Our findings show heterogeneous virological failure and illustrate that, in addition to routine access to viral load, good management of ART programs is even more critical to improve treatment outcomes in resource-limited countries.

The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays-a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)-were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n ‫؍‬ 47; median VL, 4.13 log 10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log 10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log 10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log 10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.Due to the efforts of national programs and the support of a wide range of international partners, the number of people receiving antiretroviral therapy (ART) in resource-limited countries has increased significantly over recent years, most notably in sub-Saharan Africa (24). In order to limit the emergence of resistance to antiretroviral drugs, human immunodeficiency virus (HIV) treatment should ideally be accompanied by periodic virological monitoring, such as viral load and resistance testing. However, in resource-limited countries, viral load monitoring and drug resistance tests are not yet available at an affordable cost, and infrastructure requirements limit the scale-up in access to these tests. Treatment initiation and modifications are thus guided mainly by clinical disease progression and by CD4 cell counts, when possible (13, 24). As a consequence, many people with virological failure stay on inadequate drug regimens for long periods. Rapid or uncontrolled emergence of HIV drug resistance is thus feared as a potential consequence of ART scale-up in resource-limited countries. Therefore, ART resistance monitoring is not recommended at the individual patient level in low-income countries, and the WHO has established a sys...

Virological monitoring is still feasible for DBS after 3 months of storage at 37 degrees C when VL is >5000 copies/mL, but DNA contributes largely to the final results.

h Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at ؊20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of >1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 realtime PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory performance should be monitored.T he increasing availability of antiretroviral treatment (ART) has dramatically contributed to a reduction in mortality and morbidity related to HIV/AIDS in resource-limited countries (RLC). In order to limit the emergence of resistance to antiretroviral drugs, HIV treatment should ideally be accompanied by regular virological monitoring, including viral load (VL) and genotypic drug resistance testing (1). With the recent scaling up of ART in RLC, people from semiurban and rural areas are now also receiving treatment, and recent data have shown variable levels of virological failure (VF) and drug resistance in these areas. In certain settings, VF can be Ͼ20% after 12 months of ART (2-4), stressing the urgent need for improved virological monitoring.In the majority of developing countries with limited resources, monitoring blood plasma VL poses logistical challenges since plasma preparation, storage, and/or shipment requires personnel and laboratory infrastructure that is often lacking. Today, viral load assays still require sophistic...

BackgroundmicroRNAs (miRNAs) play crucial roles in major biological processes and their deregulations are often associated with human malignancies. As such, they represent appealing candidates as targets of innovative therapies. Another interesting aspect of their biology is that they are present in various biological fluids where, advantageously, they appear to be very stable. A plethora of studies have now reported their potential as biomarkers that can be used in diagnosis, prognosis and/or theranostic issues. However, the application of circulating miRNAs in clinical practices still requires the identification of highly efficient, robust and reproducible methods for their isolation from biological samples.In that context, we performed an independent cross-comparison of three commercially available RNA extraction kits for miRNAs isolation from human blood samples (Qiagen and Norgen kits as well as the new NucleoSpin miRNAs Plasma kit from Macherey-Nagel). miRNAs were further profiled using the Taqman Low Density Array technology.ResultsWe found that, although these 3 kits had equal performances in extracting miRNAs from peripheral blood mononuclear cells, the Macherey-Nagel kit presented several advantages when isolating miRNAs from sera. Besides, our results have indicated that, depending on the quantity of the biological samples used, the extraction procedure directly impacted on the G/C composition of the miRNAs detected.ConclusionOverall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-395) contains supplementary material, which is available to authorized users.

Depleted uranium (DU) is a radioactive heavy metal coming from the nuclear industry and used in numerous military applications. Uranium inhalation can lead to the development of fibrosis and neoplasia in the lungs. As little is known concerning the molecular processes leading to these pathological effects, some of the events in terms of genotoxicity and inflammation were investigated in rats exposed to DU by inhalation. Our results show that exposure to DU by inhalation resulted in DNA strand breaks in broncho-alveolar lavage (BAL) cells and in increase of inflammatory cytokine expression and production of hydroperoxides in lung tissue suggesting that the DNA damage was in part a consequence of the inflammatory processes and oxidative stress. The effects seemed to be linked to the doses, were independent of the solubility of uranium compounds and correlating with the type of inhalation. Repeated inhalations seemed to induce an effect of potentiation in BAL cells and also in kidney cells. Comet assay in neutral conditions revealed that DNA damage in BAL cells was composed partly by double strands breaks suggesting that radiation could contribute to DU genotoxic effects in vivo. All these in vivo results contribute to a better understanding of the pathological effect of DU inhalation.

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BackgroundWith widespread use of antiretroviral (ARV) drugs in Africa, one of the major potential challenges is the risk of emergence of ARV drug-resistant HIV strains. Our objective is to evaluate the virological failure and genotypic drug-resistance mutations in patients receiving first-line highly active antiretroviral therapy (HAART) in routine clinics that use the World Health Organization public health approach to monitor antiretroviral treatment (ART) in Togo.MethodsPatients on HAART for one year (10-14 months) were enrolled between April and October 2008 at three sites in Lomé, the capital city of Togo. Plasma viral load was measured with the NucliSENS EasyQ HIV-1 assay (Biomérieux, Lyon, France) and/or a Generic viral load assay (Biocentric, Bandol, France). Genotypic drug-resistance testing was performed with an inhouse assay on plasma samples from patients with viral loads of more than 1000 copies/ml. CD4 cell counts and demographic data were also obtained from medical records.ResultsA total of 188 patients receiving first-line antiretroviral treatment were enrolled, and 58 (30.8%) of them experienced virologic failure. Drug-resistance mutations were present in 46 patients, corresponding to 24.5% of all patients enrolled in the study. All 46 patients were resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTIs): of these, 12 were resistant only to NNRTIs, 25 to NNRTIs and lamivudine/emtricitabine, and eight to all three drugs of their ARV regimes. Importantly, eight patients were already predicted to be resistant to etravirine, the new NNRTI, and three patients harboured the K65R mutation, inducing major resistance to tenofovir.ConclusionsIn Togo, efforts to provide access to ARV therapy for infected persons have increased since 2003, and scaling up of ART started in 2007. The high number of resistant strains observed in Togo shows clearly that the emergence of HIV drug resistance is of increasing concern in countries where ART is now widely used, and can compromise the long-term success of first- and second-line ART.