Comparison of different extraction techniques to profile microRNAs from human sera and peripheral blood mononuclear cells.

BACKGROUND: microRNAs (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. RESULTS: We 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. CONCLUSION: Overall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs.

Field evaluation of dried blood spots for routine HIV-1 viral load and drug resistance monitoring in patients receiving antiretroviral therapy in Africa and Asia.

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 real-time 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.

Extraordinary heterogeneity of virological outcomes in patients receiving highly antiretroviral therapy and monitored with the World Health Organization public health approach in sub-saharan Africa and southeast Asia.

BACKGROUND: The limited access to virological monitoring in developing countries is a major weakness of the current antiretroviral treatment (ART) strategy in these settings. We conducted a large cross-sectional study in Burkina Faso, Cameroon, Cote d'Ivoire, Senegal, Togo, Thailand, and Vietnam to assess virological failure and drug resistance mutations (DRMs) after 12 or 24 months of ART. METHODS: Between 2009 and 2011, we recruited adults attending ART centers 10-14 months (the M12 group) or 22-26 months (M24 group) after initiating ART. Demographic and clinical data were collected on site, and viral load was measured. Samples with a viral load of ≥ 1000 copies/mL, considered as the failure threshold, were genotyped for drug resistance assessment. RESULTS: Overall, 3935 patients were recruited (2060 at M12 and 1875 at M24). Median ages varied from 32 to 42 years. Median CD4(+) T-cell counts at ART initiation were low (99-172 cells/µL). The main ART regimens included stavudine/zidovudine plus lamivudine plus nevirapine/efavirenz. Overall, virological failure frequency was 11.1% for M12 patients and 12.4% for M24 patients, and 71.0% to 86.1% of these patients, respectively, had drug-resistant virus. Across sites, virological failure varied from 2.9% to 20.6% in M12 patients and from 3.7% to 26.0% in M24 patients. Predominant DRMs were associated with ART regimens, but virus in several patients accumulated DRMs to drugs not received, such as abacavir, didanosine, tenofovir, etravirine, and rilpivirine. CONCLUSIONS: 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.

Single real-time reverse transcription-PCR assay for detection and quantification of genetically diverse HIV-1, SIVcpz, and SIVgor strains.

Although antiretroviral treatment availability has improved, the virological monitoring of patients remains largely uneven across regions. In addition, viral quantification tests are suffering from human immunodeficiency virus type 1 (HIV-1) genetic diversity, fueled by the emergence of new recombinants and of lentiviruses from nonhuman primates. We developed a real-time reverse transcription-PCR (RT-PCR) assay that is relatively inexpensive and able to detect and quantify all circulating forms of HIV-1 and its simian immunodeficiency virus (SIV) precursors, SIVcpz and SIVgor. Primers and a probe were designed to detect all variants of the HIV-1/SIVcpz/SIVgor lineage. HIV-1 M plasma (n = 190; 1.68 to 7.78 log(10) copies/ml) representing eight subtypes, nine circulating recombinant forms, and 21 unique recombinant forms were tested. The mean PCR efficiency was 99%, with low coefficients of intra- and interassay variation (<5%) and a limit of quantification of <2.50 log(10) copies/ml, with a 200-µl plasma volume. On the studied range, the specificity and the analytical sensitivity were 100 and 97.4%, respectively. The viral loads were highly correlated (r = 0.95, P < 0.0001) with the reference method (generic HIV assay; Biocentric) and had no systematic difference, irrespective of genotype. Furthermore, 22 HIV-1 O plasmas were screened and were better quantified compared to the gold-standard RealTime HIV-1 assay (Abbott), including four samples that were only quantified by our assay. Finally, we could quantify SIVcpzPtt and SIVcpzPts from chimpanzee plasma (n = 5) and amplify SIVcpz and SIVgor from feces. Thus, the newly developed real-time RT-PCR assay detects and quantifies strains from the HIV-1/SIVcpz/SIVgor lineage, including a wide diversity of group M strains and HIV-1 O. It can therefore be useful in geographical areas of high HIV diversity and at risk for the emergence of new HIV variants.

Drug resistance mutations of HIV type 1 non-B viruses to integrase inhibitors in treatment-naive patients from sub-saharan countries and discordant interpretations.

The routine use of integrase inhibitors in sub-Saharan Africa where HIV-1 non-B viruses predominate is limited, but evaluating their effectiveness on HIV-1 subtypes and CRFs that circulate in this region is essential. We here analyzed 97 integrase sequences from HIV-1 non-B-infected individuals from African countries. Using currently available interpretation algorithms (ANRS, HIVdb, and Rega), we identified the presence of mutations at nine resistance-associated positions including L74M (3.1%), T97A (9.3%), K156N (2.1%), E157Q (5.2%), G163K (1.0%), T206S (48.5%), S230N (1.0%), D232N (1.0%), and R236K (1.0%). All but one (E157Q) were considered as accessory resistant mutation by the algorithms. E157Q identified in 5% of patients tested (5/97) was selected by the ANRS algorithm as a primary mutation, which alone can confer resistance to raltegravir. These results illustrated the need of further in vitro and clinical studies involving non-B viruses to better understand the real significance of observed mutations and harmonize interpretations.

Resistance to antiretroviral drugs in treated and drug-naive patients in the Democratic Republic of Congo.

BACKGROUND: We studied virological outcome and drug resistance in patients on antiretroviral therapy (ART) in health care centers in the Democratic Republic of Congo and looked for the presence of drug resistance in antiretroviral-naive patients attending the same clinics. METHODS: In 2008, we conducted a cross-sectional survey among patients on ART for ≥ 12 months in 4 major cities [Kinshasa (n = 289), Matadi (n = 198), Lubumbashi (n = 77), and Mbuji-Mayi (n = 103)]. Genotypic drug resistance tests were done with an in-house assay on samples with viral load >1000 copies/mL. ART-naive patients (n = 283) were also consecutively enrolled in the same clinics. RESULTS: Of the 667 patients on ART, >98% received Lamivudine + Stavudine/azidothymidine + Nevirapine/Efavirenz as first-line regimen and 74.4% were women. Median time on ART was 25 months [interquartile ratio (IQR), 19-32] in Kinshasa, 26 months (IQR, 19-32) in Matadi, 27 months (IQR, 19-44) in Lubumbashi, and 19 months (IQR, 16-24) in Mbuji-Mayi. A total of 97 patients (14.6%) had viral load >1000 copies/mL, and among the 93 successfully sequenced samples, 78 (83.9%) were resistant to at least 1 drug of their ART regimen: 68 harbored resistance mutations to nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI), 2 to NRTI only, 7 to NNRTI only, and 1 to NRTI + NNRTI + protease inhibitor. The majority of patients, 70/78 (89.7%), were resistant to at least 2 of the 3 drugs from their treatment. The use of next-generation NNRTI, etravirine was already compromised for 19.2% (15/78) of the patients and 7 patients had the K65R mutation compromising the use of tenofovir in second-line regimens. The proportion of antiretroviral-resistant patients increased over time from 8.4% to 18.6% for patients on ART for 12-23 months or >35 months (P = 0.013), respectively. Virological failure and rates of drug resistance were significantly higher among men than women, 19.9% versus 8.8%, respectively (P = 0.0001). Among the 253 recently diagnosed patients, 20 (7.9%) harbored resistance mutations. CONCLUSIONS: The accumulation of drug resistance mutations with time on ART needs further attention, and surveillance should be reinforced in ART programs in sub-Saharan Africa.

High prevalence of HIV-1 drug resistance among patients on first-line antiretroviral treatment in Lomé, Togo.

BACKGROUND: With 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. METHODS: Patients 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. RESULTS: A 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. CONCLUSIONS: In 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.

Effect of storage conditions of dried plasma and blood spots on HIV-1 RNA quantification and PCR amplification for drug resistance genotyping.

OBJECTIVES: Dried blood spots (DBS) and dried plasma spots (DPS) are easy to collect and store, and have been successfully tested as an alternative to plasma for performing virological analyses. Adequate storage conditions still need to be established and cell-associated proviral DNA in DBS can contribute to the amplified products. We evaluated these two parameters. METHODS: Residual samples from 34 HIV-1-infected patients [mean viral load (VL) = 3.93 log(10) copies/mL] were used to prepare DPS and DBS, then stored at 20 degrees C and 37 degrees C. HIV-1 nucleic acids were extracted, with or without DNase treatments, to perform HIV-1 VL quantification and nested RT-PCR to amplify the reverse transcriptase gene (798 bp). RESULTS: For DBS stored for 3 months at 20 degrees C, VL could be measured for all samples and results were comparable to plasma VL. At 37 degrees C, a slight decrease was observed after 2 and 3 months (0.16 and 0.37 log(10) copies/mL mean difference, respectively). For DPS, a significant decrease in VL (0.70 and 1.07 log(10) copies/mL after 1 and 2 months, respectively) was seen at 37 degrees C, but not at 20 degrees C. PCR amplifications from DPS were only successful for 50% of samples with an initial VL >10 000 copies/mL after 1 month at 20 degrees C. From DBS, PCR amplifications are possible until 3 months for samples with plasma VL >5000 copies/mL. VL and PCR results for DBS treated with DNase are close to results obtained for DPS. CONCLUSIONS: 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.

HIV contamination of commercial PCR enzymes raises the importance of quality control of low-cost in-house genotypic HIV drug resistance tests.

BACKGROUND: Low-cost in-house technologies for genotypic drug resistance testing use reagents with quality labels for research only. Here, we report on the results of PCR amplifications in negative-controls that were observed in two independent laboratories. METHODS: Positive PCR amplifications of protease and reverse transcriptase fragments for genotypic drug resistance testing of HIV on dried blood and/or plasma spots were observed on negative-control samples and were analysed in detail by PCR and sequence and phylogenetic analyses to identify the origin of the PCR contamination. RESULTS: Detailed analysis revealed that the RT-PCR enzymes were contaminated with an HIV-based vector commercialized by the same company. CONCLUSIONS: These observations show the need to implement quality control steps that verify for the absence of HIV in new reagent batches because this can significantly compromise molecular diagnosis of HIV and genotypic drug resistance tests using in-house protocols.

Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing.

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 degrees C but for only 1 month at 37 degrees C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37 degrees C and for 1 month at 20 degrees 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.

Heterogeneous accumulation of uranium in the brain of rats.

Recent reports suggest that uranium can accumulate not only in known target organs, that is, kidneys or bones, but also in others such as central nervous system. In the present work, the accumulation of uranium in the brain of rats was studied after repeated exposure by inhalation, chronic exposure by ingestion and acute exposure by injection. For each route of administration, the amount of uranium entering the brain was low. The results showed different accumulation in the brain areas according to the route of intake. Injection gave a rather homogeneous distribution in the different brain areas, whereas both inhalation and ingestion yielded heterogeneous but specific accumulation. These differences in distribution suggest the operation of different mechanisms of delivery of uranium to the brain tissues.

Distribution and genotoxic effects after successive exposure to different uranium oxide particles inhaled by rats.

In nuclear fuel cycle facilities, workers may inhale airborne uranium compounds that lead to internal contamination, with various exposure scenarios depending on the workplace. These exposures can be chronic, repeated, or acute, and can involve many different compounds. The effect of uranium after multiple scenarios of exposure is unknown. The aim of this study, therefore, was to investigate the genotoxic and biokinetics consequences of exposure to depleted insoluble uranium dioxide (UO2) by repeated or acute inhalation on subsequent acute inhalation of moderately soluble uranium peroxide (UO4) in rats. The results show that UO2 repeated preexposure by inhalation increases the genotoxic effects of UO4 inhalation, assessed by comet assay, in different cell types, when UO4 exposure alone has no effect. At the same time, the study of UO4 bioaccumulation showed that the UO4 biokinetics in the kidneys, gastrointestinal tract, and excreta, but not in the lungs, were slightly modified by previous UO2 exposures. All these results show that both genotoxic and biokinetics effects of uranium may depend on preexposure and that repeated exposure induces a potentiation effect compared with acute exposure.

The effect of repeated inhalation on the distribution of uranium in rats.

For the assessment of doses after inhalation of airborne uranium compounds by workers, the International Commission on Radiological Protection (ICRP) developed compartmental models that are used to calculate reference dose coefficients and retention and excretion functions. It is assumed that each acute intake has no effect on the biokinetics of later intakes. Consequently, retention and excretion after multiple or chronic exposure are predicted using the same models as after acute exposure. This assumption was tested here on rats exposed to repeated inhalation of uranium dioxide (UO2). First, excretion and organ retention were determined after a single inhalation of UO2. The follow-up of incorporated activity was used to design a biokinetic model for uranium inhaled by rats. Second, the biokinetics of uranium were monitored in two experiments of repeated inhalations of uranium dioxide under different intake patterns. For these two experiments, the organs' retention and excretion after repeated UO2 inhalation were predicted using the biokinetic model and compared to the experimental measurement. Under the two sets of experimental conditions considered, the prediction of the biokinetic model based on acute exposure data was consistent with the biokinetics observed after repeated UO2 inhalations, with the possible exception of retention in the skeleton.

Genotoxic and inflammatory effects of depleted uranium particles inhaled by rats.

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.

Bioaccumulation and behavioural effects of depleted uranium in rats exposed to repeated inhalations.

Depleted uranium has numerous industrial and military uses. Contamination by inhalation of airborne compounds is probably the most important route of exposure. In humans, there are no data clearly demonstrating neurotoxicity of uranium, yet some experimental studies suggest a link between neurological toxicity and uranium exposure. In this work, the bioaccumulation of uranium in male rats after exposure to repeated depleted uranium dioxide inhalation (30 min inhalation at 197 mgm(-3), 4 days a week for 3 weeks) has been studied, together with the behavioural effects. The uranium concentrations in the brain 1 day after the end of the exposure period varied as follows: olfactory bulb>hippocampus>frontal cortex>cerebellum, subsequently decreasing rapidly. The spontaneous locomotion activity of exposed rats was increased 1 day post exposure and the spatial working memory was less efficient 6 days post exposure, compared with control rats. These data suggest that depleted uranium is able to enter the brain after exposure to repeated inhalation, producing behavioural changes.