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SAMBA HIV semi-quantitative test, a new point-of-care viral load monitoring assay for resource-limited settingsRoutine viral load (VL) testing of HIV-infected individuals on antiretroviral therapy (ART) is used to monitor treatment efficacy. However, due to logistical challenges, implementation of VL has been difficult in resource-limited settings. The aim of this study was to evaluate the performance of the SAMBA Semi-Q Test in London, Malawi, and Uganda. The SAMBA HIV-1 Semi-Q Test can distinguish between patients with VL above or below 1000 copies/ml. The SAMBA Semi-Q was validated with diluted clinical samples and blinded plasma samples collected from HIV-1-positive individuals. SAMBA Semi-Q results were compared with results from the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0. Testing of 96 2-10 fold dilutions of four samples containing HIV-1 subtype C as well as 488 samples from patients in the United Kingdom, Malawi, and Uganda, respectively, yielded an overall accuracy for SAMBA Semi-Q of 99% (95% CI 93.8 - 99.9%) and 96.9% (95% CI 94.9 - 98.3%) respectively compared to Roche. Analysis of VL data from patients in Malawi and Uganda showed that the SAMBA cut-off of 1000 copies/ml appropriately distinguished treated from untreated individuals. Furthermore, analysis of the viral load of 232 patients on ART in Malawi and Uganda revealed similar patterns for virological control defined as either <1000 copies/ml (SAMBA cut-off) or <5000 copies/ml (WHO 2010 criterion). This study suggests that SAMBA Semi-Q has adequate concurrency with the gold standard measurements for viral load measurement. This test can allow VL monitoring of patients on ART at the point of care in resource-limited settings.