Patricia Álvarez

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.

Additional value of screening for minor genes and copy number variants in hypertrophic cardiomyopathy

Introduction Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited heart disease. Next-generation sequencing (NGS) is the preferred genetic test, but the diagnostic value of screening for minor and candidate genes, and the role of copy number variants (CNVs) deserves further evaluation. Methods Three hundred and eighty-seven consecutive unrelated patients with HCM were screened for genetic variants in the 5 most frequent genes (MYBPC3, MYH7, TNNT2, TNNI3 and TPM1) using Sanger sequencing (N = 84) or NGS (N = 303). In the NGS cohort we analyzed 20 additional minor or candidate genes, and applied a proprietary bioinformatics algorithm for detecting CNVs. Additionally, the rate and classification of TTN variants in HCM were compared with 427 patients without structural heart disease. Results The percentage of patients with pathogenic/likely pathogenic (P/LP) variants in the main genes was 33.3%, without significant differences between the Sanger sequencing and NGS cohorts. The screening for 20 additional genes revealed LP variants in ACTC1, MYL2, MYL3, TNNC1, GLA and PRKAG2 in 12 patients. This approach resulted in more inconclusive tests (36.0% vs. 9.6%, p<0.001), mostly due to variants of unknown significance (VUS) in TTN. The detection rate of rare variants in TTN was not significantly different to that found in the group of patients without structural heart disease. In the NGS cohort, 4 patients (1.3%) had pathogenic CNVs: 2 deletions in MYBPC3 and 2 deletions involving the complete coding region of PLN. Conclusions A small percentage of HCM cases without point mutations in the 5 main genes are explained by P/LP variants in minor or candidate genes and CNVs. Screening for variants in TTN in HCM patients drastically increases the number of inconclusive tests, and shows a rate of VUS that is similar to patients without structural heart disease, suggesting that this gene should not be analyzed for clinical purposes in HCM.

HIV-1 infection using dried blood spots can be confirmed by Bio-Rad Geenius™ HIV 1/2 confirmatory assay

BACKGROUND Confirmatory assays for HIV diagnosis are not well implemented in low-income countries with limited infrastructures. Geenius™ HIV 1/2 Confirmatory Assay is a single-use immunochromatographic test for the confirmation and differentiation of individual HIV-1/2 antibodies validated in venous whole blood, serum and plasma. However, dried blood specimens (DBS) are easier to collect, store and transport than plasma/serum in remote settings from limited resource countries and mobile populations. OBJECTIVES To evaluate the confirmatory assay Geenius™ HIV 1/2 for HIV diagnosis using DBS specimens. STUDY DESIGN We collected DBS from 70 Guinean women previously diagnosed as HIV-1 infected by rapid tests using whole blood samples in Equatorial Guinea and from 25 HIV-negative Guinean women and HIV-exposed infants diagnosed by molecular testing in Madrid. Geenius HIV 1/2 was performed by eluting two drops of dried blood from each patient and following the manufacturer instructions for the assay but using 40μl of the eluted blood as specimen. The results obtained were confirmed by western blot. RESULTS Geenius™ HIV 1/2 successfully confirmed the HIV-1 positive and negative infection in all tested DBS specimens, providing 100% specificity [95% Confidence Interval (CI): 86.2%-100%]. No HIV 1/2 coinfections were found in the study cohort. This is the first report that proves a good performance of Geenius™ HIV 1/2 for the HIV-1 infection confirmation using only two drops of dried blood. CONCLUSIONS Our results approve the utility of this confirmatory assay using DBS when a lack of adequate infrastructure to collect, store or transport plasma/serum is found. DBS are a practical alternative to plasma/serum for HIV serological diagnosis.

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.

Efectividad de la mupirocina frente a Staphylococcus aureus resistente a meticilina aislados en la provincia de Pontevedra

La mupirocina es un antibiótico tópico bacteriostático que presenta una excelente actividad frente a estafilococos, incluyendo Staphylococcus aureus meticilina-resistente (SARM). Recientemente, Pérez-Roth et al han comunicado la diseminación del clon internacional SARM ST36-II en Tenerife y la identificación de aislamientos pertenecientes a dicho clon con alta resistencia a la mupirocina. En un estudio que realizamos entre 1997 y 2005 en 2 hospitales de Vigo, se encontró que el 38,1% de los SARM aislados pertenecı́an al clon ST36-II, también denominado Británico-16. Por ello, se consideró importante determinar el grado de diseminación de dicho clon en la provincia de Pontevedra y la actividad de la mupirocina frente a éste. Se estudiaron 73 SARM aislados en 2004 y 2005 en 2 hospitales de Pontevedra y en un centro de especialidades de Vigo. Se aislaron 20 SARM en muestras procedentes de atención primaria y 53 muestras de los siguientes servicios: 46% medicina interna, 14% cirugı́a general, 10% urgencias, 10% urologı́a, 8% nefrologı́a, 6% uci y 8% traumatologı́a. Las muestras donde se aislaron SARM con mayor frecuencia fueron exudados de heridas (53,8%) y secreciones respiratorias (19,6%). Se determinó la sensibilidad a la oxacilina y la cefoxitina mediante el método de difusión disco-placa. La resistencia a la meticilina se confirmó mediante la amplificación del gen mecA. Para clasificar los aislamientos de SARM en distintos tipos clonales se empleó la PCR del gen de la coagulasa y digestión con CfoI (PCR-RFLP coa), comparando el patrón de bandas con el generado por los clones identificados en un estudio previo. La resistencia a la mupirocina se detectó mediante el método difusión disco-placa. En los aislamientos resistentes se determinó la CMI mediante dilución en agar, identificándose el gen mupA mediante PCR. Los SARM con resistencia de alto nivel a la mupirocina se estudiaron mediante la secuenciación de la región X del gen spa y MLST. Asimismo, se determinó el tipo de casete cromosómico estafilocócico (CCS)mec. El 60,3 (44 SARM), el 13,7 (10 SARM) y el 12,3% (9 SARM) de los aislamientos poseı́an el mismo patrón electroforético mediante PCR-RFLP coa que los clones internacionales ST36, ST5 y ST125, respectivamente (fig. 1). El resto de los aislamientos se consideró esporádico (13,7%). Solamente 1 de 73 SARM estudiados (1,3%) presentó alta resistencia a la mupirocina, detectándose en él el gen mupA (fig. 2). Se aisló en orina de un paciente procedente de la consulta de urologı́a que no habı́a recibido mupirocina. Este aislamiento se identificó con el clon epidémico Británico-16 mediante secuenciación del gen spaA (t018) y MLST (ST36). El CCSmec fue del tipo II. La diseminación de SARM se debe a unos pocos clones pandémicos. La mupirocina juega un papel muy importante en el control de la infección, permitiendo eliminar la colonización estafilocócica mediante aplicación tópica y, de esta manera, evitar la diseminación de SARM. Recientemente, Pérez-Roth et al han descrito la capacidad que tiene el clon pandémico ST36-II para adquirir distintos plásmidos de resistencia a la mupirocina. Este hecho puede convertirse en un problema para las instituciones en las que este clon sea mayoritario, tal como ocurre en Tenerife y en ARTICLE IN PRESS

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.

Diagnóstico precoz de la infección por el virus de la inmunodeficiencia humana-1 en niños: programa de prevención de la transmisión maternoinfantil en Guinea Ecuatorial

BACKGROUND Great efforts have been made in the last few years in order to implement the prevention of mother-to-child transmission (PMTCT) program in Equatorial Guinea (GQ). The aim of this study was to evaluate the rates of mother-to-child HIV transmission based on an HIV early infant diagnosis (EID) program. METHODS A prospective observational study was performed in the Regional Hospital of Bata and Primary Health Care Centre Maria Rafols, Bata, GQ. Epidemiological, clinical, and microbiological characteristics of HIV-1-infected mothers and their exposed infants were recorded. Dried blood spots (DBS) for HIV-1 EID were collected from November 2012 to December 2013. HIV-1 genome was detected using Siemens VERSANT HIV-1 RNA 1.0 kPCR assay. RESULTS Sixty nine pairs of women and infants were included. Sixty women (88.2%) had WHO clinical stage 1. Forty seven women (69.2%) were on antiretroviral treatment during pregnancy. Forty five infants (66.1%) received postnatal antiretroviral prophylaxis. Age at first DBS analysis was 2.4 months (IQR 1.2-4.9). One infant died before a HIV-1 diagnosis could be ruled out. Two infants were HIV-1 infected and started HAART before any symptoms were observed. The rate of HIV-1 transmission observed was 2.9% (95%CI 0.2-10.5). CONCLUSIONS The PMTCT rate was evaluated for the first time in GQ based on EID. EID is the key for early initiation of antiretroviral therapy and to reduce the mortality associated with HIV infection.