Leticia Martín

Most HIV Type 1 Non-B Infections in the Spanish Cohort of Antiretroviral Treatment-Naïve HIV-Infected Patients (CoRIS) Are Due to Recombinant Viruses

ABSTRACT HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected.

Trends in drug resistance prevalence in HIV-1-infected children in Madrid: 1993 to 2010 analysis.

Background: Drug resistance mutations compromise antiretroviral treatment (ART) effectiveness in HIV-1–infected children. Trends in drug resistance prevalence have not been previously evaluated in HIV-infected children in Spain. Methods: HIV-1 variants, drug resistance prevalence dynamics and drug susceptibility were analyzed from 1993 to 2010 in HIV-infected children with available pol sequence, sample or drug resistance profile. HIV-1 variants were characterized by phylogenetic analysis. Resistance mutations in pretreated and naive patients were identified according to International AIDS Society-2010 and the World Health Organization list, respectively. Results: In 232 patients, genotypic resistance profiles (n = 11) or pol sequences (n = 128) were recovered or newly generated from infected samples (n = 93). Patients were mainly in care at pediatric units (63%), were mostly Europeans (84%), with moderate AIDS symptoms (65%), on ART (91%) and infected by HIV-1 subtype B (89%). Transmitted major drug resistance mutations were selected in 6 (13.6%) of the 44 ART-naive children: 4.8%, 9.3% and 11.6%, for protease inhibitors, nucleoside reverse transcriptase inhibitors and nonnucleoside reverse transcriptase inhibitors, respectively. Overall resistance prevalence was higher (71.8%) among ART-exposed children: 39.9%, 66.5% and 35.3% for protease inhibitors, nucleoside reverse transcriptase inhibitors and nonnucleoside reverse transcriptase inhibitors, respectively. Resistance prevalence among ART-exposed children was higher in 2009 to 2010 relative to 1993 to1999 for nonnucleoside reverse transcriptase inhibitors (42% versus 6%; P = 0.006), protease inhibitors (39% versus 13%; P = 0.004) and nucleoside reverse transcriptase inhibitors (63% versus 44%; P = NS). Susceptibility to each drug in resistant viruses was predicted. The rate of non-B infections increased in the last years, mainly caused by recombinant viruses. Conclusions: The increasing resistance prevalence among the HIV-infected pediatric population in Spain highlights the importance of specific drug resistance and drug susceptibility surveillance in long-term pretreated children to optimize treatment regimens.

Drug resistance prevalence in human immunodeficiency virus type 1 infected pediatric populations in Honduras and El Salvador during 1989-2009.

Background: Emergence of viral resistance is a major obstacle for antiretroviral treatment (ART) effectiveness. Human immunodeficiency virus type-1 (HIV-1) variants and drug-resistance mutations were identified in naive and antiretroviral drug-experienced children with virologic failure, in Honduras and El Salvador. Methods: Dried blood spots (DBS) from 80 individuals (54 from Honduras, 26 from El Salvador) infected during their childhood between 1989 and 2009 were collected in 2009. The HIV pol region was amplified and sequenced to identify antiretroviral-resistant mutations according to the 2009 International AIDS Society. The genotypic drug resistance interpretation was performed using the Stanford algorithm. HIV-1 variants were characterized by phylogenetic analysis and subtyping tools. Results: HIV-1 protease and reverse transcription sequences were obtained from DBS specimens in 71 and 66 patients, respectively, of the 80 patients. All children were native Central Americans carrying subtype B, with a mean age of 9 years, most were male (65%), perinatally infected (96%), with moderate/severe AIDS symptoms (70%), and receiving first line ART at the time of sequencing (65%). Diagnostic delay was frequently observed. Infected children from Honduras presented longer ART experience and clinical outcomes, and more frequent severe symptoms. Resistant variants infected 1 of 11 naive children from El Salvador but none of the perinatally infected naive children from Honduras. Resistance was higher among ART-exposed individuals in both countries and similar for protease inhibitors (16%), nucleoside reverse transcription inhibitors (44%–52%), and nonnucleoside reverse-transcription inhibitors (66.7%). One in 10 pretreated children in each country was infected with resistant viruses to the 3 drug families. Conclusions: Our data support the need for continued surveillance of resistance patterns using DBS at national levels among naive and pretreated children to optimize the ART regimens.

Dried Blood as an Alternative to Plasma or Serum for Trypanosoma cruzi IgG Detection in Screening Programs

ABSTRACT Trypanosoma cruzi serological screening is recommended for people potentially exposed to this parasite in countries where Trypanosoma cruzi is endemic and those where it is not endemic. Blood samples on filter paper may be a practical alternative to plasma/serum for antibody detection. Using the Architect Chagas assay, we detected the presence of IgG against T. cruzi in matched serum and dried blood spots (DBS) collected from 147 patients residing in Madrid, Spain, who had potential previous exposure to T. cruzi. The κ statistic for the DBS/serum proportion of agreement for the detection of antibodies against T. cruzi was 0.803, considering an S/CO (assay result unit; chemiluminescent signal from the sample [S] divided by the mean chemiluminescent signal for the three calibrators used in the test [CO]) cutoff value of ≥1.00. The relative sensitivity of the Architect test using DBS increased from 95.2% to 98.8% when the cutoff was lowered from ≥1.00 to ≥0.88, while the relative specificity decreased from 84.1% to 71.6%. Overall, the median S/CO values for DBS were significantly lower than those for serum (2.6 versus 6.5; P < 0.001). Discrepancies that occurred with the use of DBS included 10 false positives (with low S/CO values in 9 cases [median, 2.13]) and 4 false negatives, with mean S/CO values of 0.905 (gray zone). Using DBS plus a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) may be a simple and reliable method for detecting IgG against T. cruzi when blood sampling by venipuncture is not feasible. This method may also reduce the false-negative rates observed with some rapid diagnostic tests. The lower relative sensitivity compared to the reference method may be increased by lowering the optical density threshold.

Transmitted drug-resistance in human immunodeficiency virus-infected adult population in El Salvador, Central America

El Salvador harbours one of the largest Central American human immunodeficiency virus (HIV) epidemics, but few studies have analysed it in depth. Here, we describe the presence of transmitted drug resistance (TDR) and HIV variants in the HIV-infected adult population in El Salvador. Dried blood spots from 119 HIV-infected antiretroviral-naive adults attended in El Salvador were collected in 2011. The TDR was assessed according to the list recommended by the WHO. HIV-1 variants were described using phylogeny. Pol sequences could be amplified in 88 patients (50.6% men), with a mean age of 35 years. Almost all (96.7%) were infected with HIV through sexual practice and 58.7% were recently diagnosed. The mean CD4(+) count was 474 cells/mm(3) and 43.1% and 15.5% of patients showed moderate (<500 CD4 cells) or severe (<200) immune suppression, respectively. HIV-1 viral load was >100 000 copies/mL in 24.7% of patients and <2000 copies/mL in 9.1%. Five samples (5.7%) harboured any TDR mutation: 2.3% for nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI), and 1.4% for protease inhibitor (PI). All showed only one TDR single-class resistance mutation: M184I (two cases) for NRTI, K101E and K103N for NNRTI and L23I for PI. All viruses excepting one (URF_BG) belonged to subtype B. No phylogenetic TDR networks were found. In conclusion, we report a TDR prevalence of 5.7% in El Salvador, lower than in other Central American studies. Periodical studies are essential to monitor and prevent TDR emergence in low-income and middle-income regions. Also, more efforts are needed to promote early diagnosis and prevention of infection in El Salvador.

Randomized prospective study evaluating tenofovir disoproxil fumarate prophylaxis against hepatitis B virus reactivation in anti-HBc-positive patients with rituximab-based regimens to treat hematologic malignancies: The Preblin study

Background Hepatitis B virus (HBV) reactivation in patients with resolved HBV infection (HBsAg negative, antiHBc positive) is uncommon, but potentially fatal. The role of HBV prophylaxis in this setting is uncertain. The aim of this study was to compare the efficacy of tenofovir disoproxil fumarate (TDF) prophylaxis versus close monitoring in antiHBc-positive, HBsAg-negative patients under treatment with rituximab (RTX)-based regimens for hematologic malignancy. Methods PREBLIN is a phase IV, randomized, prospective, open-label, multicenter, parallel-group trial conducted in 17 hospitals throughout Spain. Anti-HBc-positive, HBsAg-negative patients with undetectable HBV DNA were randomized to receive TDF 300 mg once daily (Group I) or observation (Group II). The primary endpoint was the percentage of patients showing HBV reactivation during 18 months following initiation of RTX treatment. Patients with detectable HBV DNA (Group III) received the same dose of TDF and were analyzed together with Group I to investigate TDF safety. Results Sixty-one patients were enrolled in the study, 33 in the TDF treatment group and 28 in the observation group. By ITT analysis, HBV reactivation was 0% (0/33) in the study group and 10.7% (3/28) in the observation group (p = 0.091). None of the patients in either group showed significant differences in liver function parameters between baseline and the last follow-up sample. TDF was generally well tolerated and there were no severe treatment-related adverse events. Conclusion In patients with hematological malignancy and resolved hepatitis B infection receiving RTX-based regimens, HBV reactivation did not occur in patients given TDF prophylaxis.

HIV-1 variability and viral load technique could lead to false positive HIV-1 detection and to erroneous viral quantification in infected specimens

OBJECTIVES Viral load (VL) testing is used for early HIV diagnosis in infants (EID) and for detecting early therapeutic failure events, but can be affected by HIV genetic variability. Dried blood samples (DBS) increase VL access and EID in remote settings and when low blood volume is available. METHODS This study compares VL values using Siemens VERSANT HIV-1 RNA 1.0 kPCR assay (kPCR) and Roche CAP/CTM Quantitative test v2.0 (CAP/CTM v2.0) in 176 DBS carrying different HIV-1 variants collected from 69 Equatoguinean mothers and their infants with known HIV-1 status (71 infected, 105 uninfected). RESULTS CAP/CTM v2.0 provided false positive VLs in 11 (10.5%) cases. VL differences above 0.5 log10 were observed in 42/49 (87.5%) DBS, and were above 1 log10 in 18 cases. CAP/CTM v2.0 quantified all the 41 specimens with previously inferred HIV-1 variant by phylogenetic analysis (68.3% recombinants) whereas kPCR only identified 90.2% of them, and was unable to detect 14.3% of 21 CRF02_AG viruses. CAP/CTM v2.0 showed higher sensitivity than kPCR (95.8% vs. 70.1%), quantifying a higher rate of viruses in infected DBS from subjects under antiretroviral exposure at sampling time compared to kPCR (94.7% vs. 96.2%, p-value<0.001). kPCR showed maximum specificity (100%) whereas for CAP/CTM v2.0 was 89.5%. CONCLUSIONS VL assays should increase their sensitivity and specificity to avoid overestimated HIV-1 quantifications, which could be interpreted as virological failure events, or false negative diagnostic results due to genetic variability. We recommend using the same VL technique for each patient during antiretroviral therapy monitoring.

HIV-1 RNA Quantification fromDried Blood Spots and PlasmaUsing the Siemens VERSANT HIV-1 RNA 1.0 Assay (kPCR)

HIV-1 RNA Quantification from Dried Blood Spots and Plasma Using the Siemens VERSANT® HIV-1 RNA 1.0 Assay (kPCR) Objective: Dried blood specimens (DBS) use simplifies the sample collection for viral load (VL) testing in some settings. We compared the VL quantification using DBS and the plasma using the Siemens VERSANT® HIV-1 RNA 1.0 (kPCR) Assay. Methods: Paired DBS/plasma samples were prepared from 62 HIV-1 infected patients harboring different HIV-1 subtypes and recombinants and HIV-1 RNA was quantified by kPCR in 62 paired specimens. DBS and plasma VL results were compared. Viraemia values in DBS were corrected for plasma volume differences assuming the hematocrit percentage in each patient. Results: The results showed a good correlation between corrected VL values in DBS and the results obtained in plasma. The intraclass correlation coefficient was 82.3%. The detection rate in DBS was 83.9%, while the sensitivity of the kPCR for DBS was 92.9%. Overall VL in DBS was, on average, 0.8 log (SD = 0.58) lower than in plasma. We observed overestimated VL in DBS specimens with less than 1,000 copies/ml in plasma. Conclusion: DBS can be used for HIV-1 quantification using kPCR and can be useful for the early therapeutic failure detection in treated subjects. However, VL in DBS can be overestimated in specimens with low VL in plasma, maybe due to a higher effect of proviral DNA in quantification. More studies including more HIV-1 variants are needed to define the kPCR performance.

Evaluation of four commercial virological assays for early infant HIV-1 diagnosis using dried blood specimens

Background:Early infant diagnosis (EID) of HIV-1 is necessary to reduce HIV-related mortality. As maternal antibodies transferred across the placenta may persist up to 18 mo, commercial virological assays (CVAs) are needed. This study compares four CVAs for EID using dried blood specimens (DBS) from HIV-1-exposed infants.Methods:DBS from 68 infants born to HIV-1-infected women were collected from November 2012 to December 2013 in Equatorial Guinea. Four CVAs were performed: Siemens VERSANT HIV-1 RNA 1.0 kPCR assay, Roche CAP/CTM Quantitative Test v2.0, CAP/CTM Qualitative Tests v1.0 and v2.0. Definitive diagnosis was established following World Health Organization (WHO) recommendations.Results:Two HIV-1-infected infants (2.9%) were detected by the four CVAs while 49 (72%) resulted negative. Discordant results were observed in 17 (25%) infants and HIV-1 infection was excluded in 14 patients when virological and serological testing was performed in additional DBS. Different false-positive rates HIV-1 were observed with Roche assays.Conclusion:CVAs using DBS were useful for EID, although discrepant results were common. Further research is required to reduce false-positive results that could result in wrong diagnosis and unneeded treatment. We propose caution with low viral load (VL) values when using VL assays. Clear guidelines are required for EID of HIV-exposed infants with discrepant virological results.

HIV-1 Variants and Drug Resistance in Pregnant Women from Bata (Equatorial Guinea): 2012-2013.

Objectives This is the first study describing drug resistance mutations (DRM) and HIV-1 variants among infected pregnant women in Equatorial Guinea (GQ), a country with high (6.2%) and increasing HIV prevalence. Methods Dried blood spots (DBS) were collected from November 2012 to December 2013 from 69 HIV-1 infected women participating in a prevention of mother-to-child transmission program in the Hospital Regional of Bata and Primary Health Care Centre María Rafols, Bata, GQ. The transmitted (TDR) or acquired (ADR) antiretroviral drug resistance mutations at partial pol sequence among naive or antiretroviral therapy (ART)-exposed women were defined following WHO or IAS USA 2015 lists, respectively. HIV-1 variants were identified by phylogenetic analyses. Results A total of 38 of 69 HIV-1 specimens were successfully amplified and sequenced. Thirty (79%) belonged to ART-experienced women: 15 exposed to nucleoside reverse transcriptase inhibitors (NRTI) monotherapy, and 15 to combined ART (cART) as first regimen including two NRTI and one non-NRTI (NNRTI) or one protease inhibitor (PI). The TDR rate was only found for PI (3.4%). The ADR rate was 37.5% for NNRTI, 8.7% for NRTI and absent for PI or NRTI+NNRTI. HIV-1 group M non-B variants caused most (97.4%) infections, mainly (78.9%) recombinants: CRF02_AG (55.2%), CRF22_A101 (10.5%), subtype C (10.5%), unique recombinants (5.3%), and A3, D, F2, G, CRF06_cpx and CRF11_cpx (2.6% each). Conclusions The high rate of ADR to retrotranscriptase inhibitors (mainly to NNRTIs) observed among pretreated pregnant women reinforces the importance of systematic DRM monitoring in GQ to reduce HIV-1 resistance transmission and to optimize first and second-line ART regimens when DRM are present.