Many studies include oral HIV-related endpoints that may be diagnosed by non-oral-health specialists (non-OHS) like nurses or physicians. Our objective was to assess the accuracy of clinical diagnoses of HIV-related oral lesions made by non-OHS compared to diagnoses made by OHS.A5254, a cross-sectional study conducted by the Oral HIV/AIDS Research Alliance within the AIDS Clinical Trial Group, enrolled HIV-1-infected adults participants from six clinical trial units (CTU) in the US  and Haiti. CTU examiners (non-OHS) received standardized training on how to perform an oral examination and make clinical diagnoses of specific oral disease endpoints. Diagnoses by calibrated non-OHS were compared to those made by calibrated OHS, and sensitivity and specificity computed.3. The overall frequency of oral mucosal disease diagnosed by OHS was 43% in US sites, and 90% in Haiti. Oral candidiasis (OC) was detected in 153 (47%) by OHS, with erythematous candidiasis (EC) the most common type (39%) followed by pseudomembranous candidiasis . The highest prevalence of OC (79%) was among participants in Haiti, and among those with CD4+ cell count ≤ 200 cells/mm3 and HIV-1 RNA > 1000 copies/mL (71%). The sensitivity and specificity of OC diagnoses by non-OHS were 90% and 92% . Sensitivity and specificity were also high for KS , but sensitivity was < 60% for HL and oral warts in all sites combined. The Candida culture confirmation of OC clinical diagnoses  was ≥ 93% for both PC and EC.Among 324 participants, the majority were black (73%), men (66%), and the median CD4+ cell count 138 cells/mmTrained non-OHS showed high accuracy of clinical diagnoses of OC in comparison with OHS, suggesting their usefulness in studies in resource-poor settings, but detection of less common lesions may require OHS. Since the beginning of the AIDS epidemic, much has been learned about human immunodeficiency virus (HIV)-related oral mucosal diseases. The occurrence of oral candidiasis (OC) and hairy leukoplakia (HL), the most common HIV-related oral diseases, are strongly associated with a low CD4+ cell count –7 and a The Oral HIV/AIDS Research Alliance (OHARA) was created in 2006 to partner with the AIDS Clinical Trials Group (ACTG) Network, which plays a major role in defining the standards of care for treatment of HIV infection and related opportunistic infections. OHARA’s Several ACTG protocols incorporate OHARA substudies and have shared oral end-points known to be associated with HIV/AIDS, that are assessed by non-oral-health clinical trial unit (CTU) examiners . Even though the diagnosis of pseudomembranous candidiasis (PC), routinely referred to as “thrush”, is very familiar to HIV clinicians, the diagnosis of erythematous candidiasis (EC) is more subtle, and may be missed in the absence of specialized training. Therefore, to standardize the measurement of oral mucosal outcomes associated with HIV/AIDS within and outside the ACTG infrastructure the OHARA clinical team developed an extensive training module. The objectives of the present study was to: 1) assess the accuracy of clinical diagnoses of HIV-related oral mucosal disease made by CTU examiners who are non-oral health specialists (OHS) compared to diagnoses made by OHS; and 2) estimate the frequency of oral mucosal disease among participants recruited as part of the ACTG within domestic and international CTUs.A5254 was a cross-sectional study that enrolled HIV-1-infected adults 18 years or older with or without prior ART from five ACTG CTUs in the US and one international CTU in Haiti. Institutional Review Boards or Ethics Committees of each participating institution approved the study, and each patient gave written informed consent. Participating Institutions were: Case Western, Cleveland; Emory University, Atlanta, New York University, New York; UCSF, San Francisco; and UNC-CH, Chapel Hill; in the US, and Centres GHESKIO, Port-au-Prince, in Haiti.At study entry, participants were stratified into four strata listed below according to their documented or screening CD4+ cell count and plasma HIV-1 viral load obtained within 60 days prior to study entry:Stratum A: CD4+ cell count ≤ 200 cells/mm³ and plasma HIV-1 viral load > 1,000 copies/mL (target N = 200).Stratum B: CD4+ cell count ≤ 200 cells/mm³ and plasma HIV-1 viral load ≤ 1,000 copies/mL (target N = 100).Stratum C: CD4+ cell count > 200 cells/mm³ and plasma HIV-1 viral load > 1,000 copies/mL (target N = 30).Stratum D: CD4+ cell count > 200 cells/mm³ and plasma HIV-1 viral load ≤ 1,000 copies/mL (target N = 30).The intent was to optimize the enrollment of individuals with more severe immunosuppression, thus a higher likelihood of HIV-related oral lesions, to provide adequate precision to our estimates of sensitivity and specificity.CTU examiners (non-OHS) received a standardized training on the performance of oral mucosal examination, and on the clinical diagnoses of specific oral disease endpoints. The training consisted of a 3-hour session that included a video of a standardized oral mucosal examination; a didactic lecture using clinical slides of oral lesions, and published case definitions for each endpoint; and a haThe OHS included four oral medicine specialists, two general dentists, one otolaryngologist, and one hygienist. All had extensive experience managing the oral health of HIV-infected patients and in diagnosing HIV-related oral diseases. The OHS watched the same presentation as that administered to the CTU examiners. Although a pre-test was not administered  oral health specialists were asked to complete a similar post test comprised of 50 oral lesions slides as described above. Again, a minimum score of 80% was required for calibration.Information regarding socio-demographic characteristics and general health-related variables, such as history of AIDS-related illnesses and current medications, were collected using a questionnaire administered during the study visit.Candida. A blood draw was performed at the time of the visit for CD4+ cell count and plasma HIV-1 viral load to be measured. The CD4+ cell count and the HIV-1 viral load assay were performed in a CLIA certified laboratory for US sites, and in a laboratory certified for protocol testing by the DAIDS Immunology Quality Assurance (IQA) Program for the Haiti site. Plasma HIV-1 viral load were performed utilizing the Abbott Realtime HIV-1 Assay.An extra-oral examination of the major salivary glands, and oral mucosal examination were performed by both a CTU examiner (non-OHS) and an OHS on each participant. Both examiners recorded their findings including descriptors of lesions with respect to location, color, and character, and a presumptive diagnosis. Examiners were blinded to each other’s findings. Oral disease endpoints explored included PC; EC; AC; HL; herpes labialis; recurrent intra-oral herpes simplex; warts; recurrent aphthous stomatitis; necrotizing gingivitis/periodontitis; necrotizing stomatitis; KS; non-Hodgkin’s lymphoma; squamous cell carcinoma; and salivary gland disease (as defined by presence/absence of parotid enlargement). A 5-minute unstimulated whole saliva (UWS) flow rate was recorded, and collected. A 1-minute oral rinse/throat wash using 10 mL of sterile saline was also collected. Both saliva and throat wash specimens were processed, frozen in aliquots at minus 80°C at the site laboratory, and shipped to the UNC-CH specimen bank unit. Before, the throat wash was processed at the sites, 2.5 mL was extracted and cultured for the presence of The 2.5 mL of oral/throat wash was centrifuged for 5 min at 3000 rpm and the supernatant discarded. The remaining pellet was resuspended by gentle tapping, with colony counts performed from the suspension and from a 1:10 diluted suspension using a calibrated 10μL loop. Cultures were performed on Sabouraud or Potato Dextrose agar plates and incubated at 37° for 48 hrs. A culture was defined as positive, and confirming the clinical diagnosis of OC, among individuals with clinical features of OC and a number of colony forming units (CFU) ≥ 1/mL.Resultant yeast colonies were subcultured onto an agar slant and sent to the Center for Medical Mycology, Cleveland, OH for identification. Strains were subsequently identified to genus and species using the BioMerieux API 20C system (these results are reported in a separate manuscript focusing on the microbiology aspect of the study).st and 3rd quartiles (Q1 and Q3) for continuous variables, and are presented for all CTUs, and for US and non-US sites separately. We compared CD4+ cell count and plasma HIV-1 viral load among participants in US versus non-US sites using the Wilcoxon rank-sum test.Sample characteristics were summarized using proportions for categorical variables, and median with 1We computed the frequency of specific oral lesions, parotid enlargement, and salivary hypofunction (defined as UWS flow rate < 0.1 mL/min)  by CD4+ We computed the percentage of clinical diagnosis for each type of OC that were confirmed by a positive culture (≥ 1 CFU/mL) of an oral/throat rinse aliquot.Seven OHS participated in the calibration across the 5 US CTUs. Prior to study initiation the median score obtained from the post-test for 6 OHS was 96% (range 92–98%), compared to one OHS who served as the reference standard. After one year, the OHS from Haiti was added to the group . The calibration at 1-year after study start-up was performed for 7 OHS  compared to the reference standard, and the median score obtained from the post-test was 98% (range: 90–100%).A total of 18 CTU examiners (non-OHS) participated in the baseline training, and the median post-test score obtained was 90% (range 75–100%). One examiner had a score of 75%, and after re-taking the training and post-test, scored 95%. Among the 18 examiners who were trained and calibrated, only 9 actually participated in the protocol all of whom repeated the training and calibration after one year, achieving a median score of 98% (range 85–100%).3 and plasma HIV-1 viral load > 1,000 copies/mL), while 105 (93%) among Haitian participants were enrolled to stratum A. So the median CD4+ cell count was substantially lower in Haiti  than in US sites . Similarly, the plasma HIV-1 viral load was much higher among Haitian than US participants. A Wilcoxon test comparing CD4+ cell count and plasma HIV-1 viral load between US and non-US sites yielded a p-value < 0.001 for both variables. Sixty six percent of all participants were on ART at study entry, and 8% had a history of an AIDS-defining illness. The proportion of Haitian participants receiving ART (53%) was slightly lower than US participants (73%), but the reported history of an AIDS-defining illness was similar in Haiti and US sites .Protocol A5254 enrolled its first participant in October 2009, and last participant in September 2012. After an interim power calculation revealed that the OC prevalence was higher than expected, the Study Monitoring Committee recommended closing the study to accrual before the target sample size of 360 was reached. The final sample size was 328 participants. Among the 328, one participant left the clinic before the oral examination could be performed, and 3 participants were seen by the CTU examiner, but not by the OHS, resulting in a final sample of 324 participants who received oral examinations by both examiners. Among these, the majority were Black (73%) including 58% of participants in US sites, and had never used injection drugs 85%; . The womp < .0001). The predominant type of OC was EC (60% among stratum A participants) followed by PC. The next most common oral lesion was HL (12%) followed by KS (10%), although KS was more frequently detected in participants in stratum A (17%) than HL (14%). Oral warts were detected in 8% of participants. While most oral mucosal lesions were significantly more common among participants in stratum A, parotid enlargement was seen in a significantly higher proportion of participants with CD4+ cell count > 200 cells/mm3 (14%) than among those with CD4+ cell count ≤ 200 cells/mm3 (stratum B: 11% and stratum A: 4%). Interestingly though, the frequency of salivary hypofunction (UWS flow rate < 0.1 mL/min) was significantly higher in stratum A (10%), than in Stratum B (1%) or C and D combined (5%). Finally, the frequency of oral mucosal disease diagnosed by OHS among participants in US sites was 43% versus 90% among participants in Haiti  compared to OHS was very high, estimated at 90% and 92%, respectively, for all sites combined . AccuracCandida culture was high among participants with clinical features of OC of all types, and for both diagnoses made by OHS and non-OHS  was also higher in Haiti than in US sites, which is not surprising given that the median CD4+ cell count was significantly lower and the plasma HIV-1 viral load significantly higher among participants in Haiti. As previously shown in other studies, there was a strong association between a high prevalence of most oral lesions and low CD4+ cell count and detectable plasma HIV-1 viral load.[Our study revealed a much higher prevalence of OC than expected  given that 66% were receiving ART. The prevalence of EC was especially high (1.5 times higher than that of PC). While the prevalence of OC was the highest among participants in Haiti (79%), it was also high among US participants (30%). More than 92% of the OC clinical diagnoses made by either OHS or non-OHS were culture-confirmed, assuming that in the presence of clinical features of OC a fungal culture exhibiting ≥ 1 CFU/mL for one or more ral load. However,The accuracy of clinical oral lesion diagnoses made by non-OHS as compared to OHS in US and non-US sites was high for the various forms of OC, which was further confirmed by the very high percentage of positive culture confirmation of the clinical diagnoses. This is an important finding, especially in resource-limited settings where OC may be a useful surrogate marker for HIV-disease stage. For example, in a recent study conducted by the OHARA/ACTG group, we found a strong association between OC and TB disease, independent of CD4+ cell count, suggesting that in resource-limited settings, OC may provide clinical evidence for increased risk of TB and contribute to TB case finding. In this The sensitivity of non-OHS diagnoses was lower for less common oral lesions, such as HL (in US sites), oral warts, and parotid enlargement, even though the specificity remained high for these conditions. A possible explanation for the low sensitivity of less common oral lesion diagnoses made by non-OHS may be frequency of detection. There was a relatively low number of cases seen over an extended period of time (it took three years to complete the study due to the difficulty in recruiting participants with low CD4+ cell count at US sites). Despite yearly trainings and online access, the CTU examiners (non-OHS) may not have been able to retain their competency with respect to the clinical diagnosis of less common conditions. This underscores the importance of repeatedly seeing oral mucosal lesions in a patient’s mouth in addition to seeing them on a picture in learning how to make a differential diagnosis. For OC, the CTU examiners had much opportunity to see the lesions clinically, and thus to learn how to differentiate one type of OC from another, whereas for less common lesions the clinical exposure was insufficient to reinforce the training acquired from clinical pictures. The higher sensitivity for the diagnosis of HL in the Haiti site (78% versus 43% in the US site), is consistent with this explanation as HL was seen more frequently in Haiti (16%) than in domestic sites. This is a difficult issue to circumvent, because clinical training sessions would require a very large number of HIV-positive patients to be able to visualize uncommon lesions. However, there is little reason to try to address this issue since the use of oral lesions as surrogate markers of HIV stage pertains mainly to common lesions like OC.3 and the median plasma HIV-1 viral load was 180,000 copies/mL, which certainly explains the high prevalence of oral opportunistic infections. This finding exemplifies the potential use of oral lesions as a marker of underlying immune suppression and ongoing HIV viral replication. While participants who were receiving ART initiated treatment at least 3 months prior to study entry (as required by the protocol), a subset of individuals may have been within their first 6 months of treatment, and may not have yet achieved optimal immune reconstitution leaving them vulnerable to opportunistic infections. The high prevalence of oral lesions among HIV-positive patients in Haiti and their persistence in the US HIV-positive populations underscores the importance of medical providers performing routine oral examinations as part of their physical examinations of HIV-infected patients. Training in the diagnosis and treatment of these conditions is critical, since most of these patients would likely not have access to care from an OHS.Our group previously established case definitions for oral mucosal manifestations of HIV/AIDS in the post HAART era. This is Candida carriage threshold at which patients develop clinical manifestations of OC.The strengths of this study include the large number of participants, which allowed us to assess estimates of sensitivity and specificity of OC diagnosis by non-OHS as compared to OHS with high precision. Further, clinical diagnoses of OC were confirmed by culture, which is the gold standard for definitive OC diagnosis. We determined that non-OHS can be effectively trained in the recognition of not only PC , but also EC, which is characterized by a discrete patchy erythema, and is often missed by non-OHS. The lower sensitivity of the non-OHS’ diagnoses of less common lesions like oral warts or HL suggests that any clinical trial or study that includes these endpoints should either rely on OHS for the diagnosis, or provide repeated training at 6-month intervals, with non-OHS spending some time in an oral medicine clinic setting where they would have the opportunity to observe these lesions. The next step for the present study is to assess the oral


In human immunodeficiency virus treatment adequate virological suppression is warranted, nevertheless for some patients it remains a challenge. We investigated factors associated with low-level viraemia (LLV) and virological failure (VF) under combined antiretroviral therapy (cART).st July 2012 and 1st July 2013 with at least one viral load (VL) measurement below the quantification limit (BLQ) in their treatment history. After a minimum of 6 months of unmodified cART, the next single VL measurement within 6 months was analysed. VF was defined as HIV RNA levels ≥200 copies/mL and all other quantifiable measurements were classified as LLV. Factors associated with LLV and VF compared to BLQ were identified by logistic regression models.We analysed patients receiving standard regimens between 1Of 2276 participants, 1972 (86.6%) were BLQ, 222 (9.8%) showed LLV and 82 (3.6%) had VF. A higher risk for LLV and VF was shown in patients with cART interruptions and in patients with boosted PI therapy. The risk for LLV and VF was lower in patients from centres using the Abbott compared to the Roche assay to measure VL. A higher risk for LLV but not for VF was found in patients with a higher VL before cART  and shorter cART duration [for <9 months: aOR (95% CI): 2.59 (1.38–4.86)]. A higher risk for VF but not for LLV was found in younger patients , people originating from high prevalence countries [aOR (95% CI): 2.20 (1.09–4.42)] and in male injecting drug users [aOR (95% CI): 2.72 (1.38–5.34)].For both VF and LLV, factors associated with adherence play a prominent role. Furthermore, performance characteristics of the diagnostic assay used for VL quantification should also be taken into consideration. The advent of combined antiretroviral therapy (cART) resulted in a significant reduction in morbidity and mortality for persons infected with the human immunodeficiency virus type 1 (HIV-1) . SuppresThe objective of this study was to investigate factors associated with LLV defined with a cutoff <200 copies/mL and VF in a cohort of HIV-1 positive patients.The Austrian HIV Cohort Study (AHIVCOS) is an open, multicentre, prospective, observational cohort study of HIV-infected individuals followed at seven HIV treatment centres in Austria. The study was initiated in 2001 and patients are enrolled actively and prospectively covering approximately 80% of all treated HIV-infected patients in Austria. Data collection includes sociodemographic information, complete history of cART as well as laboratory results with VL and CD4 cell count measurements, usually repeated every 3–6 months. The laboratory analyses were performed in each single centre and for HIV-1 VL assessment, five centres used the Roche Cobas AmpliPrep/Cobas TaqMan 2.0 assay , referred to as the Roche assay in this manuscript, while the remaining two centres used the Abbott RealTime HIV-1 assay , referred to as the Abbott assay, during the period of time analysed.Approval for this study was obtained from the local ethical committees of all participating centres: ethics committee of the Vienna Medical University (No. 898/ 2010), of the Salzburg Federal Government (No. 1159/ 2010), of the Graz Medical University (No. 21-431/ 2010), of the Innsbruck Medical University (No 283/4.4/ 2009), of the Upper Austria Federal Government (No C-3-10/ 2010) and of the Carynthian Federal State (No A-13-11/2011). Written informed consent was given by the patients for their information to be stored in the hospital database and used for research.st July 2012 and 1st July 2013 enrolled in AHIVCOS (N = 4289). Patients who were not on stable cART due to either interruptions or switches were excluded (N = 290). During these 6 months, patients had to receive cART with 2 nucleoside reverse-transcriptase inhibitors (NRTIs) and either a non-nucleoside reverse-transcriptase inhibitor (NNRTI) or a boosted protease inhibitor (PI) or an integrase inhibitor (INSTI). Individuals with other cART regimens than those mentioned above, were excluded (N = 489). A further exclusion criterion was a lacking VL below the respective limit of quantification in their treatment history. Thus, another 461 individuals were excluded from analyses (35 tested with the Abbott assay and 426 with the Taqman assay).A retrospective analysis was conducted on individuals receiving unmodified cART for more than 6 months between 1In total, 3049 patients fulfilled the inclusion criteria. 773 patients did not have any VL measurement between 6 and 12 months after initiation of the respective cART regimen and were too excluded. 2276 patients remained and their first VL measurement between 6 and 12 months after initiation of the respective cART regimen was evaluated.All quantifiable HIV VL measurements below <200 copies/mL were classified as LLV. For further analyses, LLV was divided into two categories, namely HIV RNA levels of 51–199 copies/mL and HIV RNA levels of ≤50 copies/mL. VF was defined as HIV RNA levels ≥ 200 copies/mL.Groups were compared using χ² test or Fisher´s exact test where appropriate. To investigate the association between various demographic and clinical parameters with LLV as well as VF compared to BLQ, two separate univariable and multivariable logistic regression models were performed. As reference category for both analyses, patients who were BLQ at baseline were used (N = 1972). For the first analysis, to find factors associated with LLV, 82 patients with VF were excluded. 222 (10.1%) of the remaining 2194 patients had LLV below 200 copies/mL. For the second analysis, to find factors associated with VF, 222 patients who had LLV below 200 copies/mL were excluded. 82 (4.0%) of the remaining 2054 patients had a VF. Variables considered for univariable analyses were age at VL measurement, HIV transmission category, nationality, CD4 count before cART, ever cART interruptions, type of VL assay used, diabetes, cART regimen, VL before cART and cART duration. Multivariable models were adjusted for demographic variables age at VL measurement and HIV transmission category whether or not significant in univariable analyses. Further adjustment was made for variables with p <0.05 in univariable analyses. Ever cART interruptions were defined as interruptions prior to 6 months of stable cART of the respective cART regimen. A cART discontinuation for at least 8 days after having started cART was regarded as cART interruption.In addition, different sensitivity analyses were performed for different criteria in patient selection. Another sensitivity analysis was conducted in order to analyse patients´ characteristics stratified by cART regimen. Further, we additionally did an analysis for the two outcomes LLV and VF excluding the centres measuring VL with the Abbott assay. P-values of <0.05 were considered to indicate statistical significance.All analyses were conducted using Stata software, version 13.1 .The flowchart of the patient selection is shown in As shown in A reduction in risk of VF was found in patients from 2 centres using the Abbott assay as compared to the other centres measuring VL by the Roche assay [aOR (95% CI): 0.09 (0.03–0.30)]. Younger age , male intravenous drug users [aOR (95% CI): 2.72 (1.38–5.34)] versus men who have sex with men, originating from high prevalence countries [aOR (95% CI): 2.20 (1.09–4.42)], interrupted cART [aOR (95% CI): 2.93 (1.84–4.67)] compared to uninterrupted cART and 2 NRTIs with a boosted PI as cART regimen [aOR (95% CI): 2.36 (1.45–3.83)] versus 2 NRTIs with a NNRTI or INSTI increased the risk of VF .We performed several sensitivity analyses with different criteria concerning patient selection. One analysis was performed without considering a VL measurement BLQ in treatment history in order to exclude patients with very short cART durations. Another analysis was conducted regarding a shorter recruitment period of 6 months instead of 12 months and one analysis including patients receiving unmodified cART for >9 months instead of >6 months. All additional analyses did, however, not reveal any substantial differences in ORs compared to our primary analysis (data not shown). Stratification of the patients by regimen showed that VL and age at baseline, HIV transmission category, nationality, CD4 cell count before cART, prior cART interruptions, assay used and VL before cART differ significantly see . In addind or more lines of therapy and PIs such as NNRTIs were both equally represented , followed again by INSTIs (N = 184).This study of well-defined patients on stable cART over a period of more than 6 months gives insights into the different factors associated with LLV and VF. As expected, adherence-associated factors play a predominant role. Accordingly, cART interruptions constitute an independent risk factor for both LLV and VF, while younger age, the category of male injecting drug users and originating from high prevalence regions are associated with VF alone. Additionally, PI-based regimens appear as independent markers for both groups. Several studies have already shown that NNRTI-based combinations may result in a better suppression of plasma viral load than PI-based cART –7. On thFurther, the association found between high baseline VL and the risk of developing LLV is in line with the hypothesis that the size of the cellular compartment infected before cART initiation determines the level of residual viraemia  and is iFor our group of patients on stable suppressive therapy, we found a significant risk reduction for LLV and VF according to the assay used for VL quantification. This finding is interesting since both the Abbott as well as the Roche assays are widely used in clinical settings to measure HIV RNA and differ significantly regarding their extraction system, primers and probe design. Although the overall correlation between the Roche and Abbott assay in the higher range of quantification is good ,10, signFinally, concerned by the high number of patients excluded from our study due to missing VL assessment within 6 to 12 months (N = 773), we compared patients´ characteristics of the two groups (data not shown). Individuals excluded differed significantly from included patients in some factors associated with LLV and/or VF. Excluded patients were significantly older, males were more frequently infected through injecting drug use and females through heterosexual contacts. HIV RNA of excluded individuals was more frequently measured by the Roche assay. On the other hand, VL and CD4 count before cART initiation were significantly lower and excluded patients had a shorter overall therapy duration, more cART interruptions and were more frequently on first-line therapy.In conclusion, the strengths of our study include its large sample size with similar national standards of care and our results support that adherence associated issues play a central part in LLV and VF development. The association between PI-based regimens and risk of LLV and VF remains biased due to confounding by indication. Our data seems to support the concept that performance characteristics of the diagnostic assay used has to be taken into account for evaluation of LLV and especially in cases where clinical decisions such as therapy switches are pending. The present study does not yet include clinical follow-up and predictivity. Despite its observational design, it provides implications for patient management and forms the basis for future outcome studies. We suggest that similar studies with clinical follow-up including different test systems may be able to evaluate the need for guidelines taking into account different performance characteristics of each assay to establish adequate HIV RNA cutoffs.S1 Table(DOCX)Click here for additional data file.S2 TableExclusion of the samples in which the Abbott assay was used.(DOCX)Click here for additional data file.


In a recent clinical trial (ACTG 5257), no difference in viral failure (VF) of a first-line cART containing atazanavir/r (ATV/r) or darunavir/r (DRV/r) was found . For theParticipants in the ICONA Foundation Study who started cART with 2NRTI+ ATV/r or DRV/r while ART-naïve were included. Several endpoints were evaluated: confirmed VF>200 copies/mL after six months of therapy, discontinuation of DRV/r or ATV/r for any reasons or because of intolerance/toxicity (as reported by the treating physician) and the combined endpoint of VF or stop. Survival analysis with Kaplan–Meier curves and Cox regression model stratified by clinical site was used. Patients' follow-up accrued from cART initiation to the date of the event or to the date of last available visit/viral load.894 patients starting 2NRTI+ATV/r and 686 2NRTI+DRV/r when ART-naïve on average in 2011 (IQR: 2010–2012) were studied. Most common NRTIs used were FTC/TDF (84%) and ABC/3TC (12%). Median age was 40 years, 22% females, 44% heterosexuals. Patients starting ATV/r were more likely to be hepatitis B/C infected , they started one year earlier , were more likely to be enrolled in sites located in the north of Italy , started cART less promptly after HIV diagnosis  and less likely to have started TDF/FTC . By two years of cART, 9.8% (95% CI 7.6–12.0) of those starting ATV/r experienced discontinuation due to intolerance/toxicity vs 6.5% in DRV/r group . After controlling for several potential confounders  the relative hazard (RH) for ATV/r vs DRV/r was 2.01 . There were no statistical differences detected for any of the other outcomes.Although unmeasured confounding cannot be ruled out, our results seem to be consistent with those of the ACTG 5257. When all cause discontinuations were considered, or the composite endpoint of treatment failure, there was no difference between ATV/r- and DRV/r-based regimens.


In a perspective on Habiyambere and colleagues, Peter Kilmarx and Raiva Simbi discuss the disconnect between HIV testing instrument capacity and utilization. UNAIDS has set ambitious global targets for the year 2020 of diagnosing 90% of all people living with HIV (PLWHIV), initiating antiretroviral treatment (ART) for 90% of those diagnosed with HIV infection and achieving an undetectable viral load in 90% of those on ART . SignifiLaboratory testing is a critical component in reaching these goals . MeasureIn this issue, Habiyambere and colleagues report the results of annual global surveys on availability and use of these testing technologies . QuestioWhy is there such a striking disconnect between capacity and utilization? The survey found that lack of machine installation, breakdowns, and lack of reagents were all factors, and most machines were not covered by maintenance contracts or receiving recommended service . But undThe results suggest that some programs may have overly focused on machine procurement and did not plan appropriately for the other critical aspects of a functioning national HIV laboratory monitoring system. Funding organizations must commit to supporting the full spectrum of laboratory program needs. Laboratory directors need the program management training, tools, resources, and authorities for implementation. Ensuring transparent public procurement of these complex technical products and services is also critical . Use of While Habiyambere et al. have highlighted critical shortfalls in CD4 and viral load testing, the problem is even greater when considering the gaps in the entire cascade of events, which are not captured in their survey, starting with clinicians ordering tests when appropriately indicated and ending with timely return of results and using them for appropriate clinical decision making . These gMuch has been accomplished to date, even in settings with limited laboratory capacity. In Zimbabwe, for example, nearly 900,000 people have been initiated on ART with good levels of retention and adherence by global standards, and the great majority of patients are doing well on first-line therapy . ScalingA sort of “honeymoon” period in HIV treatment in resource-poor settings, when most patients were treatment naïve, initially symptomatic, and highly adherent, and high levels of viral load suppression could be confirmed with periodic representative samples of ART patients, is coming to an end as both transmitted resistance and acquired resistance are increasing worldwide . In this


Human immunodeficiency virus (HIV) associated anemia is common and it is a challenge for prognosis of HIV positive patients. It is stated in different literature that the prevalence of anemia due to HIV before highly active antiretroviral therapy (HAART) initiation is more prevalent than after HAART initiation. Thus this study aimed to assess anemia prevalence before and after initiation of HAART among HIV patients attending university of Gondar hospital from 2008 – 2013.A retrospective study was conducted by collecting data from antiretroviral clinic of University of Gondar Hospital from January 1, 2008 to December 30, 2013. Data was collected by using data collection sheet which contains age, sex, regimen type, hematological parameters and CD4 cell count. Data were analyzed using SPSS version 20. In order to compare means paired t-test was used. P- Value <0.05 was considered as significant.Prevalence of anemia before and after HAART initiation was 21.2% and 11.5% respectively. There is a significance difference in CD4 cell count, hemoglobin and hematocrit values on patients before and after HAART initiation (P <0.001). Opportunistic infection and CD4 cell count were associated with prevalence of anemia before HAART initiation.There was a decline in the prevalence of anemia and increment of mean CD4 cell count among HIV infected patients after HAART initiation. HIV patients are recommended checking up their CD4 cell counts regularly and starting HAART when it is appropriate with regular follow-up. Anemia is the most common hematological abnormality in HIV positive patients. It has been shown to be the risk factor for early death in patients with AIDS. Anemia is not only associated with decreased quality of life but it is also independently associated with decreased survival with HIV . It is dPrevious studies have found that the incidence of anemia increases with progression of HIV infection. Several other etiologic factors may also be involved in the development of HIV-associated anemia, including micronutrient deficiencies, immunological myelosuppression, impaired erythropoietin production and blood loss from intestinal opportunistic disease .Anemia is a condition in which there is a decrease in the red blood cell count, hemoglobin and/or hematocrit values as compared to normal reference range for age, sex, race and altitude which decreases oxygen-carrying capacity of red blood cells to tissues . It is dAnemia is observed in both antiretroviral therapy treated and untreated individuals, but severity varies due to the immune status or CD4 cell levels and also the prevalence increases as the disease advanced and left untreated . If an iMost studies described the prevalence of anemia in HIV infected individuals before and after initiation of highly active antiretroviral therapy (HAART), however few studies were conducted in Ethiopia on the assessment of anemia among HIV positive patients , 12, 16.This retrospective study was conducted in the ART clinic of the University of Gondar Hospital which is found in Northwest of Ethiopia. Patients’ data with full information of sex, age, regimen type, hematological and immunological parameters, WHO clinical stage, type of regimen, opportunistic infections and cotrimoxazole were collected from follow up charts and registration books of patients who started HAART at GUH ART clinic from January 1, 2008 to December 30, 2013. Hematological and immunological parameters were collected at baseline when the HIV positive patients adhere to ART and after six month of HAART initiation. The quality of data were controlled at different levels for completeness and consistency. Whenever an error was found at any level, it was corrected by rechecking from the registration book.The data were cleaned, edited, checked for completeness and entered into SPSS version 20 for analysis. Descriptive statistics was used to get a clear picture of dependent and independent variables. In order to compare means paired t-test was used and also chi square was computed to determine association between dependent and independent variables. P- Value <0.05 was considered as significant.The study was conducted after ethical letters obtained from School of Biomedical and Laboratory Science Ethical Committee. Then permission was taken from hospital higher management. After permission was obtained, data were collected from ART clinic. To keep confidentiality codes were used and unauthorized person didn’t have access to the data.A total of 349 HIV positive patients, 218 (62.5%) females and 131 (37.5%) males, were involved in this study. The overall mean age was 34.6 ± 8.5 years, within the range of 15 – 60 years old. Majority of the patients [154 (44.1%)] were within 26 – 35 years of age. About 226 (64.8%) of participants were under WHO clinical stage I & II, 87.7% of them were taking cotrimoxazole prophylaxis therapy and about 4.3% of them were TB co-infected   before HAART initiation and 11.5% (40/114) after HAART initiation. About 40 (11.5%) had mild anemia, 27 (7.7%) had moderate anemia, and 7 (2%) had severe anemia before HAART initiation. However, the prevalence of anemia after HAART initiation was significantly decreased by 9.7%  were males and 22% (48/118) were females. Similarly 30.4% (14/46) patients at the age of <25 years old and 35.2% (50/142) of patients with CD4 cell count <200 cells/μl were developed anemia. There was a significant association between anemia with CD4 cell count and TB before HAART initiation. HIV patients with CD4 cell count <200 cells/μl before HAART initiation had higher prevalence of anemia . Similarly about 53.3% (P =0.002) patients with TB had developed anemia before HAART initiation. However, there was no association of anemia with sex, age, and WHO clinical stage  were males and 13.8% (30/118) were females. Similarly 12.3% 14/146) patients at the age of 36–45 years old and 18% (18/100) patients with CD4 cell count <200 cells/μl were developed anemia. Although, HIV patients with CD4 cell count <200 cells/μl had higher prevalence of anemia (18%) after HAART initiation, there was no significance association between anemia and CD4 cell count (P =0.051). Similarly sex, age regimen type and WHO clinical stage had no association with anemia  and 37.3%) , Hawassa% and  in this study was lower than study done in different countries, such as Germany from 2001–2002 (61%) , India iPrevalence of anemia after HAART initiation (11.5%) was lower study done in in studies from Minillik II hospital, Addis Ababa 200 (37.3%)  and HawaThe prevalence of anemia observed across sex groups before HAART initiation was 19.8% (26/131) for males and 22% (48/218) for females similarly after HAART initiation 7.6% (10/131) and 13.8% (30/218) were male and female patients were anemic respectively. This was slightly lower than a report from Minillik II hospital ART clinic, Addis Ababa Ethiopia, 70.25% vs. 29.75% in males and females before HAART initiation and 69.23% vs. 30.77% in males and females after HAART initiation respectively . PrevaleIn this study the minimum and maximum CD4 cell count before HAART initiation was 1 cell/μl and 795 cells/μl and after HAART initiation were 14 cells/μl and 969 cells/μl. Patients with CD4 cell count <200 cells/μl before HAART initiation was 142 (40.7%) while after HAART initiation was 100 (28.7%). The risk of having CD4 cell count <200 cells/μl was increased before HAART initiation, because before HAART initiation patients have less immunity towards the HIV and the virus attacks CD4 cells easily.There was a decline in the prevalence of anemia and increment of mean CD4+ T cell count among HIV infected patients after HAART initiation. Before HAART initiation, HIV patients have significantly higher prevalence of anemia than those after HAART initiation. The prevalence of anemia was significantly higher in patients with CD4 cell count <200 cell/μl. There was a significant association between prevalence of anemia with CD4 cell count and opportunistic infections before HAART initiation. But there was no association of prevalence of anemia with sex, age, regimen type and WHO clinical stage. Based on the present finding, HIV patients are recommended to check up their CD4 cell counts regularly and start HAART when it is appropriate in order to decrease the prevalence of anemia.


There are limited data from resource-limited settings on antiretroviral resistance mutations that develop in patients failing second-line PI ART.pol gene and interpreted using the 2014 IAS-USA mutation list and the Stanford algorithm. Logistic regression modelling was performed to identify predictors of VF.We performed a cross-sectional virological assessment of adults on second-line ART for ≥6 months between November 2006 and December 2011, followed by a prospective follow-up over 2 years of patients with virological failure (VF) at the Hospital for Tropical Diseases, Vietnam. VF was defined as HIV RNA concentrations ≥1000 copies/mL. Resistance mutations were identified by population sequencing of the Two hundred and thirty-one patients were enrolled in the study. The median age was 32 years; 81.0% were male, 95.7% were on a lopinavir/ritonavir-containing regimen and 22 (9.5%) patients had VF. Of the patients with VF, 14 (64%) carried at least one major protease mutation [median: 2 (IQR: 1–3)]; 13 (59%) had multiple protease mutations conferring intermediate- to high-level resistance to lopinavir/ritonavir. Mutations conferring cross-resistance to etravirine, rilpivirine, tipranavir and darunavir were identified in 55%, 55%, 45% and 27% of patients, respectively. Higher viral load, adherence <95% and previous indinavir use were independent predictors of VF. The 2 year outcomes of the patients maintained on lopinavir/ritonavir included: death, 7 (35%); worsening virological/immunological control, 6 (30%); and virological re-suppression, 5 (25%). Two patients were switched to raltegravir and darunavir/ritonavir with good HIV control.High-prevalence PI resistance was associated with previous indinavir exposure. Darunavir plus an integrase inhibitor and lamivudine might be a promising third-line regimen in Vietnam. There are few data regarding the prevalence of and risk factors for PI resistance developed on second-line ART in Asia. Significant uncertainty exists regarding the risk factors for PI resistance in programmatic settings, the contribution of HIV-1 subtypes to mutation development and the clinical outcomes in patients with PI resistance on long-term second-line ART. HIV-1 subtype CRF01_AE accounts for 99% of HIV infections in Vietnam,21 which is among the Asian countries with the highest numbers of HIV infections.23 Of the 90 000 people on ART, 3% are on second-line therapy.23Because of its costs, viral load monitoring of HIV is not performed routinely. Therefore, data on virological outcome and drug resistance in patients on second-line therapy are lacking. To this end, we aimed to generate data on antiretroviral resistance profiles of HIV-1 CRF01_AE-infected patients with viraemia on second-line PI therapy at the largest HIV treatment centre in Vietnam. Our objectives were: (i) to identify the risk factors for resistance development; (ii) to describe the long-term clinical outcomes of patients with resistance maintained on a failing second-line regimen; and (iii) to investigate cross-resistance to second-generation NNRTIs and PIs to inform national policy on third-line therapy.The WHO endorses ritonavir-boosted PI (PIr)-based ART as an efficacious second-line treatment after failure of NNRTI-based first-line therapy in resource-limited settings.24 In 2011, tenofovir disoproxil fumarate replaced stavudine as a preferred NRTI backbone drug. Patients in the national programme were required to attend monthly appointments for clinical and adherence evaluation. CD4 cell count was performed every 6 months. HIV load testing was performed to confirm treatment failure when the WHO's defined clinical and/or immunological failure criteria were met.26 HIV genotyping was performed to diagnose antiretroviral resistance prior to therapy switch, and results were reported to treating clinicians. Second-line therapy included nelfinavir prior to 2006 and lopinavir/ritonavir thereafter, in combination with tenofovir disoproxil fumarate and/or zidovudine plus lamivudine.28The study was conducted at the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City (HCMC). The HTD is the largest centre for HIV care in southern Vietnam, providing ART for more than 5000 patients according to the national ART programme. Until the de-centralization of care in 2011–12, the HTD had been the primary provider of second-line ART for patients living in the 17 southern provinces of Vietnam. First-line therapy was administered according to national and international guidelines and at the time of the study consisted of two NRTIs (lamivudine in combination with either zidovudine or stavudine) and one NNRTI—either nevirapine or efavirenz. Indinavir was generically and locally produced  during this time and was prescribed (without ritonavir boosting) in public and private settings for patients with treatment failure or intolerance on nevirapine before efavirenz became available in 2004.This study consisted of a cross-sectional survey of adult patients (age ≥15 years) who had been on second-line ART for at least 6 months and were on active care to identify those with VF and their drug resistance development, followed by a prospective follow-up over 2 years of patients with VF. VF was defined as at least two viral loads ≥1000 copies/mL measured 1–3 months apart after intensive adherence counselling. The patients who had been on second-line therapy for <6 months at the time of study assessment, who died or who switched therapy due to drug intolerance were excluded. The study was conducted between December 2011 and June 2014.Clinical data, including demographic information, HIV risk factors, ART history, CD4 counts, HIV viral load, genotyping results at the time of therapy switch (if available), AIDS events and therapy adherence, were obtained both retrospectively and prospectively from patients' charts and from one-on-one interviews.26 Additionally, for this study, adherence was evaluated over the 6 months preceding the time of study assessment using a simple self-reported visual analogue scale (VAS).29 For analysis, sub-optimal adherence was defined as having at least one adherence score of <95% by pill count or by the VAS and/or receiving at least one qualitative adherence assessment of ‘average’ or ‘poor’ over the preceding 6 months prior.Treating clinicians routinely assessed adherence according to the national guidelines at all clinic visits; adherence was recorded either as an estimated percentage of pills taken or as a qualitative assessment of ‘good’, ‘average’ or ‘poor’, corresponding to ≥95%, 80%–94% or <80% adherence, respectively.18 The sequences were analysed using SeqScape (Applied Biosystems). Nucleotide changes were determined by comparison with the consensus sequence pNL4-3 for HIV-1 subtype B (GenBank accession number M19921). Antiretroviral resistance mutations were identified based on the 2014 IAS-USA mutation list.30 The antiretroviral resistance profile of each patient was predicted using the Stanford resistance interpretation algorithm (http://hivdb.stanford.edu). The Rega HIV-1 subtyping tool was used to determine the HIV-1 subtype of each patient sample.31At the time of enrolment, 5 mL of EDTA blood was collected for viral load measurement using the Abbott m2000rt Real Time HIV-1 assay (limit of detection of 150 copies/mL) . Antiretroviral resistance testing was performed for patients with VF using an in-house population sequencing and sequence analysis protocol as previously described, with bidirectional coverage of the complete protease gene and reverse transcriptase codons 10–300.10-transformed) HIV RNA load at therapy switch, history of indinavir use, second-line therapy delay (defined as time in months from first detection of failure of first-line ART to time of second-line therapy initiation) and an overall measure of therapy adherence (<95% versus ≥95%). The chosen covariates were either established risk factors for ART outcome36 or were based on clinicians' observations (i.e. previous indinavir use). Both univariate and multivariable analyses were performed.The following pre-defined covariates were included in the logistic regression model: CD4 cell count and . Nelfinavir was the starting PI in 10 (4.3%) patients, but it was replaced by lopinavir/ritonavir within 12 months for all patients. A total of 36 (17.1%) patients had a history of treatment with indinavir, which was frequently prescribed at 800 mg twice daily or 400 mg three times daily. Sub-optimal adherence was identified in 12.1% patients.Figure Because of cost constraints, HIV genotyping was performed only for 173 of 231 (74.9%) patients prior to therapy switch. Figure The predicted susceptibilities to the national second-line regimens containing tenofovir disoproxil fumarate, lamivudine and lopinavir/ritonavir were evaluated for the 173 patients who had genotype results using the Stanford HIV Drug Resistance algorithm. Intermediate- to high-level resistance to tenofovir disoproxil fumarate was present in 120 of 173 (69.4%), to lamivudine in 165 of 173 (95.4%) and to lopinavir/ritonavir in 2 of 173 (1.2%).The numbers of patients predicted to receive one, two and three fully active drugs were 138 of 173 (79.8%), 25 of 173 (14.5%) and 4 of 173 (2.3%), respectively.10 copies/mL (IQR: 3.92–5.01 log10 copies/mL). Five of 231 (2.2%) patients had HIV RNA concentrations between 400 and <1000 copies/mL, 4 (1.7%) patients had HIV RNA concentrations between 150 and <400 copies/mL and the remaining 200 (86.6%) patients had undetectable viral loads.The virological outcomes of the 231 patients are shown in Figure Of the 22 patients with VF, 21 (95%) were infected with HIV-1 subtype CRF01_AE; a single patient was infected with HIV-1 CRF01_AE/B recombinant. Table Figure P = 0.039; 7.8 (95% CI: 2.1–31.0), P = 0.002; and 12.8 (95% CI: 3.7–49.8), P < 0.001, respectively]. Multivariate analysis shown in Table Table ad hoc univariate and multivariate analyses of factors associated with the development of PI resistance. The three covariates identified to be independent predictors of VF were entered into the logistic regression model. Higher viral load and previous indinavir exposure remained independent predictors of PI resistance in both univariate  and multivariate  analyses. Adherence did not predict PI resistance in the univariate or multivariate analyses.We performed 3 (range: 384–942 cells/mm3). Of these five patients, two had no major PI resistance mutations and three had only one major mutation—V82A—at VF. The remaining six patients had persistent viral replication . Table 3, range: 0–177 cells/mm3); however, there were no AIDS events over the 24 months of follow-up.The clinical outcomes of the 22 patients with VF are shown in Figure 37 Ritonavir-boosted PIs are known to have a high genetic barrier to resistance.38 The minimum plasma concentrations of ritonavir-boosted PIs far exceed the levels required to inhibit WT virus replication,40 making PIs a durable class of antiretroviral drug to be used across different patient populations. A high prevalence of PI resistance has been reported in four studies, two of which studied populations from Asia, specifically, from Cambodia 15 and India .16 The other two study populations were from West Africa: Mali 37 and Nigeria .41 Except for the study from India, where indinavir/ritonavir and atazanavir/ritonavir were commonly used, the studies in these other countries used lopinavir/ritonavir for second-line therapy. Previous exposure to generically produced un-boosted indinavir and nelfinavir was implicated in the observed high prevalence of PI resistance mutations in the reports from Asia and Nigeria, although formal analyses were lacking. Our study is the first to systematically link previous PI exposure to VF and PI resistance.We report the antiretroviral resistance profiles of patients failing second-line PI-based therapy in Vietnam. The major finding was that amongst the patients experiencing VF, 64% harboured at least one major PI resistance mutation and 60% had mutations that conferred intermediate- to high-level resistance to lopinavir/ritonavir. This level of PI resistance is significantly higher than has been previously reported in either resource-rich or resource-poor settings.42 Further, the most common PI resistance mutations detected in our cohort—M46I/L, I54V, V82A and L90M—were shown to be the first mutations to be sequentially selected by indinavir therapy.43 Cheap generically made indinavir, nelfinavir and saquinavir were available in India, China and south-east Asian countries during the same time.44 This availability likely explains the higher prevalence of PI resistance reported in the studies from Cambodia and India and suggests that the scope of PI exposure and resistance in Asia might be larger than is currently appreciated. Another reason for the high level of PI resistance observed in our study as well as these other studies is the lack of viral load monitoring, which leads to late detection of VF and accumulation of PI resistance mutations. Better understanding of the extent and determinants of PI resistance in developing countries is needed.Indinavir was generically produced in Vietnam during the early 2000s. The correct dosing was 800 mg three times daily; however, because of the high rate of side effects, many Vietnamese clinicians prescribed it at 400 mg three times daily or 800 mg twice daily. A combination of high pill burden, short half-life, food restriction, high rate of side effects and inadequate dosing likely led to inadequate plasma drug concentrations and increased the risk of PI resistance in patients. Low plasma indinavir concentration has been shown to increase the risk of developing PI resistance mutations in patients experiencing early VF.41 One reason for the observed high-level etravirine cross-resistance is programmatic. The lack of virological monitoring led to prolonged periods of undetected VF in the presence of the low-genetic-barrier drugs nevirapine and efavirenz and accumulation of resistance mutations. This effect was shown by the extensive NNRTI resistance mutations in our cohort (94% of patients with ≥1 and 32% with ≥3 major NNRTI resistance mutations). NNRTI resistance mutations have been shown to persist up to 45 months after the discontinuation of NNRTI therapy. This finding is due to the low fitness costs of these mutations on viral replication, thus explaining the slow reversion of these mutant viruses to WT in the absence of drug pressure.46 The presence of ≥3 IAS-USA-defined NNRTI resistance mutations has been associated with decreased virological response to etravirine in the DUET trials.48Among the next-generation NNRTIs and PIs potentially available as third-line drugs, there was evidence of probable intermediate or high levels of cross-resistance to etravirine, rilpivirine and tipranavir in ∼50% of patients. Cross-resistance to darunavir was less frequent (27%) and was observed only at the intermediate level. These prevalences are noticeably higher than those found in studies in similar settings.49 However the most frequent NNRTI resistance mutations selected in subtype CRF01_AE virus by nevirapine and efavirenz exposure are Y181C and G190A/S, rather than K103N.51 In the DUET trials, the presence at baseline of these substitutions was associated with impaired virological response to etravirine.48 High prevalence of cross-resistance to etravirine (60%) has been reported in several studies of CRF01_AE-infected patients in Thailand for whom first-line NNRTI-based therapy was failing.54 As efficacy data of etravirine use in south-east Asia are lacking, phenotypic assays investigating the in vitro susceptibilities of these clinical isolates would be helpful. Until then, etravirine and rilpivirine should probably be avoided as third-line drugs for patients infected with subtype CRF01_AE in south-east Asia. Our data do not support the 2010 and 2013 WHO recommendations to use etravirine in a third-line ART regimen in resource-limited settings.25 The phenotypic susceptibility of tipranavir is not as well predicted as that of darunavir by most genotypic interpretation algorithms, in particular for non-B subtypes.55 However, based on our predicted cross-resistance data, darunavir/ritonavir plus a brand-new class of antiretroviral drug, such as integrase strand transfer inhibitors (INSTIs), combined with lamivudine is a reasonable third-line option for Vietnam. As the need for third-line therapy is imminent in the developing world, clinical trials evaluating cost-effective third-line treatment strategies and regimens are needed.Another reason for high etravirine cross-resistance is the inherent genetic variability of the HIV-1 subtype CRF01_AE in south-east Asia. Etravirine was designed to work against HIV containing the NNRTI signature mutation—K103N—which is highly prevalent in HIV-1 subtype B.41 However, virological re-suppression and good immune response were achieved in five patients; these patients either had no or only one major PI resistance mutation at VF detection. A strategy combining adherence intervention and close monitoring of patients failing second-line therapy before switching to third-line therapy would be cost saving yet effective in resource-poor settings.Among the 20 patients who were maintained on the failing lopinavir/ritonavir regimen, death or worse virological/immunological control occurred for 13 patients, with accumulation of resistance mutations occurring in those who remained alive at 24 months. This finding is consistent with that of a study from Nigeria showing accumulation of PI resistance mutations in patients maintained on failing second-line therapy.Our study has limitations. The study captured VF at one point in time and only in patients who were in active follow-up. The unavoidable exclusion of the 12% who had died and the 14% who had been transferred to their respective resident provinces reduces the power of our observations. Further, PI resistance might be underestimated due to the lack of data from those who had died. Nevertheless, the study site is the largest centre for second-line therapy in Vietnam. The highly uniform HIV care system along with standardized ART regimens in the national programme allow for reasonable generalizability of our findings. We did not sequence the integrase gene in this cohort, as INSTIs are not yet available in Vietnam.In conclusion, we identified a significantly higher prevalence of PI resistance in patients failing second-line therapy in Vietnam, which was associated with previous indinavir exposure. The widespread availability of generically made PIs in Asia suggests that the scope of PI resistance might be underestimated in this region. Our data emphasize the need for viral load monitoring to limit the accumulation of NRTI and NNRTI resistance mutations, thus improving second-line treatment outcome and preserving the limited third-line therapy options. Significant cross-resistance to etravirine is common in subtype CRF01_AE-infected patients failing NNRTI therapy, suggesting that etravirine should be avoided as a third-line therapy drug. Research on cost-effective strategies and timing of third-line therapy switch are now needed.Wellcome Trust.This work was supported by the None to declare.Study concept and design: T. L., V. M. Q., C. S., S. D. and J. F. Obtaining funding: T. L., S. D., J. D. and J. F. Acquisition of data: V. P. T., V. M. Q., N. T. C., N. V. V. C. and T. L. Analysis and interpretation of the data: V. P. T. and T. L. Drafting the manuscript: V. P. T. and T. L. Critical revision of the manuscript for important intellectual content: V. M. Q., N. T. C., C. S., J. F., J. D., N. V. V. C., S. D. and G. T. All authors contributed to and approved the final manuscript.


In the Mitra plus study of prevention of mother-to-child transmission of HIV-1, which included 501 women in Dar es Salaam, Tanzania, triple antiretroviral therapy (ART) was given from late pregnancy throughout breastfeeding up to 6 months postnatally. Here we report findings in a sub-cohort of women with ≤200 CD4cells/μL at enrolment who were continued on ART for life and followed up during 24 months after delivery to determine virologic and immunologic responses, drug resistance and mortality.Blood samples for viral load and CD4 counts testing were collected at enrolment and at 3, 6, 12 and 24 months postpartum. HIV drug resistance testing was performed at 12 months. Data analysis included descriptive statistics and multivariate analysis using Generalized Estimated Equations of 73 women with at least two postpartum assessments. The mortality analysis included 84 women who had delivered.The proportion of women with a viral load ≥400 copies/mL was 97% (71/73) at enrolment, 16% (11/67), 22% (15/69), 61% (36/59) and 86% (48/56) at 3, 6, 12 and 24 months postpartum, respectively. The proportion of women with immunologic failure was 12% (8/69), 25% (15/60) and 41% (24/58) at 6, 12 and 24 months, respectively. At 12 months, drug resistance was demonstrated in 34% (20/59), including 12 with dual-class resistance. Self-report on drug adherence was 95% (64/68), 85% (56/66), 74% (39/53) and 65% (30/46) at 3, 6, 12 and 24 months, respectively. The mortality rate was 5.9% (95% CI 2.5-13.7%) at 24 months. The probability of virologic and immunologic failure was significantly higher among women who reported non-perfect adherence to ART at month 24 postpartum.Following an initial decline of viral load, virologic failure was common at 12 and 24 months postpartum among women initiated on ART for life during pregnancy because of low CD4 cell counts. A high proportion of viremic mothers also had resistance mutations. However, at 24 months follow-up, the mortality rate was still fairly low. Continuous adherence counseling and affordable means of monitoring of the virologic response are crucial for successful implementation of the WHO Option B+ guidelines to start all HIV-infected pregnant women on ART for life. By the end of 2011, women made up 58% of the estimated 23.5 million people living with HIV infection in sub-Saharan Africa (SSA), home to 92% of pregnant women living with HIV worldwide [In Tanzania the national HIV prevalence among women 15–49 years of age is 6.2%, but higher in Dar es Salaam, 6.9% -17.The World Health Organization (WHO) estimates that pregnant women with CD4 counts ≤ 350 cells/μL account for about 38% of all HIV-infected pregnant women and for up to 85% of MTCT of HIV . There iThe virologic efficacy of ART in SSA is not routinely monitored by plasma viral load assessment except in research environments . IncreasThis study is nested within the Mitra Plus PMTCT study  and aimeThe Mitra Plus was an open-label, non-randomized, prospective cohort study as described previously . EnrolmeThe Mitra Plus study population consisted of 501 ARV treatment naïve HIV-1 infected pregnant women recruited from four antenatal clinics providing antenatal care services, one each from the three municipals of Dar es Salaam and from the antenatal clinic at the Muhimbili National Hospital in Dar es Salaam. Prior to enrolment, all women signed a written consent form to agree or disagree to participate. For those who could not read and write, fingerprints of the right thumb were used. Out of the 501 women enrolled in the Mitra Plus study, 86 (17%) had a CD4 cell count of ≤200 cells /μL at enrolment. Among these 86 women, two were lost before delivery (one died and the other one withdrew from the study), 8 women who were included in the mortality analysis were excluded from the treatment response analysis due to early loss to follow-up before the month 3 assessment. Three women who died early before they could give a second blood sample post-delivery were also excluded from the treatment response analysis. Hence 84 women were included in the mortality analysis (excluding the two women lost before delivery) and 73 women were included in the longitudinal analysis  150 mg twice daily + nevirapine (NVP) 200 mg lead dose for 14 days which was thereafter increased to 400 mg twice daily. Nelfinavir replaced NVP in women who developed side effects and towards the end of enrolment also for those with a CD4 cell count >200/μL. All women were advised to exclusively breastfeed and to wean abruptly between 5 and 6 months. ART was stopped at 6 months except for the subgroup of women with ≤200 CD4 cells/μL at enrolment who continued ART and this is analyzed in this paper.Quality of life was determined at enrolment by trained study doctors using the Karnofsky’s score instrument . The scaPostnatal follow-up of mother-child pairs was done at weeks 1, 3 and 6, and at months 3, 6, 9, 12, 18, 21 and 24 after delivery. At each visit we performed a clinical examination of the mothers and children and recorded adverse events. Blood samples were drawn for complete blood count, viral load and CD4 cell count assessments at enrolment, month 3, 6, 12, and 24 postpartum. Drug refills and drug adherence counseling was done after every 28 days.Study nurses conducted home visits to all women who had missed two consecutive postnatal visits to find out and record their reasons for non-attendance and to ask them to come back to the clinic for continuum of care if willing. At the end of the study follow-up women were discharged from the study clinic and referred to care and treatment clinics nearest to their place of residence.Women came for follow up visits and ARV drug refills every two weeks followed by weekly visits after gestational week 36. After delivery women were given a 30-day drug refill with an appointment after 28 days, providing a two day-margin for drug refill. A self report on drug adherence was given at every clinic visit and categorized as “drugs taken as prescribed yes/no”. Only women who reported not having missed a single dose during the past month were regarded as adherent (“taken drugs as prescribed”).Screening for HIV antibodies in the pregnant women was done at the recruitment site by trained nurse counselors or health laboratory technologists using the CapillusHIV-1/2 assay  for initial testing followed by testing of reactive samples on the Determine HIV-1/2 assay (Abbott Laboratories). Before recruitment into the study, a second sample was collected for confirmatory HIV antibody testing by two consecutive anti-HIV Enzyme-Linked Immunosorbent Assays (ELISAs), Enzygnost anti-HIV 1/2 Plus ELISA  and Vironostika HIV uniform II plus O ELISA  in the Department of Microbiology and Immunology at MUHAS. Sera reactive on both ELISAs were considered HIV-1 antibody positive. Those that gave discordant results between the two ELISA were resolved by a Western blot assay.Plasma viral load was quantified by the Amplicor HIV-1 Monitor assay version 1.5 . The detection limits of the assay using standard protocol testing were 400–750,000 copies/mL. In this study, virologic failure was defined as a detectable HIV-1 RNA load of ≥400 copies/mL at any time during follow up after three months on ART.Determination of T-lymphocyte subsets was done using the SimulSET flow cytometry method  as described previously . ImmunolDrug resistance testing was carried out on plasma RNA at 12 months after delivery using an in-house method as described previously ,30. ResiData analysis was performed using the STATA software 11 . Mean, median, interquartile range and standard deviation were used for descriptive analysis of numerical variables. Frequencies and percentages were used for categorical variables. Generalized estimating equations were used to estimate the mean viral load and CD4 change across 5 measurement occasions namely: month 0, 3, 6, 12, and 24 and also the association between virologic and immunologic failure with the baseline and clinical characteristics at these time points. The command ‘xtgee’ in STATA was used to run the regression models. The following variables were tested in the regression models; mother’s age (years), mother’s education (years in school), gravidity (number of pregnancies), parity (number of term deliveries), hemoglobin (g/dl), quality of life (Karnofsky’s score 1–100), marital status , disclosure of HIV status to partner (no/yes), partners HIV status , and self-reported adherence at 6, 12 and 24 months postpartum (taken as prescribed yes/no). Selection of the mean model and variance-covariance structure was done based on quasi likelihood under the independence model criterion (QIC). Multivariate analysis for drug resistance and mortality were not performed due to small numbers of mothers with this endpoint. For both the mean viral load and CD4 cell change, virologic and immunologic failure models, p-values less than 0.05 were considered significant.The Mitra Plus study protocol was approved by the Institutional Review Boards of Tanzania; National Institute of Medical Research (NIMR) NIMR/HQ/R.Sa/Vol.IX/273, Muhimbili University of Health and Allied Sciences (MUHAS) reference number MU/01/1022 Vol. VXXII/11 which was also renewed in March 2009 with reference number MU/RP/AEC/Vol.XIII/20, and Karolinska Institutet, Stockholm, Sweden (reference number 03–404).Table Table At month 12 postpartum, 42% (25/59) had only virologic failure, 7% (4/59) had only immunologic failure, 19% (11/59) had both virologic and immunologic failures while 32% (19/59) had neither virologic nor immunologic failure. At month 24, 46% (26/56) had only virologic failure, 2% (1/56) had only immunologic failure, 39% (22/56) had both virologic and immunologic failure and 13% (7/56) had neither virologic nor immunologic failure.Table At 12 months postpartum, drug resistance mutations could be detected in 34% of 59 women who were available for follow-up at this time point, corresponding to 56% (20/36) of all women with detectable viral load. Among those with detectable drug resistance mutations, 60% (12/20) had resistance to both NRTIs and NNRTIs while 40% (8/20) had resistance to NNRTIs only . The mortality rate was 5.9% (95% CI 2.5-13.7%) at 24 months.Table We found an unexpectedly high rate of treatment failure and drug resistance in this cohort of HIV-infected women initiated on ART for life during pregnancy in urban Dar es Salaam, Tanzania. The lowest proportion with virologic treatment failure (defined as ≥400 copies/mL), 16% was found 3 months post-delivery, but thereafter increased to 22% at month 6, 61% at month 12 and 86% at 24 months after delivery. Thus, the great majority of women initiated on ART during pregnancy failed to stay on treatment despite needing it for their own health. A high proportion (56%) of mothers with detectable viral load also had resistance mutations at 12 months postpartum. Despite of this, there was a significantly lower two-year maternal mortality rate in this study, 5.9% (95% CI 2.5-13.7%), compared to the 29.9% (95% CI 13.1-46.9%) mortality rate among mothers with ≤200 CD4 cells/μL at enrolment in the previous Petra PMTCT trial at the Dar es Salaam site who only received short-course ART perinatally . The lonA review of 89 studies on virologic outcomes among adult African patients on ART showed an overall treatment success defined as a viral load of <400 copies/mL of 78% after 6 months of ART, in 76% after 12 months and in 67% after 24 months , but thePrevious studies of HIV-infected women in resource-limited countries have found high attrition between diagnosis of HIV infection in pregnancy-related services and initiation of ART among those eligible for ART for life -35. A meWe defined virologic failure as a viral load ≥400 copies/mL while previous studies of virologic failure in resource-limited settings often have used a cut-off of 1000 copies/mL . On the As expected virologic failure preceded immunologic failure as a warning indicator of treatment failure and was also associated with self-reported adherence. Several other African studies have also found that CD4 cell count monitoring is a poor indicator for treatment failure -43. A stIn our study, self-reported adherence correlated but also seriously underestimated the proportion with virologic failure at month 24 postpartum. Several studies have shown that virologic failure in resource-limited countries is associated with incomplete adherence -46 and iThe majority of the women with drug resistance in our study had multiple resistance mutations, most commonly M184V, K103N and Y181C. The same resistant mutations were also found in infants of women in the Kisumu PMTCT study . In the Despite close monitoring and follow-up as part of our research study including home visits to all clients who had missed more than two consecutive appointments, 21% (15/73) of the women on ART for life and eligible for follow up after delivery did not return for their 24-month visit. Loss to follow-up and sub-optimal postpartum ART adherence has been major drawbacks to the efforts of combating MTCT of HIV in resource-limited regions -36. ThisThus, there is a clear need for rapid and affordable viral load and drug resistance testing and finding feasible ways to improve drug adherence and minimize loss to follow-up in resource-limited settings.The main limitation of this study is the small sample size limiting us from observing significant associations so as to draw more firm conclusions. A major strength of the study is the prospective follow-up for 24 months and the availability of viral load and drug resistance information.In summary, our findings show that a very high proportion of Tanzanian HIV-infected women with low CD4 cell count who started ART during pregnancy, failed virologically after the breastfeeding period with high risk of drug resistance to standard ART regimens. Counseling for adherence together with use of viral load assessments to check for adherence and treatment response is crucial for achieving successful treatment outcomes. This is especially relevant now when the current WHO guidelines to start HIV-infected pregnant women on ART for life irrespective of CD4 cell count in resource-limited settings (Option B+) are being implemented in several resource-poor countries in SSA including in Tanzania.


Since 2009, seven countries in the Organization of Eastern Caribbean States (OECS), Antigua & Barbuda, Dominica, Grenada, Montserrat, St. Kitts & Nevis, Saint Lucia, and St. Vincent & the Grenadines, have been utilizing a laboratory referral service for HIV-1 viral load (VL) offered by The Ladymeade Reference Unit (LRU) Laboratory, Barbados. The objective of this study was to evaluate 5 year VL trends in the six larger OECS countries participating in this regional referral service.Blood samples were collected in source countries and transported to Barbados as frozen plasma according to a standardized protocol. Plasma specimens were amplified by RT PCR on a Roche TaqMan 48 analyser . VL was considered optimally suppressed below a threshold level of < 200 HIV-1 copies/mL of blood. The same threshold was used as a binary indicator in an analysis of the secular change in VL suppression. Montserrat was excluded due to insufficient number of samples.A steady rise in VL referrals from OECS countries was recorded, rising from 312 samples in 2009 to 1,060 samples in 2013. A total of 3,543 samples were tested, with a sample rejection rate (9.2%) mostly due to breaks in the cold chain. Aggregate VL data showed the odds of VL suppression in the Eastern Caribbean improved by 66% for each additional year after 2009 .We demonstrate the feasibility of establishing a regional laboratory referral service for HIV VL monitoring in the Eastern Caribbean. Aggregate VL trends showed a significant year-on-year improvement in VL suppression, implying public health benefits through treatment as prevention in the OECS. VL provides a powerful monitoring & evaluation tool for strengthening HIV programs at country level among the small island states participating in this regional referral network. The Caribbean has the second highest HIV prevalence in the world (0.9%) outside that of sub-Saharan Africa, with 200,000 persons estimated to be living with HIV . HIV/AIDThere is much interest in the HIV field on the public health benefits of antiretroviral therapy, a paradigm known as “Treatment as Prevention”. Cohort studies, critical reviews and randomized control trials have all suggested that sexual transmission of HIV is virtually eliminated in persons receiving ART with demonstrated VL suppression –10. TreaA threshold value for optimal VL suppression has been proposed by the Centers for Disease Control and Prevention (CDC) at ≤200 copies/ml . Using tMany small island developing states lack the necessary infrastructure to conduct the VL test, despite its recognized role in the monitoring of HIV treatment programs ;19. NoneHere we review uptake of the VL referral service and report five year trends of VL suppression in OECS countries between 2009–2013.A total of 3,543 coded patient samples were received by The Ladymeade Reference Unit (LRU) Laboratory from OECS countries over a five year period 2009–2013. Samples were sent as frozen plasma in batches from national HIV treatment centres or from treating physicians in OECS countries. Anonymized viral load data was aggregated by year and country for analysis. The secondary analysis of aggregated data performed in this study from a coded dataset containing no personal identifiers was waived by the chairman of the joint Ministry of Health/University of the West Indies Ethical Review Board (ERB) from further consideration of the ERB. The Ministry of Health does not require explicit consent from patients accessing healthcare facilities for permission to use aggregate laboratory indicators collected as part of routine patient care for the purposes of disease surveillance or programmatic monitoring and evaluation. This stance is supported by the joint Ministry of Health/University of the West Indies Ethical Review Board.Two 4ml samples of EDTA anticoagulated blood were collected from patients in source countries and plasma was separated by centrifugation at 800-1600xg for 20 minutes at room temperature (18–25°C). Centrifuged plasma was transferred into two sterile cryotubes, which were frozen at -80C prior to shipment in batches on ice. Samples were rejected by the receiving laboratory if they had warmed to ambient temperature, if there was any leakage, or if they were incorrectly identified. All aspects of blood collection and shipping were governed by a standard operating procedure (SOP) prepared by the LRU Laboratory: document ‘LRU-M02A-Shipping’.Viral load determinations were performed in Barbados using the Roche AmpliPrep and TaqMan 48 Analysers (Roche Diagnostics), with a lowest detection limit of 20 copies/ml.Many patients were tested repeatedly during the study period and we present the number of patients, the number of viral load measurements, and the average number of measurements per patient, stratified by country and year of measurement. We present log viral load summaries by country and year of measurement . The thresholds for VL suppression (≤ 200 copies/ml) was adopted from the CDC document: “Guidance on Community Viral Load: A Family of Measures, Definitions, and Method for Calculation” . To examTable 1 shows the distribution of patients and samples tested across the laboratory referral network by country and by year. Antigua and St. Vincent collectively comprised 53% of participants and 50% of the submitted samples, with St. Lucia (22% and 20%), Grenada (12% and 12%), Dominica (7% and 12%), and St. Kitts & Nevis (6% and 7%) submitting the remaining samples. There were just 21 samples in total tested from Montserrat (data not shown), too few to include in a country level analysis. Among the six larger OECS countries there were a total of 3,543 samples tested from 1,654 patients between August 2009 and December 2013.Table 1 and across all years, Antigua submitted 925 samples from 446 patients (2.18 samples per patient), St.Vincent submitted 847 from 426 patients (2.00 samples per patient), St. Lucia 707 from 368 (1.93 samples per patient), Grenada 406 from 201 (2.02 samples per patient), Dominica 427 from 120 (3.58 samples per patient), and St. Kitts 231 from 93 (2.49 samples per patient). St. Vincent has evolved from being the smallest referral country in 2009 to being the largest in 2013.From  The data highlight a general male testing excess , a particularly large male excess in Dominica , but country variation in this pattern, with St. Lucia and St. Kitts & Nevis having more female samples .From 3,543 samples, 325 (9.2%) were spoiled (mainly due to breaks in cold chain during shipping) and so were not tested, leaving 3,216 samples tested for viral load. Among tested samples, the sex of the patient was available for 2,957 (91.9%) of the samples, and the distribution of samples by country, year of test and sex is presented in Table 3). Using a threshold value of 200 copies/ml to define optimal virological suppression [Table 3 and Fig 1A). Assuming a linear effect of measurement year on VL suppression, the odds ratio for each one-year change was 1.66  (p<0.001), meaning that the odds of VL suppression in the Eastern Caribbean increased by 66% for each additional year after 2009. The median (logged) VL counts across the OECS network decreased from 2.7 in 2009 to 1.8 in 2013  in 2013 [Despite the quality assurance provided by international accreditation through CAP , there r in 2013  noted th in 2013 ;29. To m in 2013 .There are some limitations to this study. While the reference laboratory is able to report aggregate VL trends for the purpose of programmatic strengthening within a country, comparisons between countries may not be valid. The anonymization process renders the reference laboratory blind to the sample source, which may derive from adults, children, antenatal screening, high risk groups, clients failing on HAART, or a combination of some or all of the above. For example, the male testing excess in Dominica or the female testing excess in St. Lucia may reflect different epidemic patterns across the islands, but they may equally represent differential testing regimes among healthcare providers or differences in patient groups. We also remain cautious interpreting the strength of temporal trends within countries, which may be confounded by relatively low sample numbers.Chlamydia trichomonas and Neisseria gonorrhea (CT/NG) [Nonetheless, the referral service has proven the principle that a Caribbean laboratory network is feasible and can thrive. It provides a template to encourage further expansion of laboratory services within the Caribbean region, particularly those utilizing expensive molecular platforms, and the LRU laboratory is already handling other regional referrals for early infant diagnosis for HIV by DNA PCR, HIV drug resistance testing, and molecular testing for  (CT/NG) . This na (CT/NG) .S1 DatasetThis sensitive HIV dataset has been anonymized to protect the identity of patients and countries participating in the laboratory referral service.(XLSX)Click here for additional data file.


The current goal of antiretroviral treatment (ART) is to achieve and maintain virological suppression below limits of detection (<50 copies/mL). Despite a potent ART, some patients experience persistently low viral loads (VL), between 50-1000 copies/mL. The long-term consequences of persistent low-level viremia (LLV) are negative, usually predicting a virological failure.A cohort, retrospective study was conducted in Adult Clinic I of the National Institute for Infectious Diseases "Prof. Dr. Matei Balş", Bucharest, over a 6 year-period (01.2008-12.2013). The main inclusion criteria were: HIV-positive patients stable on ART (>6 months) at their first regimen, good adherence and absence of other medication susceptible for drug-drug interactions with ART.We recorded the demographical data (age/gender), the HIV transmission route, CDC stage, the baseline immune-virological status  and ART regimen. There were also registered subsequently CD4 count and HIV-VL . We then analyzed the management of the patients found with LLV: maintaining the current ART regimen and close monitoring, ART intensification or ART switch. LLV was defined as VL >50 and <1000 copies/mL in at least 2 determinations over a 24 week-period, after at least 24 weeks of stable ART.10 copies/mL . Two patients had detectable baseline mutations. All ART regimens contained 2 NRTI plus one as following: boosted-lopinavir (11 patients), efavirenz (10 patients), boosted-darunavir (6 patients), boosted-atazanavir (5 patients) and raltegravir (3 patients). Of 11 patients (31.4%) who had detectable VL at 6 months, 5 met the LLV definition criteria. Their median VL was 267 copies/mL and the median CD4 count was 551/cmm. None of them had had baseline mutations. They didn’t have changes in the current ART regimen, except one patient in whom we increased darunavir dose to 1200 mg/day. At 12 months their median CD4 count raised to 743/cmm and the median VL declined to 150 copies/mL.Of 61 patients screened, 35 met the inclusion criteria. The median age was 38 years,  and 71.4% (n=25) were male. According to 1993 CDC classification, 42.8% (n=15) were A2 and 22.8% (n=8) were C3. Sixty percent (n=21) were heterosexually infected. The median baseline CD4 count was 292 cells/cmm , and median VL was 5.1 logAlthough none of the patients with LLV became undetectable at 12 months of treatment, their VL levels decreased progressively in line with the increase in CD4 count in the absence of ART changing.