Strategies for the successful implementation of viral laboratory automation.

It has been estimated that more than 70% of all medical activity is directly related to information providing analytical data. Substantial technological advances have taken place recently, which have allowed a previously unimagined number of analytical samples to be processed while offering high quality results. Concurrently, yet more new diagnostic determinations have been introduced - all of which has led to a significant increase in the prescription of analytical parameters. This increased workload has placed great pressure on the laboratory with respect to health costs. The present manager of the Clinical Laboratory (CL) has had to examine cost control as well as rationing - meaning that the CL's focus has not been strictly metrological, as if it were purely a system producing results, but instead has had to concentrate on its efficiency and efficacy. By applying re-engineering criteria, an emphasis has had to be placed on improved organisation and operating practice within the CL, focussing on the current criteria of the Integrated Management Areas where the technical and human resources are brought together. This re-engineering has been based on the concepts of consolidating and integrating the analytical platforms, while differentiating the production areas (CORE Laboratory) from the information areas. With these present concepts in mind, automation and virological treatment, along with serology in general, follow the same criteria as the rest of the operating methodology in the Clinical Laboratory.

Clinical and Virological Efficacy of Etravirine Plus Two Active Nucleos(t)ide Analogs in an Heterogeneous HIV-Infected Population.

Etravirine (ETV) is recommended in combination with a boosted protease inhibitor plus an optimized background regimen for salvage therapy, but there is limited experience with its use in combination with two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs). This multicenter study aimed to assess the efficacy of this combination in two scenarios: group A) subjects without virologic failure on or no experience with non-nucleoside reverse-transcriptase inhibitors (NNRTIs) switched due to adverse events and group B) subjects switched after a virologic failure on an efavirenz- or nevirapine-based regimen. The primary endpoint was efficacy at 52 weeks analysed by intention-to-treat. Virologic failure was defined as the inability to suppress plasma HIV-RNA to <50 copies/mL after 24 weeks on treatment, or a confirmed viral load >200 copies/mL in patients who had previously achieved a viral suppression or had an undetectable viral load at inclusion. Two hundred eighty seven patients were included. Treatment efficacy rates in group A and B were 88.0% (CI95, 83.9-92.1%) and 77.4% (CI95, 65.0-89.7%), respectively; the rates reached 97.2% (CI95, 95.1-99.3%) and 90.5% (CI95, 81.7-99.3), by on-treatment analysis. The once-a-day ETV treatment was as effective as the twice daily dosing regimen. Grade 1-2 adverse events were observed motivating a treatment switch in 4.2% of the subjects. In conclusion, ETV (once- or twice daily) plus two analogs is a suitable, well-tolerated combination both as a switching strategy and after failure with first generation NNRTIs, ensuring full drug activity. TRIAL REGISTRATION ClinicalTrials.gov NCT01437241.

Infección por virus pandémico (H1N1) 2009 en Andalucía

In April 2009, in response to the WHO's alert due to the existence of human infection cases with a new AH1N1 influenza virus, known as swine flu, Andalusian Health Authorities trigger an specific action plan. The surveillance actions developped provided us with appropriate clinical, epidemiological and virological characteristics of the disease. During the first few days, contingency plans were set up based on epidemiological surveillance and outbreak control measures were adopted through early alert and rapid response systems. After phase 6 was declared, influenza sentinel and severe cases surveillance were used in order to plan healthcare services, to reduce transmission and to identify and protect the most vulnerable population groups. Behaviour of pandemic influenza in Andalusia was similar to that observed in the rest of the world. Atack rate was similar to a seasonal flu and the peak was reached at the 46th/2009 week. Most of them were mild cases and affected particularly to young people. The average age of hospitalised patients was 32. Prior pulmonary disease, smoking and morbid obesity (BMI>40) were the most common pathologies and risk factors in severe cases. An impact scenario of pandemic wave in Andalusia, with an expected attack rate from 2 to 5%, was prepared considering watt observed in the southern hemisphere. Characteristics of the epidemic concerning its extent, severity and mortality rate were adjusted to this scenario.

Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

Effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children: 3 years of follow-up. Long-term response to nelfinavir in children.

BACKGROUND Antiretroviral treatment (ART) in children has special features and consequently, results obtained from clinical trials with antiretroviral drugs in adults may not be representative of children. Nelfinavir (NFV) is an HIV-1 Protease Inhibitor (PI) which has become as one of the first choices of PI for ART in children. We studied during a 3-year follow-up period the effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children. METHODS Forty-two vertically HIV-infected children on HAART with NFV were involved in a multicentre prospective study. The children were monitored at least every 3 months with physical examinations, and blood sample collection to measure viral load (VL) and CD4+ cell count. We performed a logistic regression analysis to determinate the odds ratio of baseline characteristics on therapeutic failure. RESULTS Very important increase in CD4+ was observed and VL decreased quickly and it remained low during the follow-up study. Children with CD4+ <25% at baseline achieved CD4+ >25% at 9 months of follow-up. HIV-infected children who achieved undetectable viral load (uVL) were less than 40% in each visit during follow-up. Nevertheless, HIV-infected children with VL >5000 copies/ml were less than 50% during the follow-up study. Only baseline VL was an important factor to predict VL control during follow-up. Virological failure at defined end-point was confirmed in 30/42 patients. Along the whole of follow-up, 16/42 children stopped HAART with NFV. Baseline characteristics were not associated with therapeutic change. CONCLUSION NFV is a safe drug with a good profile and able to achieve an adequate response in children.

Efficacy, safety and pharmacokinetic of once-daily boosted saquinavir (1500/100 mg) together with 2 nucleos(t)ide reverse transcriptase inhibitors in real life: a multicentre prospective study.

BACKGROUND. Ritonavir-boosted saquinavir (SQVr) is nowadays regarded as an alternative antiretroviral drug probably due to several drawbacks, such as its high pill burden, twice daily dosing and the requirement of 200 mg ritonavir when given at the current standard 1000/100 mg bid dosing. Several once-daily SQVr dosing schemes have been studied with the 200 mg SQV old formulations, trying to overcome some of these disadvantages. SQV 500 mg strength tablets became available at the end of 2005, thus facilitating a once-daily regimen with fewer pills, although there is very limited experience with this formulation yet. METHODS. Prospective, multicentre study in which efficacy, safety and pharmacokinetics of a regimen of once-daily SQVr 1500/100 mg plus 2 NRTIs were evaluated under routine clinical care conditions in either antiretroviral-naïve patients or in those with no previous history of antiretroviral treatments and/or genotypic resistance tests suggesting SQV resistance. Plasma SQV trough levels were measured by HPLV-UV. RESULTS. Five hundred and fourteen caucasian patients were included (47.2% coinfected with hepatitis C and/or B virus; 7.8% with cirrhosis). Efficacy at 52 weeks (plasma RNA-HIV <50 copies/ml) was 67.7% (CI95: 63.6 - 71.7%) by intention-to-treat, and 92.2% (CI95: 89.8 - 94.6%) by on-treatment analysis. The reasons for failure were: dropout or loss to follow-up (18.4%), virological failure (7.8%), adverse events (3.1%), and other reasons (4.6%). The high rate of dropout may be explained by an enrollment and follow-up under routine clinical care condition, and a population with a significant number of drug users. The median SQV Cmin (n = 49) was 295 ng/ml (range, 53-2172). The only variable associated with virological failure in the multivariate analysis was adherence (OR: 3.36; CI95, 1.51-7.46, p = 0.003). CONCLUSIONS. Our results suggests that SQVr (1500/100 mg) once-daily plus 2 NRTIs is an effective regimen, without severe clinical adverse events or hepatotoxicity, scarce lipid changes, and no interactions with methadone. All these factors and its once-daily administration suggest this regimen as an appropriate option in patients with no SQV resistance-associated mutations.

Influence of IL28B polymorphisms on response to a lower-than-standard dose peg-IFN-α 2a for genotype 3 chronic hepatitis C in HIV-coinfected patients.

BACKGROUND Data on which to base definitive recommendations on the doses and duration of therapy for genotype 3 HCV/HIV-coinfected patients are scarce. We evaluated the efficacy of a lower peginterferon-α 2a dose and a shorter duration of therapy than the current standard of care in genotype 3 HCV/HIV-coinfected patients. METHODS AND FINDINGS Pilot, open-label, single arm clinical trial which involved 58 Caucasian HCV/HIV-coinfected patients who received weekly 135 µg peginterferon-α 2a plus ribavirin 400 mg twice daily during 20 weeks after attaining undetectable viremia. The relationships between baseline patient-related variables, including IL28B genotype, plasma HCV-RNA, ribavirin dose/kg, peginterferon-α 2a and ribavirin levels with virological responses were analyzed. Only 4 patients showed lack of response and 5 patients dropped out due to adverse events related to the study medication. Overall, sustained virologic response (SVR) rates were 58.3% by intention-to-treat and 71.4% by per protocol analysis, respectively. Among patients with rapid virologic response (RVR), SVR and relapses rates were 92.6% and 7.4%, respectively. No relationships were observed between viral responses and ribavirin dose/kg, peginterferon-α 2a concentrations, ribavirin levels or rs129679860 genotype. CONCLUSIONS Weekly 135 µg pegIFN-α 2a could be as effective as the standard 180 µg dose, with a very low incidence of severe adverse events. A 24-week treatment duration appears to be appropriate in patients achieving RVR, but extending treatment up to just 20 weeks beyond negativization of viremia is associated with a high relapse rate in those patients not achieving RVR. There was no influence of IL28B genotype on the virological responses.

Laboratory detection of respiratory viruses by automated techniques.

Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection.

Long-term assessment of didanosine, lamivudine, and efavirenz in antiretroviral-naive patients: 3-year follow-up.

The aim of this study was to evaluate the long-term efficacy and safety of didadosine (ddI), lamivudine (3TC), and efavirenz (EFV). This was a follow-up to the VESD study, a 12-month open-label, observational, multicenter study of adult patients with HIV infection who started antiretroviral treatment with the ddI-3TC-EFV once-daily regimen. Of the 167 patients originally included, 106 patients remained on the same triple therapy at the end of the study (1 year), and they were offered an extra 24 months of follow-up; 96 were enrolled in this study (VESD-2). Seventy patients out of the initial cohort were still on the same regimen at month 36, with 97% of them with plasma viral load <50 copies /ml. An intention-to-treat analysis showed that the percentage of patients with plasma viral load <50 copies/ml was 73% at 36 months. CD4 cell counts increased 344 cells/microl over the 36 months. Safety and tolerance were good with no unexpected long-term toxicity. After 3 years of treatment with ddI-3TC-EFV, more than 40% of the patients were still receiving the initial antiretroviral therapy with sustained, durable immunovirological benefit and good acceptance. Long-term toxicity and virological failure were low.

Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Syphilis in HIV-infected patients: Predictors for serological failure and serofast state.

Purpose: HIV-infected patients treated for syphilis may be at increased risk for serological failure and serofast state. Our aim was to analyse serological response to treatment in HIV-infected patients diagnosed with syphilis, and factors associated with serological cure and serofast state. Methods: Open-label, no controlled study of a series of HIV- patients diagnosed with syphilis during 2004-2011. Patients were categorized by rapid plasma reagin titer (RPR) into success (4-fold decrease in RPR by 12 or 24 months after treatment of early or late syphilis), serofast (success with persistently stable reactive RPR), and failure/ re-infection ( failure to decrease 4-fold in RPR by 12 or 24 months after treatment or sustained 4-fold increase in RPR after treatment response). Results: 141 HIV- patients were diagnosed with syphilis during the study period (104 early syphilis, 36 late or indeterminate latent syphilis). The mean age was 36.3 years, 98.5% were male, and 87.2% homosexual men. In 46 (32.6%) cases, HIV and syphilis infection diagnosis were coincident (mean CD4 457/mm3 and HIV-VL 4.72 log10). Among patients with prior known HIV infection, 65 were on antiretroviral therapy (ART) at syphilis diagnosis (mean CD4 469/ mm3, 76.9% undetectable HIV-VL). 116 patients satisfied criteria for serological response analysis (89 early, 24 late/indeterminate). At 12 months of early syphilis treatment (89.2% penicillin) there were 16 (18%) failures, and at 24 months of late/indeterminate syphilis (91.7% penicillin) there were 5 (18.5%) failures. Overall, 36 (31.0%) patients presented serofast state. Treatment failure was related with lower CD4 count (295 vs 510/μL; p=0.045) only in patients with coincident diagnosis. Serofast state was related with older age (41 vs 36 years; p=0.024), and lower CD4 count (391 vs 513/mm3; p=0.026). Conclusions: In this series of HIV-infected patients, with many patients on ART and with good immunological and virological parameters, serological failure and serofast state were frequent. Immunological status, and age could influence on serological response to syphilis treatment in HIV-infected patients.

Evaluation of three automated systems for susceptibility testing of enterobacteria containing qnrB, qnrS, and/or aac(6')-Ib-cr.

The accuracy of the MicroScan WalkAway, BD Phoenix, and Vitek-2 systems for susceptibility testing of quinolones and aminoglycosides against 68 enterobacteria containing qnrB, qnrS, and/or aac(6 ')-Ib-cr was evaluated using reference microdilution. Overall, one very major error (0.09%), 6 major errors (0.52%), and 45 minor errors (3.89%) were noted.