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1.  Broad Phenotypic Cross-Resistance to Elvitegravir in HIV-Infected Patients Failing on Raltegravir-Containing Regimens 
The failure of raltegravir (RAL) is generally associated with the selection of mutations at integrase position Y143, Q148, or N155. However, a relatively high proportion of failures occurs in the absence of these changes. Here, we report the phenotypic susceptibilities to RAL and elvitegravir (EVG) for a large group of HIV-infected patients failing on RAL-containing regimens. Plasma from HIV-infected individuals failing on RAL-containing regimens underwent genotypic and phenotypic resistance testing (Antivirogram v2.5.01; Virco). A control group of patients failing on other regimens was similarly tested. Sixty-one samples were analyzed, 40 of which belonged to patients failing on RAL-containing regimens. Full RAL susceptibility was found in 20/21 controls, while susceptibility to EVG was diminished in 8 subjects, with a median fold change (FC) of 2.5 (interquartile range [IQR], 2.1 to 3.1). Fourteen samples from patients with RAL failures showed diminished RAL susceptibility, with a median FC of 38.5 (IQR, 10.8 to 103.2). Primary integrase resistance mutations were found in 11 of these samples, displaying a median FC of 68.5 (IQR, 23.5 to 134.3). The remaining 3 samples showed a median FC of 2.5 (IQR, 2 to 2.7). EVG susceptibility was diminished in 19/40 samples from patients with RAL failures (median FC, 7.71 [IQR, 2.48 to 99.93]). Cross-resistance between RAL and EVG was high (R2 = 0.8; P < 0.001), with drug susceptibility being more frequently reduced for EVG than for RAL (44.3% versus 24.6%; P = 0.035). Susceptibility to RAL and EVG is rarely affected in the absence of primary integrase resistance mutations. There is broad cross-resistance between RAL and EVG, which should preclude their sequential use. Resistance to EVG seems to be more frequent and might be more influenced by integrase variability.
PMCID: PMC3370736  PMID: 22450969
2.  Comparison of HIV-1 RNA Measurements Obtained by Using Plasma and Dried Blood Spots in the Automated Abbott Real-Time Viral Load Assay 
Journal of Clinical Microbiology  2012;50(3):569-572.
Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIV-infected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 μl of blood onto filter paper cards and were stored desiccated at −20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R = 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic.
PMCID: PMC3295109  PMID: 22170904
3.  HIV-1 drug resistance testing from dried blood spots collected in rural Tanzania using the ViroSeq HIV-1 Genotyping System 
To assess whether the commercial ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, IL, USA) can be used in conjunction with dried blood spots (DBS) for clinical monitoring of drug resistance in patients who fail antiretroviral treatment (ART) in rural Tanzania.
Patients and methods
Patients at Haydom Lutheran Hospital with confirmed treatment failure (viral load >1000 copies/mL) of a first-line ART regimen were selected for resistance testing. DBS were stored with desiccant at −20°C for a median of 126 days (range 0–203) and shipped at ambient temperature for 20 days. After manual extraction of nucleic acids, the ViroSeq kit was used for amplification and sequencing. DBS-derived genotypes were compared with those of a plasma-based assay.
Seventeen of 36 (47%) DBS specimens were successfully genotyped. Only 2 of 16 (13%) DBS with a viral load <10 000 copies/mL could be amplified, compared with 15 of 20 (75%) DBS with a viral load >10 000 copies/mL (P = 0.001). In samples that yielded a sequence, all 23 clinically significant reverse transcriptase (RT) mutations in plasma were also detected in DBS. One RT mutation was found in DBS only. In the protease region, 77 polymorphisms were found in plasma, of which 70 (91%) were also detected in DBS. Sixteen of 17 (94%) patients had identical resistance profiles to antiretroviral drugs in plasma and DBS.
The ViroSeq kit performed well in patients with a high viral load, but failed to genotype most DBS with a viral load <10 000 copies/mL. In DBS that yielded a genotype, there was high concordance with a plasma-based assay.
PMCID: PMC3019084  PMID: 21115444
HIV infections; antiretroviral therapy; molecular diagnostic techniques; sub-Saharan Africa
4.  Correlation between Human Immunodeficiency Virus Type 1 (HIV-1) RNA Measurements Obtained with Dried Blood Spots and Those Obtained with Plasma by Use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV Load Tests ▿  
Journal of Clinical Microbiology  2009;47(4):1031-1036.
The plasma human immunodeficiency virus (HIV) RNA load is used in the clinical routine for the monitoring of HIV infection and the patient's response to antiretroviral therapy. Other body fluids or dried blood spots (DBS) can be used, however, to assess the level of viremia. The use of DBS may be especially helpful for the monitoring of HIV-infected patients in resource-poor settings, where access to adequate laboratory facilities is often difficult. However, the correlation between the HIV RNA levels in plasma and those in DBSs has not been well established. Paired plasma and DBS samples obtained from HIV type 1 (HIV-1)-infected patients were tested for HIV RNA copy numbers by using two different commercial assays, the Nuclisens EasyQ HIV-1 (version 1.1) test (the Nuclisens test; Biomerieux) and the m2000rt RealTime HIV test (the m2000rt test; Abbott). Nucleic acid extraction was performed manually by using either the Nuclisens isolation kit (which uses the Boom methodology) or the m2000rt sample preparation kit (an iron particle-based method). A total of 103 paired plasma and DBS samples were tested. Viral load results were obtained for 97 (94.2%) samples with the Nuclisens isolation kit and 81 (78.6%) samples with the m2000rt kit. The overall correlation between the RNA loads in plasma and DBS was good, although better results were obtained by the Nuclisens test (R2 = 0.87, P < 0.001) than by the m2000rt test (R2 = 0.70, P < 0.001). While the specificities were excellent and similar for both the Nuclisens and the m2000rt tests (97.1% and 100%, respectively), the sensitivity was greater by the Nuclisens test than by the m2000rt test (75.8% and 56.6%, respectively). Overall, the viral loads in DBS tended to be lower than those in plasma, with mean differences of 0.3 log unit (standard deviation, 0.5 log unit) and 0.76 log unit (standard deviation, 0.8 log unit) for the Nuclisens and the m2000rt tests, respectively. The levels of agreement between the measurements in plasma and DBS were assessed by using the Bland-Altman plot for each assay. The Nuclisens test gave results within its defined limits (−0.65 to 1.26) for 95.9% of the samples, while the m2000rt test gave results within its limits (−0.83 to 2.33) for 100% of the samples. In summary, the HIV-1 load can accurately be quantified by testing DBS by either the Nuclisens or the m2000rt test, although the Nuclisens test may outperform the m2000rt test when nucleic acids are extracted manually.
PMCID: PMC2668340  PMID: 19193847
5.  Evaluation of Eight Different Bioinformatics Tools To Predict Viral Tropism in Different Human Immunodeficiency Virus Type 1 Subtypes▿  
Journal of Clinical Microbiology  2008;46(3):887-891.
Human immunodeficiency virus type 1 (HIV-1) tropism can be assessed using phenotypic assays, but this is quite laborious, expensive, and time-consuming and can be made only in sophisticated laboratories. More accessible albeit reliable tools for testing of HIV-1 tropism are needed in view of the prompt introduction of CCR5 antagonists in clinical practice. Bioinformatics tools based on V3 sequences might help to predict HIV-1 tropism; however, most of these methods have been designed by taking only genetic information derived from HIV-1 subtype B into consideration. The aim of this study was to evaluate the performances of several genotypic tools to predict HIV-1 tropism in non-B subtypes, as data on this issue are scarce. Plasma samples were tested using a new phenotypic tropism assay (Phenoscript-tropism; Eurofins), and results were compared with estimates of coreceptor usage using eight different genotypic predictor softwares (Support Vector Machine [SVM], C4.5, C4.5 with positions 8 to 12 only, PART, Charge Rule, geno2pheno coreceptor, Position-Specific Scoring Matrix X4R5 [PSSMX4R5], and PSSMsinsi). A total of 150 samples were tested, with 115 belonging to patients infected with non-B subtypes and 35 drawn from subtype B-infected patients, which were taken as controls. When non-B subtypes were tested, the concordances between the results obtained using the phenotypic assay and distinct genotypic tools were as follows: 78.8% for SVM, 77.5% for C4.5, 82.5% for C4.5 with positions 8 to 12 only, 82.5% for PART, 82.5% for Charge Rule, 82.5% for PSSMX4R5, 83.8% for PSSMsinsi, and 71.3% for geno2pheno. When clade B viruses were tested, the best concordances were seen for PSSMX4R5 (91.4%), PSSMsinsi (88.6%), and geno2pheno (88.6%). The sensitivity for detecting X4 variants was lower for non-B than for B viruses, especially in the case of PSSMsinsi (38.4% versus 100%, respectively), SVMwetcat (46% versus 100%, respectively), and PART (30% versus 90%, respectively). In summary, while inferences of HIV-1 coreceptor usage using genotypic tools seem to be reliable for clade B viruses, their performances are poor for non-B subtypes, in which they particularly fail to detect X4 variants.
PMCID: PMC2268339  PMID: 18199789
6.  HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C 
Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at −20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year.
A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4°C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT–PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment.
Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10 000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant.
We show that drug resistance genotyping from DBSs stored at 4°C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4°C may represent a suitable temperature for long-term storage of DBSs.
PMCID: PMC2386080  PMID: 18344550
resistance testing; ViroSeq assay; 903 filter paper

Results 1-6 (6)