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1.  Hepatitis B, C, and D and HIV Infections among Immigrants from Equatorial Guinea Living in Spain 
A total of 1,220 subjects from Equatorial Guinea living in Spain (median age = 41 years; 453 male and 767 female) was examined for antibodies to human immunodeficiency virus (HIV) and Hepatitis B (HBV), C (HCV), and D (HDV) viruses. Extracted RNA and DNA from the positive samples were used to quantify viral load. The prevalence of HIV antibodies, HCV RNA, and HBV surface antigen (HBsAg) was 10.8% (N = 132), 11.6% (N = 141), and 7.9% (N = 96), respectively. The most prevalent HIV variant was CRF02_AG (38.5%; N = 40). HCV genotype 4 (60%; N = 36) and HBV genotype A3 (32%; N = 8) were the hepatitis variants most frequently found. Superinfection with HDV was seen in 20.9% (N = 24) of HBsAg carriers. A control group of 276 immigrants from other sub-Saharan countries showed similar rates of HIV and HBsAg, although no HCV cases were found. Immigrants constitute a major source of HIV and hepatitis viruses in Spain; therefore, it is important that control measures are intensified.
doi:10.4269/ajtmh.12-0319
PMCID: PMC3617871  PMID: 23339201
2.  Short Communication: RNASEL Alleles and Susceptibility to Infection by Human Retroviruses and Hepatitis Viruses 
AIDS Research and Human Retroviruses  2012;28(10):1259-1261.
Abstract
RNASEL seems to function as an intracellular restriction factor blocking the establishment of infections caused by viral agents. Herein, we investigated whether allelic variants at the RNASEL gene might influence the susceptibility to viral infections or conditions potentially linked to viral agents. The allelic distribution at codon 462 was 139 (33.9%), 204 (49.8%), and 67 (16.3%) for RR, RQ, and QQ, respectively, in 410 individuals in Spain. There were no significant differences comparing 105 blood donors and 71 patients with HIV-1 infection, 27 with chronic hepatitis C, 67 with prostate cancer, and 107 with chronic fatigue syndrome. In contrast, two-thirds of 18 patients with HTLV-1 infection and 15 with chronic hepatitis B harbored RR. Thus, polymorphisms at the RNASEL gene do not seem to influence the susceptibility to common viral infections or conditions potentially of viral etiology. The role in influencing the susceptibility to HTLV-1 or HBV chronic infection warrants further examination in larger patient populations.
doi:10.1089/aid.2012.0014
PMCID: PMC3448100  PMID: 22356654
3.  Evaluation of In-house Genotyping Assay Performance Using Dried Blood Spot Specimens in the Global World Health Organization Laboratory Network 
In resource-limited settings, there is increased demand for human immunodeficiency virus type 1 drug resistance testing. Because preservation of plasma specimens is often not feasible in resource-limited settings, use of dried blood spots (DBSs) is being adopted. We used 2 panels of DBSs for genotyping assay validation and proficiency testing in selected laboratories in the World Health Organization laboratory network in 14 countries. An amplification sensitivity of 1000 copies/mL was achieved by 2 laboratories. Reproducibility and accuracy of nucleotide sequence determination and resistance-associated mutation identification from DBSs was similar to that previously determined for plasma. International shipping at ambient temperature had no significant effect on amplification success. These studies indicate that DBS-based genotyping is equally reproducible and reliable, although slightly less sensitive, compared with plasma.
doi:10.1093/cid/cir982
PMCID: PMC3338305  PMID: 22544187
4.  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.
doi:10.1128/AAC.06170-11
PMCID: PMC3370736  PMID: 22450969
5.  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.
doi:10.1128/JCM.00418-11
PMCID: PMC3295109  PMID: 22170904
6.  Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy 
Retrovirology  2012;9:68.
Background
Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex.
Results
The cross-sectional study carried out with >200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations.
Conclusions
Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations.
doi:10.1186/1742-4690-9-68
PMCID: PMC3468358  PMID: 22889300
7.  Molecular Surveillance of HIV-1 in Madrid, Spain: a Phylogeographic Analysis ▿  
Journal of Virology  2011;85(20):10755-10763.
The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europe's main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set (n = 2,792) from 11 Madrid hospitals of viral pol sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.
doi:10.1128/JVI.00454-11
PMCID: PMC3187488  PMID: 21795343
8.  Trends in the prevalence and distribution of HTLV-1 and HTLV-2 infections in Spain 
Virology Journal  2012;9:71.
Background
Although most HTLV infections in Spain have been found in native intravenous drug users carrying HTLV-2, the large immigration flows from Latin America and Sub-Saharan Africa in recent years may have changed the prevalence and distribution of HTLV-1 and HTLV-2 infections, and hypothetically open the opportunity for introducing HTLV-3 or HTLV-4 in Spain. To assess the current seroprevalence of HTLV infection in Spain a national multicenter, cross-sectional, study was conducted in June 2009.
Results
A total of 6,460 consecutive outpatients attending 16 hospitals were examined. Overall, 12% were immigrants, and their main origin was Latin America (4.9%), Africa (3.6%) and other European countries (2.8%). Nine individuals were seroreactive for HTLV antibodies (overall prevalence, 0.14%). Evidence of HTLV-1 infection was confirmed by Western blot in 4 subjects (prevalence 0.06%) while HTLV-2 infection was found in 5 (prevalence 0.08%). Infection with HTLV types 1, 2, 3 and 4 was discarded by Western blot and specific PCR assays in another two specimens initially reactive in the enzyme immunoassay. All but one HTLV-1 cases were Latin-Americans while all persons with HTLV-2 infection were native Spaniards.
Conclusions
The overall prevalence of HTLV infections in Spain remains low, with no evidence of HTLV-3 or HTLV-4 infections so far.
doi:10.1186/1743-422X-9-71
PMCID: PMC3337814  PMID: 22444832
HTLV; Spain; Seroprevalence; Epidemiology; HTLV-3; HTLV-4
12.  Drug Resistance Mutations in HIV-Infected Patients in the Spanish Drug Resistance Database Failing Tipranavir and Darunavir Therapy▿  
The presence of resistance mutations in patients failing tipranavir or darunavir was examined at the national drug resistance database of the Spanish AIDS Research Network. Although mutations emerging during tipranavir and darunavir failures differed considerably, cross-resistance was found in up to half of the patients tested. Interestingly, mutation 54L, which is associated with tipranavir hypersusceptibility, was selected in half of the darunavir failures. Thus, resistance testing seems mandatory to ensure the benefit of the sequential use of these drugs.
doi:10.1128/AAC.00160-10
PMCID: PMC2897318  PMID: 20479204
13.  HIV-1 drug resistance testing from dried blood spots collected in rural Tanzania using the ViroSeq HIV-1 Genotyping System 
Objectives
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.
Results
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.
Conclusions
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.
doi:10.1093/jac/dkq433
PMCID: PMC3019084  PMID: 21115444
HIV infections; antiretroviral therapy; molecular diagnostic techniques; sub-Saharan Africa
14.  Use of Different Inhibitory Quotients To Predict Early Virological Response to Tipranavir in Antiretroviral-Experienced Human Immunodeficiency Virus-Infected Patients▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4153-4158.
Information about the relationship between pharmacological parameters and an early virological response to tipranavir (TPV) is scarce. Human immunodeficiency virus (HIV)-infected patients who had received TPV as part of a salvage regimen were analyzed retrospectively. A virological response was defined as a decline in the HIV RNA level of ≥1 log unit or to <50 copies/ml between weeks 4 and 12 of therapy. The virtual inhibitory quotient (vIQ) was calculated as the ratio of the TPV plasma trough concentration (Ctrough)/virtual change in the 50% inhibitory concentration. Three genotypic inhibitory quotients (gIQs) were calculated by using different TPV resistance mutation scores (from the International AIDS Society—USA [IAS-USA], Randomized Evaluation of Strategic Intervention in Multidrug-Resistant Patients with Tipranavir [RESIST], and Agence Nationale de Recherches sur le Sida et les Hépatites Virales [ANRS] trials). The sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratios for a positive result (LHR+) and a negative result (LHR−) [LHR+ = sensitivity/(1 − specificity); LHR− = (1 − sensitivity)/specificity] were calculated. A total of 57 HIV-infected patients were analyzed. A virological response was achieved by 77% of the patients. TPV resistance mutations, TPV Ctrough, vIQs, and gIQs were all significantly associated with a virological response. The vIQ had the best PPV and NPV (97% and 78%, respectively). The values of the LHR+ were 7.8 for vIQ, 3.4 for the RESIST gIQ, 3.3 for the IAS-USA gIQ, 3.1 for the ANRS gIQ, 2.2 for TPV Ctrough, and 1.3 for the IAS-USA and RESIST scores. The values of LHR− were 0 for the RESIST score, 0.07 for vIQ, 0.09 for the IAS-USA score, 0.27 for the RESIST gIQ, 0.32 for the IAS-USA gIQ, 0.37 for the ANRS gIQ, and 0.48 for TPV Ctrough. HIV-infected patients who initiate a salvage regimen based on TPV may benefit from baseline drug resistance testing and TPV plasma concentration determination, as vIQ is the best predictor of a virological response.
doi:10.1128/AAC.00041-09
PMCID: PMC2764193  PMID: 19596874
15.  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.
doi:10.1128/JCM.02099-08
PMCID: PMC2668340  PMID: 19193847
16.  Tracing the HIV-1 subtype B mobility in Europe: a phylogeographic approach 
Retrovirology  2009;6:49.
Background
The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced.
Results
In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred.
Conclusion
Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.
doi:10.1186/1742-4690-6-49
PMCID: PMC2717046  PMID: 19457244
17.  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.
doi:10.1128/JCM.01611-07
PMCID: PMC2268339  PMID: 18199789
18.  HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1093/jac/dkn100
PMCID: PMC2386080  PMID: 18344550
resistance testing; ViroSeq assay; 903 filter paper
19.  Predictive factors of virological success to salvage regimens containing protease inhibitors in HIV-1 infected children 
Background
The impact of HIV drug resistance mutations in salvage therapy has been widely investigated in adults. By contrast, data available of predictive value of resistance mutations in pediatric population is scarce.
Methods
A multicenter, retrospective, observational study was conducted in children who received rescue salvage antiretroviral therapy after virologic failure. CD4 counts and viral load were determined at baseline and 6 months after rescue intervention. Genotypic HIV-1 resistance test and virtual phenotype were assessed at baseline.
Results
A total of 33 children met the inclusion criteria and were included in the analysis. The median viral load (VL) and median percentage of CD4+ at baseline was 4.0 HIV-RNA log copies/ml and 23.0% respectively. The median duration that children were taking the new rescue regimen was 24.3 weeks (23.8–30.6). Overall, 47% of the 33 children achieved virological response at 24 weeks. When we compared the group of children who achieved virological response with those who did not, we found out that mean number of PI related mutations among the group of responders was 3.8 vs. 5.4 (p = 0.115). Moreover, the mean number of susceptible drugs according to virtual phenotype clinical cut-off for maximal virologic response was 1.7 vs. 0.8 and mean number of susceptible drugs according to virtual phenotype cut-off for minimal virlologic response was 2.7 vs. 1.3 (p < 0.01 in all cases). Eighteen children were rescued with a regimen containing a boosted-PI and virological response was significantly higher in those subjects compared with the others (61.1% vs. 28.6%, p < 0.01).
Conclusion
Salvage treatment containing ritonavir boosted-PIs in children with virological failure was very efficient. The use of new tools as virtual phenotype could help to improve virologic success in pediatric population.
doi:10.1186/1471-2334-7-55
PMCID: PMC1896165  PMID: 17559687
20.  Impact of Human Immunodeficiency Virus Type 1 (HIV-1) Genetic Diversity on Performance of Four Commercial Viral Load Assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT 
Journal of Clinical Microbiology  2005;43(8):3860-3868.
Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions.
doi:10.1128/JCM.43.8.3860-3868.2005
PMCID: PMC1233972  PMID: 16081923
21.  Different Viral Rebound following Discontinuation of Antiretroviral Therapy in Cases of Infection with Viruses Carrying L74V or Thymidine-Associated Mutations 
Journal of Clinical Microbiology  2004;42(2):862-866.
A total of 76 patients discontinued treatment with didanosine plus hydroxyurea after 1 year of maintenance therapy. The greatest human immunodeficiency virus (HIV)-RNA rebounds were seen in 10 patients harboring an L74V mutation, and the presence of viruses with this mutation rapidly waned. In contrast, viral rebounds were significantly less pronounced (P < 0.01) in 12 subjects harboring thymidine-associated mutations; these mutations persisted in all instances. Thus, selection of an L74V mutation during didanosine therapy may compromise HIV replication in vivo.
doi:10.1128/JCM.42.2.862-866.2004
PMCID: PMC344444  PMID: 14766874
22.  Role of Baseline Human Immunodeficiency Virus Genotype as a Predictor of Viral Response to Tenofovir in Heavily Pretreated Patients 
Journal of Clinical Microbiology  2003;41(9):4421-4423.
Human immunodeficiency virus (HIV)-infected patients (n = 153) failing antiretroviral therapy after exposure to compounds from all three drug families were monitored for 6 months after beginning a rescue intervention program including tenofovir (TDF). At 3 months, levels of HIV RNA in plasma dropped by a mean of 0.9 log10 and the mean CD4 count increased by 52 cells/μl. At 6 months, HIV RNA levels had dropped by a mean of 1.06 log10 and the mean CD4 count had increased by 49 cells/μl. Only five (3.7%) patients discontinued TDF use due to adverse events. In the multivariate analysis, the presence of M41L and/or L210W at baseline was the only viral determinant of a lower response to TDF.
doi:10.1128/JCM.41.9.4421-4423.2003
PMCID: PMC193778  PMID: 12958282
23.  Changes in the Human Immunodeficiency Virus p7-p1-p6 gag Gene in Drug-Naive and Pretreated Patients 
Journal of Clinical Microbiology  2003;41(3):1245-1247.
Resistance to antiretroviral agents often results from mutations within the human immunodeficiency virus (HIV) pol gene. Moreover, insertions within the p6 gag-pol region have recently been found to be involved with resistance to nucleoside analogs. Overall, we found that 21% of 156 specimens collected from HIV-infected individuals (17.6% from 74 drug-naive patients and 24.4% from 82 pretreated patients) harbored these insertions. Insertions around the KQE (Lys-Gln-Glu) motif were found in 12.2% of the pretreated patients but in none of the drug-naive subjects (P = 0.002). In contrast, insertions around the PTAP (Prol-Thre-Ala-Prol) motif were seen at similar rates (∼15%) among drug-naive and pretreated patients, which supports the idea that they may be natural polymorphisms.
doi:10.1128/JCM.41.3.1245-1247.2003
PMCID: PMC150303  PMID: 12624058
24.  Evaluation of the Abbott LCx Quantitative Assay for Measurement of Human Immunodeficiency Virus RNA in Plasma 
Journal of Clinical Microbiology  2002;40(4):1518-1521.
Plasma human immunodeficiency virus RNA in 491 clinical specimens was measured by LCx. There was a strong correlation with the results provided by other methods (r2 values of 0.93 with Cobas-Monitor version 1.5 and of 0.95 with Quantiplex version 3.0). However, values were uniformly higher with LCx than with Quantiplex when non-B subtypes were tested.
doi:10.1128/JCM.40.4.1518-1521.2002
PMCID: PMC140332  PMID: 11923386

Results 1-24 (24)