A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART).Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4 ؉ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n ‫؍‬ 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P ‫؍‬ 0.0003 and P ‫؍‬ 0.0004, respectively), while the frequency of CD4 ؉ T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. IMPORTANCESince the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

In cattle and other ruminants, infection with the intracellular pathogen

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at ؊20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at ؊20°C or at ؊70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that ؊20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.The introduction of highly active antiretroviral therapy and the demonstration of dramatic improvements in human immunodeficiency virus (HIV)-and AIDS-related mortality and morbidity in North America and Europe have fueled international efforts to expand access to care and treatment in lessdeveloped countries. Several major initiatives to provide treatment in resource-limited settings, including the U.S. President's Emergency Plan for AIDS Relief and the Global Fund against AIDS, TB and Malaria, are now in progress (16). The implementation of these programs requires the development of appropriate and effective patient-monitoring systems, including surveillance for antiretroviral drug resistance. Sentinel drug resistance surveillance systems are important public health tools that can provide information on trends in the prevalence of resistance at the population level and can be used to modify treatment guidelines.Plasma and serum are considered the preferred specimen types for HIV type 1 (HIV-1) drug resistance testing. However, these types of specimens are not optimal in resource-limited settings where the equipment necessary for PCR amplification and sequencing may not be available at collection sites and resistance testing requires transportation of the samples to a refer...

: We demonstrated a high concordance between resistance genotypes from plasma and DBS, and that resistance testing from DBS can achieve sensitive levels similar to those seen using plasma. Our results indicate that DBS may represent a feasible alternative to plasma for drug resistance testing in treated individuals.