Expanding Xpert MTB/RIF Ultra® and LF-LAM testing for diagnosis of tuberculosis among HIV-positive adults admitted to hospitals in Tanzania and Mozambique: a randomized controlled trial (the EXULTANT trial).

INTRODUCTION: Tuberculosis (TB) is an important cause of morbidity and mortality among people living with HIV (PLHIV). Current WHO-recommended strategies for diagnosing TB among hospitalized PLHIV rely on symptom screening and disease severity to assess eligibility for urine lipoarabinomannan lateral flow (LF-LAM) and molecular testing. Despite these recommendations, autopsy studies show a large burden of undiagnosed TB among admitted PLHIV. The EXULTANT trial aims to assess the impact of an expanded screening strategy using three specimens (sputum, stool, and urine) for TB diagnosis among PLHIV admitted to hospitals in two high HIV and TB burden African countries. METHODS: This is a multicenter, pragmatic, individually randomized controlled trial conducted across eleven hospitals in Tanzania and Mozambique. Participants in the intervention arm will be tested with Xpert MTB/RIF Ultra® from expectorated sputum, stool, and urine samples, with additional urine LF-LAM testing in the first 24 h after hospital admission, irrespective of the presence of the symptoms. The control arm will implement the WHO standard of care recommendations. Hospitalized adults (≥ 18 years) with a confirmed HIV-diagnosis, irrespective of antiretroviral (ART) therapy status or presence of TB symptoms will be assessed for eligibility at admission. Patients with a pre-existing TB diagnosis, those receiving anti-tuberculosis therapy or tuberculosis preventive treatment in the 6 months prior to enrolment, and those transferred from other hospitals will not be eligible. Also, participants admitted for traumatic reasons such as acute abdomen, maternal conditions, scheduled surgery, having a positive SARS-CoV2 test will be ineligible. The primary endpoint is the proportion of participants with microbiologically confirmed TB starting treatment within 3 days of enrolment. DISCUSSION: The EXULTANT trial investigates rapid implementation after admission of a new diagnostic algorithm using Xpert MTB/RIF Ultra® in several non-invasive specimens, in addition to LF-LAM, in hospitalized PLHIV regardless of TB symptoms. This enhanced strategy is anticipated to detect frequently missed TB cases in this population and is being evaluated as an implementable and scalable intervention. TRIAL REGISTRATION: Trial reference number: NCT04568967 (ClinicalTrials.gov) registered on 2020-09-29.

Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique.

Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted.

Nevirapine or efavirenz for tuberculosis and HIV coinfected patients: exposure and virological failure relationship.

OBJECTIVES: We describe nevirapine and efavirenz exposure on and off tuberculosis treatment and consequences for virological efficacy and tolerance in patients included in the ANRS 12146/12214-CARINEMO trial. METHODS: Participants were randomly selected to receive either nevirapine at 200 mg twice daily (n = 256) or efavirenz at 600 mg daily (n = 270), both combined with two nucleoside analogues. Blood samples were drawn 12 h after nevirapine or efavirenz administration, while on tuberculosis treatment and after tuberculosis treatment discontinuation. In 62 participants, samples taken 12 h after drug administration were drawn weekly for the first month of ART. Sixteen participants participated in an extensive pharmacokinetic study of nevirapine. Concentrations were compared with the therapeutic ranges of 3000-8000 ng/mL for nevirapine and 1000-4000 ng/mL for efavirenz. RESULTS: Nevirapine concentrations at the end of the first week of treatment (on antituberculosis drugs) did not differ from concentrations off tuberculosis treatment, but declined thereafter. Concentrations at steady-state were 4111 ng/mL at week 12 versus 6095 ng/mL at week 48 (P < 0.0001). Nevirapine concentrations <3000 ng/mL were found to be a risk factor for virological failure. Efavirenz concentrations were higher on than off tuberculosis treatment (2700 versus 2450 ng/mL, P < 0.0001). CONCLUSIONS: The omission of the 2 week lead-in dose of nevirapine prevented low concentrations at treatment initiation but did not prevent the risk of virological failure. Results support the WHO recommendation to use efavirenz at 600 mg daily in patients on rifampicin-based antituberculosis therapy.

Performance of two plasma separation devices for HIV-1 viral load measurement in primary healthcare settings.

IMPORTANCE: Burnett and HemaSpot are two novel technologies that allow whole blood collection and plasma separation and stabilization at room temperature without the need of additional equipment. Hence, these devices are potential alternatives to fresh plasma as a suitable specimen for viral load scale-up to monitor antiretroviral therapy in resource-limited settings.

Stability of HIV-1 Nucleic Acids in cobas Plasma Separation Card for Viral Load Measurement.

OBJECTIVES: Our study aimed to evaluate the stability of human immunodeficiency virus 1 (HIV-1) RNA on cobas plasma separation card (PSC) specimens for viral load (VL) testing after being exposed to varied temperatures and storage times. METHODS: For this purpose, venous PSC specimens were collected and stored at 25ºC to 42ºC for a period of up to 28 days. Plasma VL at baseline was used as reference, against which PSC VL was compared at different time points. RESULTS: From the 30 patients included in the study, 600 PSC and 30 fresh plasma specimens were obtained. Plasma VL at baseline was fewer than 1,000 copies/mL in 16 patients, and 99.4% of PSCs from these patients yielded nonquantifiable VL at all temperature ranges and time points. During the study period, minor variation of VL was observed in PSCs obtained from 13 patients with plasma VL fewer than 1,000 copies/mL at baseline. For the patient with plasma VL at 1,000 copies/mL, the PSC VL varied from undetectable to 1,670 copies/mL. CONCLUSIONS: Our results show minor variation of VL in PSC specimens in the study conditions. HIV RNA is stable in PSCs exposed to high temperatures for up to 28 days.

Nevirapine hair and plasma concentrations and HIV-1 viral suppression among HIV infected ante-partum and post-partum women attended in a mother and child prevention program in Maputo city, Mozambique.

INTRODUCTION: Prevention of mother to child transmission of HIV (PMTCT) is frequently challenged by irregular access to more effective anti-retroviral therapy. Nevirapine single dose (sdNVP), sdNVP+AZT+3TC for MTCT prophylaxis and NVP+ AZT+3TC for treatment and PMTCT were withdrawn due to low genetic resistance barrier and low efficacy. However current PMTCT lines in Mozambique include DTG+3TC+TDF, TDF+3TC+EFV, DTG +ABC+3TC, and AZT + NVP syrup prophylaxis for exposed babies. We assessed NVP hair and plasma concentrations and association with HIV-1RNA suppression among HIV+ ante-partum and post-partum women under PMTCT in Maputo, Mozambique. METHODS: From December 2013 to November 2014, prospectively were enrolled 200 HIV+ ante-partum women on 200mg nevirapine and zidovudine 300 plus lamivudine 150mg twice daily at least with 3 months treatment and seen again at 24 weeks post-partum. Self-reported pill-taking adherence, NVP concentrations in hair, plasma, hemoglobin, CD4 cell count, HIV-1 RNA load was evaluated. NVP concentration in hair and plasma was analyzed as categorical quartile variable based on better data fit. NVP concentration was set between ≤3.77 ng/ml in plasma and ≤17,20 ng/mg in hair in quartile one to ≥5.36 ng/ml in plasma and ≥53.21 ng/mg in hair in quartile four. Logistic regression models for repeated measures were calculated. Following the World Health Organization (WHO) guidelines we set viral suppression at HIV-1RNA < 1000 c/mL. Outcome was HIV-1 RNA<1000 copies/ml. Predictor was NVP concentration in hair categorized in quartiles. RESULTS: In total 369 person-visits (median of 1.85) were recorded. Self-reported adherence was 98% (IQR 97-100%) at ante-partum. In 25% person visits, NVP concentrations were within therapeutic levels (3.77 ng/ml to 5.35 ng/ml) in plasma and (17.20 ng/mg to 53.20 ng/mg) in hair. In 50% person visits NVP concentrations were above 5.36 ng/ml in plasm and 53.21 ng/mg in hair. HIV-1 RNA suppression was found in 34.7% of women with two viral loads, one at enrollment and another in post-partum. Odds of HIV-1 RNA suppression in quartile 4, was about 6 times higher than in quartile 1 (p-value = 0.006) for NVP hair concentration and 7 times for NVP plasma concentration (p-value = 0.012). CONCLUSIONS: The study results alert for potential low efficacy of current PMTCT drug regimens in use in Mozambique. Affordable means for individual monitoring adherence, ART plasma and hair levels, drug resistant and HIV-1 RNA levels monitoring are recommended for prompt identification of inadequate drug regimens exposure patterns and adjust accordingly.

Frequency of human immunodeficiency virus type-2 in hiv infected patients in Maputo City, Mozambique.

The HIV/AIDS pandemic is primarily caused by HIV-1. Another virus type, HIV-2, is found mainly in West African countries. We hypothesized that population migration and mobility in Africa may have facilitated the introduction and spreading of HIV-2 in Mozambique. The presence of HIV-2 has important implications for diagnosis and choice of treatment of HIV infection. Hence, the aim of this study was to estimate the prevalence of HIV-2 infection and its genotype in Maputo, Mozambique.HIV-infected individuals (N = 1,200) were consecutively enrolled and screened for IgG antibodies against HIV-1 gp41 and HIV-2 gp36 using peptide-based enzyme immunoassays (pepEIA). Specimens showing reactivity on the HIV-2 pepEIA were further tested using the INNO-LIA immunoblot assay and HIV-2 PCR targeting RT and PR genes. Subtype analysis of HIV-2 was based on the protease gene.After screening with HIV-2 pepEIA 1,168 were non-reactive and 32 were reactive to HIV-2 gp36 peptide. Of this total, 30 specimens were simultaneously reactive to gp41 and gp36 pepEIA while two samples reacted solely to gp36 peptide. Only three specimens containing antibodies against gp36 and gp105 on the INNO-LIA immunoblot assay were found to be positive by PCR to HIV-2 subtype A.The proportion of HIV-2 in Maputo City was 0.25% (90%CI 0.01-0.49). The HIV epidemic in Southern Mozambique is driven by HIV-1, with HIV-2 also circulating at a marginal rate. Surveillance program need to improve HIV-2 diagnosis and consider periodical survey aiming to monitor HIV-2 prevalence in the country.

Inpatient Point-of-Care HIV Early Infant Diagnosis in Mozambique to Improve Case Identification and Linkage to Antiretroviral Therapy.

INTRODUCTION: Novel approaches to case identification and linkage to antiretroviral therapy (ART) are needed to close gaps in early infant diagnosis (EID) of HIV. Point-of-care (POC) EID is a recent innovation that eliminates the long turnaround times of conventional EID that limit patient management in the inpatient setting. The initial deployment of POC EID in Mozambique focused primarily on outpatient clinics; however, 2 high-volume tier-4 pediatric referral hospitals were also included. METHODS: To assess the impact of inpatient POC EID, a retrospective review of testing and care data from Hospital Central de Beira (HCB) and Hospital Central de Maputo (HCM) was performed for the period September 2017 to July 2018, with comparison to the 8-month pre-POC period when dried blood spots were used for conventional EID. RESULTS: Monthly testing volume increased from 8.5 tests pre-POC to 17.6 tests with POC (P<.001). Among 511 children with POC testing, the median age was 5 months, there was ongoing breastfeeding in 326 (63.8%), and 136 (26.6%) of mothers and 146 (28.6%) of infants had not received ART or antiretroviral prophylaxis, respectively. POC tests were positive in 152 (29.7%) infants, and 52 (37.5%) had a previous negative DNA polymerase chain reaction through the conventional outpatient EID program. Linkage to ART for infants with HIV-positive tests improved 64% during the POC period (P=.002). Inpatient mortality for infected infants during the POC period was 28.2%. Excluding these deaths, 61.2% of eligible infants initiated ART as inpatients, but only 29.8% of those discharged without ART were confirmed to have initiated as outpatients. CONCLUSIONS: Inpatient wards are a high-yield site for EID and ART initiation that have historically been overlooked in programming for prevention of mother-to-child transmission. POC platforms represent a transformative opportunity to increase inpatient testing, make definitive diagnoses, and improve timely linkage to ART. Scale-up plans should prioritize pediatric wards.

Performance of a True Point-of-Care Assay for HIV-1/2 Viral Load Measurement at Antenatal and Postpartum Services.

BACKGROUND: Timely viral load (VL) results during pregnancy and the postpartum period are crucial for HIV disease management and for preventing mother-to-child transmission. Point-of-care (POC) VL testing could reduce turnaround times and streamline patient management. We evaluated the diagnostic performance of the novel m-PIMA HIV-1/2 VL assay (Abbott, Chicago, IL) in Mozambique. SETTING: The study was conducted in prenatal and postpartum consultation rooms in 2 primary health care clinics. Sample collection and testing on m-PIMA were performed by trained nurses. METHODS: HIV-infected pregnant and postpartum women on antiretroviral treatment (ART) or ART naive were tested using both on-site m-PIMA POC and referral laboratory-based real-time VL assays. Linear regression analysis and Bland-Altman plots were used to calculate the agreement between both. FINDINGS: Correlation between venous blood plasma POC and plasma laboratory-based VL was strong (r2 = 0.850, P < 0.01), with good agreement between the methods [overall bias 0.202 log copies/mL (95% CI: 0.366 to 0.772 log copies/mL)]. Using the threshold of 1000 copies/mL, which is used to determine ART failure, the sensitivity and specificity of the POC VL assay were 95.0% (95% CI: 91.6% to 97.3%) and 96.5% (95% CI: 94.2% to 98.0%), respectively. The correlation coefficient between the venous and capillary sample types was 0.983 (r2 = 0.966). CONCLUSIONS: On-site, nurse-performed POC VL testing is feasible and accurate in resource-limited primary health care settings. The operational challenge of plasma separation within clinics for POC testing was successfully overcome using minicentrifuges. The use of capillary blood could simplify the execution of the assay in a clinical environment.

Viral load assay performs comparably to early infant diagnosis assay to diagnose infants with HIV in Mozambique: a prospective observational study.

INTRODUCTION: Viral load testing is essential to manage HIV disease, especially in infants and children. Early infant diagnosis is performed using nucleic-acid testing in children under 18 months. Resource-limited health systems face severe challenges to scale-up both viral load and early infant diagnosis to unprecedented levels. Streamlining laboratory systems would be beneficial to improve access to quality testing and to increase efficiency of antiretroviral treatment programmes. We evaluated the performance of viral load testing to serve as an early infant diagnosis assay in children younger than 18 months. METHODS: This study was an observational, prospective study, including children between one and 18 months of age who were born to HIV-positive mothers in 134 health facilities in Maputo City and Maputo Province, Mozambique. Dried blood spot specimens from heel or toe pricks were collected between January and April 2018, processed using SPEX buffer for both assays, and tested for routine EID and VL testing using the Roche CAP/CTM HIV-1 Qualitative v2 and Roche CAP/CTM HIV-1 Quantitative v2 assays respectively. The sensitivity, specificity and positive and negative predictive values were estimated using the EID results as the reference standard. RESULTS: A total of 1021 infants were included in the study, of which 47% were female. Over 95% of mothers and children were on antiretroviral treatment or received antiretroviral prophylaxis respectively. The sensitivity and specificity of using the viral load assay to detect infection were 100% (95% CI: 96.2 to 100%) and 99.9% (95% CI: 99.4 to 100%). The positive and negative predictive values were 99.0% (95% CI: 94.3 to 100%) and 100% (95% CI: 99.6 to 100%). The McNemar's test was 1.000 and Cohen's kappa was 0.994. CONCLUSIONS: The comparable performance suggests that viral load assays can be used as an infant diagnostic assay. Infants with either low levels of viraemia or high cycle threshold values should be repeat tested to ensure the result is truly positive prior to treatment initiation, regardless of assay used. Viral load assays could replace traditional early infant diagnosis testing, substantially streamlining molecular laboratory services for children and lowering costs, with the additional advantage of providing baseline viral load results for antiretroviral treatment management.

Accurate HIV viral load measurement in primary health care settings using the cobas® plasma separation card.

INTRODUCTION: Plasma is considered the gold standard for HIV viral load (VL) testing, however its use is challenging due to the need for phlebotomy and centrifugation services, as well as cold chain for transporting to laboratories for testing. The use of Dried Blood Spot (DBS) specimen has allowed a rapid expansion of antiretroviral therapy (ART) monitoring in remote areas in many African countries, however, the VL in DBS may overestimate the copies of viral RNA result at the clinically relevant range of 1000 copies/ml, due to proviral DNA and intracellular RNA. The characteristics of the cobas® Plasma Separation Card (PSC) specimen are similar to fresh plasma (gold standard), so a better performance of HIV VL is expetected in PSC specimen and can be an alternative to DBS. This study aims to evaluate the performance of cobas® PSC for VL testing at primary health care facilities in Mozambique. METHODOLOGY: HIV-1 infected adults on ART were enrolled consecutively in two health facilities in Mozambique, between August 2018 and October 2018. Capillary and venous cobas® PSC, DBS and fresh plasma specimens were collected from each patient. All specimens were tested for VL using CAP/CTM v2.0. Sensitivity and specificity of viral load using DBS, capillary and venous PSC specimens were estimated. Viral load obtained in fresh plasma specimen was used as reference and a threshold of 1000 copies/ml was considered for the analyses. RESULTS: From the total 613 patients included for the study, 2444 specimens including DBS (613), plasma (613), venous cobas® PSC (609) and capillary cobas® PSC (609) were collected and 2407 results were obtained. Sensitivity and specificity of the VL using venous cobas®PSC specimen at 1000 copies/ml threshold were 99.8% and 98.1% respectively, whereas for capillary cobas® PSC sensitivity was 99.6% and specificity was 97.2%. For DBS VL, sensitivity was 96.9% and specificity was 81.8%. Misclassification rate was more prominent in DBS specimens (5.9%), but lower in venous and capillary cobas®PSC with a rate of 0.3% and 0.7% respectively. CONCLUSION: The cobas® PSC specimen has improved performance over DBS for more accurate VL testing aligned with plasma testing. The use of this specimen type can increase the rates of reliable VL results and this will improve the quality of VL monitoring of HIV-positive patients in low-income settings.

Proceedings from the CIHLMU occupational safety and health symposium 2019 "Protecting workers' health: global challenges and opportunities in work-related respiratory diseases".

The international CIHLMU Occupational Safety and Health Symposium 2019 was held on 16th March, 2019 at the Ludwig-Maximilians-Universität Munich, Germany. About 60 participants from around the world representing occupational health and safety professionals, students, instructors from several institutions in Germany and abroad, attended the symposium.The main objective of the symposium was to create awareness on global challenges and opportunities in work-related respiratory diseases. One keynote lecture and six presentations were made. While the keynote lecture addressed issues on occupational diseases in the twenty-first century, the six presentations were centered on: Prevention and control of work-related respiratory diseases, considerations; Occupational health and safety in Mining: Respiratory diseases; The prevention of TB among health workers is our collective responsibility; Compensation and prevention of occupational diseases and discussion on how artificial intelligence can support them: Overview of international approaches; Work-related Asthma: Evidence from high-income countries; and The role of imaging in the diagnosis of work- related respiratory diseases. A panel discussion was conducted following the presentations on the importance and challenges of data acquisition which is needed to have a realistic picture of the occupational safety and health status of workers at different levels. The current summary is an attempt to share the proceedings of the symposium.

Effect of point-of-care early infant diagnosis on antiretroviral therapy initiation and retention of patients.

OBJECTIVE: We measured the effect of point-of-care (POC) early infant HIV testing on antiretroviral therapy initiation rates and retention in care among infants in Mozambique. DESIGN: A cluster-randomized trial was conducted in 16 primary healthcare centres providing either on-site POC arm (n = 8) or referred laboratory [standard-of-care (SOC) arm; n = 8] infant HIV testing. METHODS: The primary outcomes were the proportion of HIV-positive infants initiating antiretroviral therapy within 60 days of sample collection, and the proportion of HIV-positive infants who initiated antiretroviral therapy that were retained in care at 90 days of follow-up. RESULTS: The proportion of HIV-positive infants initiating antiretroviral therapy within 60 days of sample collection was 89.7% (157 of 175) for the POC arm and 12.8% (13 of 102) for the SOC arm [relative risk (RR)(adj) 7.34; P < 0.001]. The proportion of HIV-positive infants who initiated antiretroviral therapy that were retained in care at 90 days of follow-up was 61.6% (101 of 164) for the POC arm and 42.9% (21 of 49) for the SOC arm [RR(adj) 1.40; P < 0.027]. The median time from sample collection to antiretroviral therapy initiation was less than 1 day (interquartile range: 0-1) for the POC arm and 127 days (44-154; P < 0.001) for the SOC arm. CONCLUSION: POC infant HIV testing enabled clinics to more rapidly diagnose and provide treatment to HIV-infected infants. This reduced opportunities for pretreatment loss to follow-up and enabled a larger proportion of infants to receive test results and initiate antiretroviral therapy. The benefits of faster HIV diagnosis and antiretroviral treatment may also improve early retention in care.

Performance of point-of-care birth HIV testing in primary health care clinics: An observational cohort study.

BACKGROUND: Failure to timely diagnose HIV in infants is a major barrier for scaling-up paediatric antiretroviral treatment (ART). WHO recommends birth testing for earlier diagnosis and to improve test coverage, but current diagnosis takes 2-3 weeks to complete, thereby limiting the ability of care givers to provide follow-on care, especially in low-resource settings. We evaluated the benefit of implementing rapid diagnosis of HIV at birth in primary health care maternity wards in Mozambique. METHODS AND FINDINGS: Infants born to HIV-infected mothers delivering consecutively at eight primary health care clinics were tested within 24 hours of delivery using on-site POC (Alere q HIV1/2 Detect) and standard laboratory (Roche COBAS AmpliPrep/TaqMan HIV-1 qualitative assay v2.0) testing. Infants were also tested at 4-6 weeks of age with both assays. Of 2,350 HIV-exposed infants enrolled in this implementation research study, 33 tested HIV-positive at birth on both assays. Sensitivity and specificity of POC testing compared with laboratory testing at birth were 100% (95% CI 89·4-100·0) and 100% (95% CI 99·8-100·0), respectively. At 4-6 weeks of age, 61 infants were identified as HIV-positive; of these 29 (47·5%) had a positive test at birth. Testing at both birth and 4-6 weeks identified 71 HIV-positive infants compared with 61 infants by testing at 4-6 weeks alone, a 16% increase. Two infants tested positive at birth but tested HIV-negative during follow-up. CONCLUSIONS: Adding POC birth testing to the 4-6 week screen may increase access to HIV diagnosis and expedite ART initiation in primary health care settings within low resource settings. Guidance on appropriate confirmatory HIV testing algorithms for birth testing is needed.

Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 µg (n = 10; 2 × 0.1 ml) or 1,200 µg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 108 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 µg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

Point-Of-Care p24 Infant Testing for HIV May Increase Patient Identification despite Low Sensitivity.

The long delay in returning test results during early infant diagnosis of HIV (EID) often causes loss-to-follow-up prior to antiretroviral treatment (ART) initiation in resource-limited settings. A point-of-care (POC) test may help overcome these challenges. We evaluated the performance of the LYNX p24 Antigen POC test in Mozambique. 879 HIV-exposed infants under 18 months of age were enrolled consecutively at three primary healthcare clinics (PHC). Lancet heel-drawn blood was tested on-site by nurses using a prototype POC test for HIV Gag p24 antigen detection. Results of POC testing were compared to laboratory-based nucleic acid testing on dried blood spots. A comparison of the effect of sensitivity and timely test results return on successful diagnosis by POC and laboratory-based platforms was also calculated. The sensitivity and specificity of the LYNX p24 Ag test were 71.9%; (95% confidence interval [CI]: 58.5-83.0%) and 99.6% (95% CI: 98.9-99.9%), respectively. The predictive value of positive and negative tests were 93.2% (95% CI: 81.3-98.6%) and 97.9% (95% CI: 96.8-98.8%), respectively. Overall agreement was high (Cohen Kappa = 0.80; 95% CI: 0.71-0.89). Despite its lower sensitivity, the POC test had the potential to provide test results to up to 81% more patients compared to the laboratory-based test. This prototype POC p24 assay was feasible for use in PHCs but demonstrated low sensitivity for HIV detection. POC EID technologies that perform below standard recommendations may still be valuable diagnostic tools in settings with inefficient EID networks.

Stability of HIV-1 Nucleic Acids in cobas Plasma Separation Card for Viral Load Measurement

Objectives: Our study aimed to evaluate the stability of human immunodeficiency virus 1 (HIV-1) RNA on cobas plasma separation card (PSC) specimens for viral load (VL) testing after being exposed to varied temperatures and storage times. Methods: For this purpose, venous PSC specimens were collected and stored at 25ºC to 42ºC for a period of up to 28 days. Plasma VL at baseline was used as reference, against which PSC VL was compared at different time points. Results: From the 30 patients included in the study, 600 PSC and 30 fresh plasma specimens were obtained. Plasma VL at baseline was fewer than 1,000 copies/mL in 16 patients, and 99.4% of PSCs from these patients yielded nonquantifiable VL at all temperature ranges and time points. During the study period, minor variation of VL was observed in PSCs obtained from 13 patients with plasma VL fewer than 1,000 copies/mL at baseline. For the patient with plasma VL at 1,000 copies/mL, the PSC VL varied from undetectable to 1,670 copies/mL. Conclusions: Our results show minor variation of VL in PSC specimens in the study conditions. HIV RNA is stable in PSCs exposed to high temperatures for up to 28 days.

Performance Evaluation of the MyT4 Technology for Determining ART Eligibility.

BACKGROUND: In resource-limited countries, CD4 T-cell (CD4) testing continues to be used for determining antiretroviral therapy (ART) initiation eligibility and opportunistic infection monitoring. To support expanded access to CD4 testing, simple and robust technologies are necessary. We conducted this study to evaluate the performance of a new Point-of-Care (POC) CD4 technology, the MyT4, compared to conventional laboratory CD4 testing. METHODS: EDTA venous blood from 200 HIV-positive patients was tested in the laboratory using the MyT4 and BD FACSCalibur™. RESULTS: The MyT4 had an r2 of 0.82 and a mean bias of 12.3 cells/µl. The MyT4 had total misclassifications of 14.7% and 8.8% when analyzed using ART eligibility thresholds of 350 and 500 cells/µl, respectively. CONCLUSIONS: We conclude that the MyT4 performed well in classifying patients using the current ART initiation eligibility thresholds in Mozambique when compared to the conventional CD4 technology.

Viral load assay performs comparably to early infant diagnosis assay to diagnose infants with HIV in Mozambique: a prospective observational study

Introduction: Viral load testing is essential to manage HIV disease, especially in infants and children. Early infant diagnosis (EID) is performed using nucleic-acid testing in children under 18 months. Resource-limited health systems face severe challenges to scale-up both viral load and EID to unprecedented levels. Streamlining laboratory systems would be beneficial to improve access to quality testing and to increase efficiency of antiretroviral treatment programmes. We evaluated the performance of viral load testing to serve as an EID assay in children younger than 18 months. Methods: This study was an observational, prospective study, including children between one and 18 months of age who were born to HIV-positive mothers in 134 health facilities in Maputo City and Maputo Province, Mozambique. Dried blood spot specimens from heel or toe pricks were collected between January and April 2018, processed using SPEX buffer for both assays, and tested for routine EID and viral load testing using the Roche CAP/CTM HIV-1 Qualitative v2 and Roche CAP/ CTM HIV-1 Quantitative v2 assays respectively. The sensitivity, specificity and positive and negative predictive values were estimated using the EID results as the reference standard. Results: A total of 1021 infants were included in the study, of which 47% were female. Over 95% of mothers and children were on antiretroviral treatment or received antiretroviral prophylaxis respectively. The sensitivity and specificity of using the viral load assay to detect infection were 100% (95% CI: 96.2 to 100%) and 99.9% (95% CI: 99.4 to 100%). The positive and negative predictive values were 99.0% (95% CI: 94.3 to 100%) and 100% (95% CI: 99.6 to 100%). The McNemar's test was 1.000 and Cohen's kappa was 0.994. Conclusions: The comparable performance suggests that viral load assays can be used as an infant diagnostic assay. Infants with either low levels of viraemia or high cycle threshold values should be repeat tested to ensure the result is truly positive prior to treatment initiation, regardless of assay used. Viral load assays could replace traditional EID testing, substantially streamlining molecular laboratory services for children and lowering costs, with the additional advantage of providing baseline viral load results for antiretroviral treatment management.

Quality assurance for point-of-care testing in Mozambique's National Health Service

Medical care in Mozambique is mostly provided through the national health service of the Ministry of Health. All primary healthcare and HIV-related services are provided free of charge. There are over 1500 public sector health facilities in Mozambique and most of these are primary healthcare centres. Although all hospitals have a laboratory, only a quarter of the health centres have a formal laboratory. In this context, point-of-care (POC) testing and syndromic management of diseases play an important role in the health system. Both communicable and non-communicable diseases are prevalent in the Mozambican population. Mozambique has a population of 28 million and is among the nine countries with the highest HIV prevalence in the world.1 HIV prevalence in the country among people aged 15­49 years is 11.5%, ranging from 3.7% in the Niassa province in the north to 25.1% in the Gaza province in the south.2,3 HIV prevalence is higher among women (13.1%) than among men (9.2%), and higher in urban areas (15.9%) compared with rural areas (9.2%).2,3 Among children aged between 0 and 11 years, HIV prevalence is 1.4%, and 2.3% in those younger than one year.2,3 It is estimated that 102 new infections in children occur daily in Mozambique (Ministry of Health, unpublished data). Demographic impact studies show that an estimated 1.6 million Mozambicans were living with HIV in 2009.