter. If the patient also interrupted ART, this should be re-initiated 4–6 weeks after induction antifungal treatment is re-started. Antiretroviral treatment may need to be adjusted if there is a concern that there has been virological failure.40 IRIS occurs on average 6 weeks after ART is commenced but delayed cases (> 1 year after ART initiation) have been described.41 Cryptococcal meningitis IRIS-associated mortality may be substantial.42 The most frequent manifestation is recurrence of symptoms of meningitis often with raised intracranial pressure. Typically, the CSF fungal culture is negative at the time of IRIS presentation – IRIS represents an immunological reaction rather than a microbiological recurrence. However, in cases where induction therapy was recent (< 2 months), the CSF fungal culture may still be positive. Other cryptococcal IRIS manifestations include lymphadenitis and cryptococcomas.40Paradoxical cryptococcal IRIS occurs among patients treated for cryptococcal disease who start ART and develop a recurrence or worsening of clinical manifestations of cryptococcal disease. Immune reconstitution inflammatory syndrome is thought to be the result of an immunopathological reaction directed at residual fungal antigens at sites of disease.In all patients with suspected paradoxical CM-IRIS, an LP should be performed to measure pressure and obtain a fungal culture incubated for up to 14 days. It is not possible to make a diagnosis of IRIS with certainty prior to excluding microbiological relapse on CSF fungal culture.If the symptoms are mild, the panel recommends performing therapeutic LPs if there is raised intracranial pressure, providing analgesia and increasing the fluconazole dose to 1200 mg daily with regular review and follow-up of the CSF fungal culture result. If the CSF fungal culture is subsequently confirmed as negative, the dose of fluconazole can be reduced to what it was (800 mg or 200 mg daily depending on the timing of the CM-IRIS event).If patients have severe symptoms or deteriorate with the approach above, the panel recommends treating with amphotericin B deoxycholate 1 mg/kg/day plus either flucytosine (100 mg/kg/day divided into four doses per day) or fluconazole 1200 mg daily until the CSF culture is confirmed as negative. If the CSF culture is still negative after 7 days’ incubation, amphotericin B can be stopped. If the fungal culture is positive by 7 days, then amphotericin B should be continued for 14 days if it is given with fluconazole . Daily therapeutic LPs may be required if opening pressure is raised. A CT brain scan should be considered as mass lesions and cerebral oedema can occur with IRIS. Analgesia should be provided. Among patients with severe IRIS who do not respond to the above treatment, corticosteroids should be considered: prednisone 1 mg/kg/day or an equivalent dose of dexamethasone for up to 2 weeks or until clinical improvement occurs, tapered over 2–6 weeks. Longer treatment may be required depending on the symptom response. The panel recommends that corticosteroids preferably should be used among patients with IRIS who are documented to be CSF fungal culture-negative and when other aetiologies  are excluded. However, if there is life-threatening neurological deterioration, corticosteroids should be started immediately.Cryptococcus, subsequent management should be discussed with a medical practitioner experienced in the treatment of CM. Such patients should receive induction therapy with amphotericin B again. Consolidation and maintenance options will depend on the fluconazole MIC and may include high doses of fluconazole, voriconazole or posaconazole with or without weekly amphotericin B infusions.25Among patients with meningitis caused by fluconazole-resistant Among patients with multiple relapses, it is important to document conversion of CSF from culture-positive to culture-negative before stopping amphotericin B and such cases should be discussed with a medical practitioner experienced in CM management and fluconazole susceptibility testing should be performed.65 However, a positive CrAg test or fungal culture is diagnostic for CM, and concomitant TBM is then less likely.66 All patients with CM relapse or cryptococcal IRIS should have diagnostic tests performed for concomitant TB with CSF and sputum GeneXpert Ultra assay, urinary lipoarabinomannan assay and chest radiograph .Distinguishing CM relapse or IRIS from TBM: Immune-suppressed HIV-seropositive adults are at high risk of disseminated TB, including TBM. Cerebrospinal fluid cell profiles and protein levels in CM are similar to those seen in TBM.


Failure to retain HIV-positive pregnant women on antiretroviral therapy (ART) leads to increased mortality for the mother and her child. This study evaluated different retention measures for women’s engagement along the continuum of care for prevention of mother-to-child transmission (PMTCT) option B+ services in Mozambique.We compared ‘point’ retention (patient’s presence in care 12-month post-ART initiation or any time thereafter) with the following definitions: alive and in care 12 month post-ART initiation ; attendance at a health facility up to 15-month post-ART initiation ; alive and in treatment at 1-, 2-, 3-, 6-, 9-, and 12-month post-ART initiation ; and alive and in care 12-month post-ART initiation with ≥75% appointment adherence during follow-up (i.e. ‘appointment adherence’ retention) or with ≥75% of appointments met on time during follow-up (i.e. ‘on-time adherence’ retention). Kaplan-Meier survival curves were produced to assess variability in retention rates. We used ‘on-time adherence’ retention as our reference to estimate sensitivity, specificity, and proportion of misclassified patients.Considering the ‘point’ retention definition, 16,840 HIV-positive pregnant women enrolled in option B+ PMTCT services were identified as ‘retained in care’ 12-month post-ART initiation. Of these, 60.3% (95% CI 59.6–61.1), 84.8% (95% CI 84.2–85.3), and 16.4% (95% CI 15.8–17.0) were classified as ‘retained in care’ using MOH, WHO, and IATT definitions, respectively, and 1.2% (95% CI 1.0–1.4) were classified as ‘retained in care’ using the ‘≥75% on-time adherence’ definition. All definitions provided specificity rates of ≥98%. The sensitivity rates were 3.0% with 78% of patients misclassified according to the WHO definition and 4.3% with 54% of patients misclassified according to the MOH definition. The ‘point’ retention definition misclassified 97.6% of patients. Using IATT and ‘appointment adherence’ retention definitions, sensitivity rates  were also low; however, the proportion of misclassified patients was smaller .More stringent definitions indicated lower retention rates for PMTCT programs. Policy makers and program managers should include attendance at follow-up visits when measuring retention in care to better guide planning, scale-up, and monitoring of interventions. In 2015, the World Health Organization (WHO) recommended that all HIV-positive pregnant women be provided with lifelong antiretroviral therapy (ART) regardless of their CD4 count and WHO staging, also called “option B+” . Women’s retention status was classified into a binary outcome (retained vs. not retained in care) using a 75% threshold for clinical attendance. Each woman contributed to the analyses from the date of ART initiation to the first occurrence of a failure event, as described in Table Kaplan-Meier survival curves were produced to estimate retention in care at 3, 6, 9, and 12 months post-ART initiation using each of the definitions described above. We assessed variability in the different measures of retention between respective definitions used. We did not consider ‘appointment adherence’ retention for the survival analysis, as this definition does not contemplate a time event, but rather a total number of visits completed during the observation period.We assessed sensitivity, specificity, and the proportion of misclassified women for each alternative definition of retention compared to the reference of an ‘ideal case scenario’ of full ‘on-time adherence’ retention, defined as a woman alive and in care 12 months post-ART initiation and ≥ 75% of scheduled visits attended on time (+/− 15 days). Sensitivity and specificity values were calculated with 95% confidence intervals.Of 31,186 HIV-positive women enrolled in the PMTCT option B+ program during the study period, 18,739 were considered as being retained in care at 12 months post-ART initiation according to the ‘point’ retention definition. We excluded 1899 women with a follow-up ART time of < 12 months. Finally, 16,840 women were included in this study.Of 16,840 women included, only 2764 (16%) and 407 (2%) were considered retained at 12 months post-ART initiation when considering regularity and timeliness of appointment attendance, respectively . The IATT, ‘appointment’, and ‘on-time adherence’ retention definitions reflect a more continuous engagement in care, in which follow-up visits between initiation of ART and the endpoint date are considered.As expected, we found that the more stringent the definition, the lower the probability for women to be defined as retained in care; when levels of attendance at visits during follow-up were considered, estimates were even lower (85 and 60% when using the WHO and MOH definitions compared to 16 and 1% when using the IATT and ‘on-time adherence’ retention definitions). At the end of the first year, most women starting ART had not adhered to their scheduled visits, with median appointment adherence at 66% and median on-time adherence at 40% among the 16,840 women considered as retained in care using the ‘point’ retention definition. Of these, only 16 and 2% were actually defined as retained in care 12 months post-ART initiation if a threshold of ≥75% for appointment and on-time attendance were considered, respectively.Initial visits in the first year of ART initiation are critical to ensure successful engagement in care, as they convey important preventive healthcare and risk reduction messages, involve intensive counseling, peer support, and monitor patterns of healthier behavior for the women and their exposed children. This interaction between the health system and the HIV-infected woman determines the survival likelihood of the pair and the mother-to-child transmission of HIV. Adding the frequency and regularity of visits into the concept of retention increases the quality of the PMTCT program evaluations, enabling a distinction to be made between women who attend for all, or some care and treatment, and those who completely fail to attend scheduled visits and are therefore considered not to be engaged in care.Table When considering ‘on-time adherence’ retention as the reference, all other definitions provided very low sensitivity rates to accurately detect patients retained in care, with high rates of misclassified patients. The WHO and MOH retention definitions provided the lowest sensitivity rates, of 3 and 4%, respectively. For the ‘point’ retention, WHO, and MOH definitions, which do not consider continuous follow-up in their calculations, the proportion of misclassified patients ranged from 54 to 97%. The ‘appointment adherence’ definition provided the highest sensitivity rate (11.7%), with a fairly low proportion of misclassified patients (18.3%) compared to all other definitions analyzed. We found that the median time to first failure of correct follow-up was short (≤3 months), if visit attendance during follow-up was considered, which demonstrates the need to implement early measures to prevent patients in the PMTCT program from disengaging from care. Such strategies should focus on already well-known barriers to regular attendance, such as a lack of disclosure, poor staff attitudes, competing personal priorities, medication side-effects, or transportation difficulties .While our definition of ‘on-time adherence’ retention represents the ideal situation, in which an HIV-positive pregnant woman who enrolls in a PMTCT program and starts ART under option B+ should be considered as fully engaged in care to ensure an optimal viral response, it is perhaps too stringent for PMTCT program evaluations. This definition is also time consuming to use on a routine basis and may not be compatible with current PMTCT service settings. Therefore, we recommend using either the IATT or the ‘appointment adherence’ retention definitions to better measure levels of engagement in care for mother-infant pairs.Our analysis had several limitations. To accurately evaluate attendance at scheduled visits, it is desirable to distinguish visits canceled in advance (either by the patient or the care provider) from ‘no show’ visits that are missed by the patient . Our datSome women, although categorized as having full attendance at scheduled visits, did not follow per se the recommended schedule of visits according to national guidelines. More frequent visits may indicate a more advanced HIV disease, problems in counseling that need to be addressed, or drug or laboratory reagent stock-outs . Less frFinally, we analyzed data from health facilities with ePLD in two provinces in Mozambique. Although these two provinces are among the most populated in the country, with large volumes of patients seen at health facilities, our results may not be generalizable to other regions in Mozambique or to other resource-limited settings.Our findings suggest that women’s engagement to care cannot be precisely assessed without taking into account their presence at key time-points along the continuum of care. Lack of agreed definition hinders comparison of findings from different programs. We recognize that capturing timeliness and regularity of women’s clinical attendance with routine data can be a complex task and that applying more stringent definitions is likely to result in lower rates of retention in care in PMTCT programs. Nonetheless, HIV services should improve its monitoring systems to ensure that clinical decision be made based on sound and timely assessment of women’s retention and key indicators related to maternal viral load, infant prophylaxis or infant HIV serostatus determination. A consensus on universal definition is urgently needed; however, data availability, diverse clinical practices in different settings, and local analytical capacities should be taken into account to ensure practicability of the definition. Since retention estimates are used for the modeling of vertical transmission rates of HIV, lack of precision could lead to false interpretation of program achievements in relation to elimination of mother-to-child transmission of HIV. This consideration may become particularly important as recent data seems to indicate a stagnation in the achievements of PMTCT programs worldwide, which suggests the need for revised global, national, and local strategies to end the HIV epidemic by 2030.