Effective Rapid Diagnosis of Bacterial and Fungal Bloodstream Infections by T2 Magnetic Resonance Technology in the Pediatric Population.

Children are prone to bloodstream infections (BSIs), the rapid and accurate diagnosis of which is an unmet clinical need. The T2MR technology is a direct molecular assay for identification of BSI pathogens, which can help to overcome the limits of blood culture (BC) such as diagnostic accuracy, blood volumes required, and turnaround time. We analyzed results obtained with the T2Bacteria (648) and T2Candida (106) panels in pediatric patients of the Bambino Gesù Children's Hospital between May 2018 and September 2020 in order to evaluate the performance of the T2Dx instrument with respect to BC. T2Bacteria and T2Candida panels showed 84.2% and 100% sensitivity with 85.9% and 94.1% specificity, respectively. The sensitivity and specificity of the T2Bacteria panel increased to 94.9% and 98.7%, respectively, when BC was negative but other laboratory data supported the molecular result. T2Bacteria sensitivity was 100% with blood volumes <2 mL in neonates and infants. T2Bacteria and T2Candida provided definitive microorganism identification in a mean time of 4.4 and 3.7 h, respectively, versus 65.7 and 125.5 h for BCs (P < 0.001). T2 panels rapidly and accurately enable a diagnosis of a pediatric BSI, even in children under 1 year of age and for very small blood volumes. These findings support their clinical use in life-threatening pediatric infections, where the time to diagnosis is of utmost importance, in order to improve survival and minimize the long-term sequalae of sepsis. The T2 technology could be further developed to include more bacteria and fungi species that are involved in the etiology of sepsis.

SARS-CoV-2 RNA in plasma samples of COVID-19 affected individuals: a cross-sectional proof-of-concept study.

BACKGROUND: Recent studies showed that plasma SARS-CoV-2 RNA seems to be associated with worse COVID-19 outcome. However, whether specific population can be at higher risk of viremia are to date unexplored. METHODS: This cross-sectional proof-of-concept study included 41 SARS-CoV-2-positive adult individuals (six affected by haematological malignancies) hospitalized at two major hospital in Milan, for those demographic, clinical and laboratory data were available. SARS-CoV-2 load was quantified by ddPCR in paired plasma and respiratory samples. To assess significant differences between patients with and patients without viremia, Fisher exact test and Wilcoxon test were used for categorical and continuous variables, respectively. RESULTS: Plasma SARS-CoV-2 RNA was found in 8 patients (19.5%), with a median (IQR) value of 694 (209-1023) copies/mL. Viremic patients were characterized by an higher mortality rate (50.0% vs 9.1%; p = 0.018) respect to patients without viremia. Viremic patients were more frequently affected by haematological malignancies (62.5% vs. 3.0%; p < 0.001), and had higher viral load in respiratory samples (9,404,000 [586,060-10,000,000] vs 1560 [312-25,160] copies/mL; p = 0.002). CONCLUSIONS: Even if based on a small sample population, this proof-of-concept study poses the basis for an early identification of patients at higher risk of SARS-CoV-2 viremia, and therefore likely to develop severe COVID-19, and supports the need of a quantitative viral load determination in blood and respiratory samples of haematologic patients with COVID-19 in order to predict prognosis and consequently to help their further management.

Viral Respiratory Infections: New Tools for a Rapid Diagnosis.

Respiratory tract infection is one of the most common diseases in human worldwide. Many viruses are implicated in these infections, including emerging viruses, such as the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Identification of the causative viral pathogens of respiratory tract infections is important to select a correct management of patients, choose an appropriate treatment, and avoid unnecessary antibiotics use. Different diagnostic approaches present variable performance in terms of accuracy, sensitivity, specificity, and time-to-result, that have to be acknowledged to be able to choose the right diagnostic test at the right time, in the right patient. This review describes currently available rapid diagnostic strategies and syndromic approaches for the detection of viruses commonly responsible for respiratory diseases.

Sex differences in electrolyte imbalances caused by SARS-CoV-2: A cross-sectional study.

BACKGROUND: Since SARS-CoV-2 spread, evidence regarding sex differences in progression and prognosis of COVID-19 have emerged. Besides this, studies on patients' clinical characteristics have described electrolyte imbalances as one of the recurrent features of COVID-19. METHODS: We performed a cross-sectional study on all patients admitted to the emergency department (ED) from 1 March to 31 May 2020 who had undergone a blood gas analysis and a nasopharyngeal swab test for SARS-CoV-2 by rtPCR. We defined positive patients as cases (n = 710) and negatives as controls (n = 619), for a total number of patients of 1.329. The study was approved by the local ethics committee Area 3 Milan. Data were automatically extracted from the hospital laboratory SQL-based repository in anonymised form. We considered as outcomes potassium (K+ ), sodium (Na+ ), chlorine (Cl- ) and calcium (Ca++ ) as continuous and as categorical variables, in their relation with age, sex and SARS-CoV-2 infection status. RESULTS: We observed a higher prevalence of hypokalaemia among patients positive for SARS-CoV-2 (13.7% vs 6% of negative subjects). Positive patients had a higher probability to be admitted to the ED with hypokalaemia (OR 2.75, 95% CI 1.8-4.1, P < .0001) and women were twice as likely to be affected than men (OR 2.43, 95% CI 1.67-3.54, P < .001). Odds ratios for positive patients to manifest with an alteration in serum Na+ was (OR 1.6, 95% CI 1.17-2.35, P < .001) and serum chlorine (OR 1.6, 95% CI 1.03-2.69, P < .001). Notably, OR for positive patients to be hypocalcaemic was 7.2 (95% CI 4.8-10.6, P < .0001) with a low probability for women to be hypocalcaemic (OR 0.63, 95% CI 0.4-0.8, P = .005). CONCLUSIONS: SARS-CoV-2 infection is associated with a higher prevalence of hypokalaemia, hypocalcaemia, hypochloraemia and sodium alterations. Hypokalaemia is more frequent among women and hypocalcaemia among men.

Quantification of 1,3-ß-d-glucan by Wako ß-glucan assay for rapid exclusion of invasive fungal infections in critical patients: A diagnostic test accuracy study.

OBJECTIVES: Rapid and reliable exclusion of invasive fungal infections (IFI) by markers able to avoid unnecessary empirical antifungal treatment is still a critical unmet clinical need. We investigated the diagnostic performance of a newly available ß-d-Glucan (BDG) quantification assay, focusing on the optimisation of the BDG cut-off values for IFI exclusion. METHODS: BDG results by Wako ß-glucan assay (lower limit of detection [LLOD] = 2.16 pg/mL, positivity ≥ 11 pg/mL) on two consecutive serum samples were retrospectively analysed in 170 patients, admitted to haematological wards (N = 42), intensive care units (ICUs; N = 80), or other wards (N = 48), exhibiting clinical signs and/or symptoms suspected for IFI. Only patients with proven IFI (EORTC/MSG criteria) were considered as true positives in the assessment of BDG sensitivity, specificity and predictive values. RESULTS: Patients were diagnosed with no IFI (69.4%), proven IFI (25.3%) or probable IFI (5.3%). Two consecutive BDG values < LLOD performed within a median of 1 (interquartile range: 1-3) day were able to exclude a proven IFI with 100% sensitivity and negative predictive value (primary study goal). Test's specificity improved by using two distinct positivity and negativity cut-offs (7.7 pg/mL and LLOD, respectively), but remained suboptimal in ICU patients (50%), as compared to haematological or other patients (93% and 90%, respectively). CONCLUSIONS: The classification of Wako's results as negative when < LLOD, and positive when > 7.7 pg/mL, could be a promising diagnostic approach to confidently rule out an IFI in both ICU and non-ICU patients. The poor specificity in the ICU setting remains a concern, due to the difficulty to interpret positive results in this fragile population.

HCV resistance compartmentalization within tumoral and non-tumoral liver in transplanted patients with hepatocellular carcinoma.

BACKGROUND & AIMS: We investigated the HCV-RNA amount, variability and prevalence of resistance-associated substitutions (RASs), in plasma, hepatic tumoral and non-tumoral tissue samples in patients undergoing liver-transplant/hepatic-resection (LT/HR), because of hepatocellular carcinoma and/or cirrhosis. METHODS: Eighteen HCV-infected patients undergoing LT/HR, 94.0% naïve to direct-acting antivirals (DAAs), were analysed. HCV-RNA was quantified in all compartments. NS3/NS5A/NS5B in plasma and/or in tumoral/non-tumoral tissues were analysed using Sanger and Ultra-deep pyrosequencing (UDPS, 9/18 patients). RASs prevalence, genetic-variability and phylogenetic analysis were evaluated. RESULTS: At the time of LT/HR, HCV-RNA was quantifiable in all compartments of DAA-naïve patients and was generally lower in tumoral than in non-tumoral tissues (median [IQR] = 4.0 [1.2-4.3] vs 4.3[3.1-4.9] LogIU/µg RNA; P = 0.193). The one patient treated with sofosbuvir + ribavirin represented an exception with HCV-RNA quantifiable exclusively in the liver, but with higher level in tumoral than in non-tumoral tissues (51 vs 7 IU/µg RNA). RASs compartmentalization was found by Sanger in 4/18 infected-patients, and by UDPS in other two patients. HCV-compartmentalization resulted to be associated with HBcAb-positivity (P = 0.013). UDPS showed approximately higher genetic-variability in NS3/NS5A sequences in all compartments. Phylogenetic-analysis showed defined and intermixed HCV-clusters among/within all compartments, and were strongly evident in the only non-cirrhotic patient, with plasma and non-tumoral sequences generally more closely related. CONCLUSIONS: Hepatic compartments showed differences in HCV-RNA amount, RASs and genetic variability, with a higher segregation within the tumoral compartment. HBV coinfection influenced the HCV compartmentalization. These results highlight HCV-strain diversifications within the liver, which could explain some of the failures occurring even today in the era of DAAs.

Hepatitis C virus drug resistance associated substitutions and their clinical relevance: Update 2018.

Nowadays, due to the development of potent Direct-Acting Antiviral Agents (DAAs) that specifically target NS3, NS5A and NS5B viral proteins, several new and highly efficacious options to treat chronic Hepatitis C virus (HCV) infection are available. The natural presence of resistance associated substitutions (RASs), as well as their rapid emergence during incomplete drug-pressure, are intrinsic characteristics of HCV that greatly affect treatment outcome and the chances to achieve a virolgical cure. To date, a high number of RASs in NS3, NS5A, and NS5B have been associated in vivo and/or in vitro with reduced susceptibility to DAAs, but no comprehensive RASs list is available. This review thus provides an updated, systematic overview of the role of RASs to currently approved DAAs or in phase II/III of clinical development against HCV-infection, discriminating their impact in different HCV-genotypes and DAAs, providing assistance for a fruitful use of HCV resistance testing in clinical practice.

Italian expert panel consensus statements on two-drug antiretroviral regimens to treat naïve and virologically suppressed HIV-1 infected patients.

New strategies for HIV treatment able to reduce drug-exposure, medium-long term toxicities and costs are a recognized clinical need. The availability of newer drugs with improved potency and tolerability, as well as high genetic barrier to resistance, makes antiretroviral-sparing strategies with two-drug regimens (2DRs) particularly attractive. Substantial evidence has been generated over the last few years supporting 2DR in virologically suppressed HIV infected patients for whom a therapy switch is planned. More recently, very promising data on 2DR in naïve patients have also been reported. The main purpose of this consensus is to provide an overview of guideline indications and recommendations, and the most recent data from clinical studies of 2DR in both naïve and virologically suppressed patients. As an expert consensus, suggestions and indications on the use and management of 2DR are also provided.

HCV very late relapse following an atypical viral kinetics in a HIV patient treated for hepatitis C with direct-acting antivirals.

Direct-acting antivirals (DAAs) for the treatment of HCV have dramatically increased the rate of sustained virological response: patients not achieving sustained virological response represent a challenge and rates of late recurrent viremia are very low. We describe here the first case of a very late HCV relapse, following an atypical kinetics (characterized by a spontaneous but transient HCV clearance after an early virological relapse), in a HIV co-infected patient treated with DAAs. Optimal adherence to the therapy was well documented and a phylogenetic analysis ruled out a possible reinfection from a different HCV strain. In conclusion, our case underlines the importance of a long follow-up (> 48 weeks) after DAAs therapies in HCV-HIV co-infected patients who might benefit the most from a very rigorous virological surveillance.

Implications of hepatitis C virus subtype 1a migration patterns for virus genetic sequencing policies in Italy.

BACKGROUND: In-depth phylogeographic analysis can reveal migration patterns relevant for public health planning. Here, as a model, we focused on the provenance, in the current Italian HCV subtype 1a epidemic, of the NS3 resistance-associated variant (RAV) Q80K, known to interfere with the action of NS3/4A protease inhibitor simeprevir. HCV1a migration patterns were analysed using Bayesian phylodynamic tools, capitalising on newly generated and publicly available time and geo-referenced NS3 encoding virus genetic sequence data. RESULTS: Our results showed that both immigration and local circulation fuel the current Italian HCV1a epidemic. The United States and European continental lineages dominate import into Italy, with the latter taking the lead from the 1970s onwards. Since similar migration patterns were found for Q80K and other lineages, no clear differentiation of the risk for failing simeprevir can be made between patients based on their migration and travel history. Importantly, since HCV only occasionally recombines, these results are readily transferable to the genetic sequencing policy concerning NS5A RAVs. CONCLUSIONS: The patient migration and travel history cannot be used to target only part of the HCV1a infected population for drug resistance testing before start of antiviral therapy. Consequently, it may be cost-effective to expand genotyping efforts to all HCV1a infected patients eligible for simeprevir-based therapies.

Optimal cure rate by personalized HCV regimens in real-life: a proof-of-concept study.

BACKGROUND: Pretreatment evaluation of HCV-infected patients is a complex interplay between multiple clinical and viral parameters, leading to a tailored approach that may bring real-life sustained virological response (SVR) rates close to 98%-99%. OBJECTIVES: As proof-of-concept, we evaluated the efficacy of all-oral direct-acting antiviral (DAA) regimens in patients whose personalization included pre-therapy evaluation of natural resistance-associated substitutions (RASs), in addition to international guideline recommendations. METHODS: One hundred and thirty-one patients who started a first-line all-oral DAA regimen between April 2015 and December 2016 were tested for baseline NS3 and NS5A RASs by Sanger sequencing. SVR12 was defined as HCV-RNA undetectability 12 weeks after treatment discontinuation. RESULTS: Compatibly with a real-life context, 74.0% (97 of 131) of patients presented ≥2 pretreatment risk factors for failure to achieve SVR12 (infection by GT-1a/GT-3a; cirrhosis; previous treatment experience; HCV-RNA ≥800 000 IU/mL) and 33.6% had ≥3 risk factors. Natural RASs were frequently detected (32.1% prevalence), with substantial prevalence of NS5A RASs (15.3%), mostly represented by Y93H in GT-1b (3 of 36, 8.3%) and GT-3a (3 of 25, 12.0%) and F28C in GT-2c (2 of 11, 18.2%). Overall, personalized treatment led to 100% SVR12, even in those patients for whom treatment strategy was either strengthened (by ribavirin inclusion and/or duration increase) or simplified (by ribavirin exclusion and/or duration reduction), thanks to baseline RAS evaluation. CONCLUSIONS: Even with newer DAA regimens, an integrated interpretation of clinical and virological pretreatment parameters, including natural RASs, may play a relevant role in bringing SVR rates close to the highest achievable. Treatment tailoring can be foreseen in 'hard-to-treat' patients, but also in 'easy' patients with favourable indicators, whereby a simplification/shortening of recommended regimens can be indicated.

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies.

BACKGROUND & AIMS: Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures. METHODS: Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70). RESULTS: The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001). Interestingly, 57.1% of DAA IFN-free non-responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major-RAS-S282T, while RAS-L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RASs, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure. CONCLUSIONS: In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring.

May some HCV genotype 1 patients still benefit from dual therapy? The role of very early HCV kinetics.

When treating HCV patients with conventional dual therapy in the current context of rapidly evolving HCV therapy, outcome prediction is crucial and HCV kinetics, as early as 48 hours after the start of treatment, may play a major role. We aimed at clarifying the role of HCV very early kinetics. We consecutively enrolled mono-infected HCV patients at 7 treatment sites in Central Italy and evaluated the predictive value of logarithmic decay of HCV RNA 48 hours after the start of dual therapy (Delta48). Among the 171 enrolled patients, 144 were evaluable for early and sustained virological response (EVR, SVR) prediction; 108 (75.0%) reached EVR and 84 (58.3%) reached SVR. Mean Delta 48 was 1.68 ± 1.22 log10 IU/ml, being higher in patients with SVR and EVR. Those genotype-1 patients experiencing a Delta 48 >2 logs showed a very high chance of success (100% positive predictive value), even in the absence of rapid virological response (RVR). Evaluation of very early HCV kinetics helped identify a small but significant proportion of genotype-1 patients (close to 10%) in addition to those identified with RVR, who could be treated with dual therapy in spite of not reaching RVR. In the current European context, whereby sustainability of HCV therapy is a crucial issue, conventional dual therapy may still play a reasonable role in patients with good tolerance and early prediction of success.

Beyond the distance between juridic and scientific decision-making process: An evidence-based prediction algorithm for professional liability assessment in healthcare-associated infections.

BACKGROUND: HAIs (Healthcare-Acquired-Infections) have been recently the subject of judgment n. 6386 pronounced on 3rd March 2023 by the Italian Supreme Court. This sentence provided three criteria to determine whether a health facility is responsible for the patient contracting a nosocomial infection, i.e. time criterion, topographical criterion and clinical criterion. Accordingly, the healthcare facility is obliged to prove the fulfillment of a series of preventive hygiene measures specifically detailed by the legislator. Herein, the positive predictive value of these criteria ("juridic criteria") in the identification of professional liability for nosocomial infections was evaluated in comparison with clinical criteria reviewed by Infectious Disease specialists ("Infectious-Disease criteria", i.e. presence of a Multidrug Resistant Organism (MDRO); development of surgical site infection; inadequate antibiotic therapy; inadequate disinfection). METHODS: Two retrospective cohorts were compared from the Portal of Telematic Services of the Ministry of Justice; 51 patients were extrapolated from Italian judgments concerning claims for Gram-negative nosocomial infections in the three-year period 2020-2022. On the other side, from the electronic database of University Hospital of Bari we extracted 349 patients affected by Gram-negative infections in the same timespan. Both "juridic" criteria and "Infectious-Disease" criteria were then applied to the full cohort after stratification for cohort of origin and after stratification for nosocomial or non-nosocomial infections. Predictive value of criteria was evaluated through receiver operating characteristic (ROC) curves and area under the curve (AUC). RESULTS: Overall, the incidence of definite nosocomial infections (according to final judgement or clinical records discharge letter) was 84 % in juridic cohort and 46 % in "real-world" series. Data suggested that the presence of all three juridic criteria [ROC AUC = 0.944 (95%CI = 0.924-0.963)] or the four clinical criteria [ROC AUC = 0.948 (95%CI = 0.928-0.969)] predicted well a case of nosocomial infection with professional liability. Moreover, by summarizing both criteria in a single classification system, the generated ROC curve (was the one with the highest AUC [0.9488 (95%CI = 0.928-0.969)]. Accordingly, further tests were performed, evaluating the predictive value of one juridic criterium plus at one of more Infectious-Disease criteria. Interestingly, the ROCs curves demonstrated that the presence of at least 1 juridic criteria plus at least 2 Infectious Disease criteria reached a predictive value comparable to 2 or 3 juridic criteria. CONCLUSIONS: The results highlight the efficiency of new criteria laid down in the judgment of the Italian Supreme Court to attribute liability for nosocomial infection despite the disputed distance between juridic and scientific decision-making process. In addition, the use of a combined score combining "juridic" and "Infectious-Disease" criteria provides a high-quality tool to be used by technical consultants to make up for lack of clinical documentation by passing judgments concerning litigation about professional liability in case of nosocomial infections. This sheds light on the possibility to face worldwide judicial inquiries with scientific rigor.

Safety and effectiveness of fifth generation cephalosporins for the treatment of methicillin-resistant staphylococcus aureus bloodstream infections: a narrative review exploring past, present, and future.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) is a major issue in healthcare, since it is often associated with endocarditis or deep site foci. Relevant morbidity and mortality associated with MRSA-BSIs forced the development of new antibiotic strategies; in particular, this review will focus the attention on fifth-generation cephalosporins (ceftaroline/ceftobiprole), that are the only ß-lactams active against MRSA. AREAS COVERED: The review discusses the available randomized controlled trials and real-world observational studies conducted on safety and effectiveness of ceftaroline/ceftobiprole for the treatment of MRSA-BSIs. Finally, a proposal of MRSA-BSI treatment flowchart, based on fifth-generation cephalosporins, is described. EXPERT OPINION: The use of anti-MRSA cephalosporins is an acceptable choice either in monotherapy or combination therapy for the treatment of MRSA-BSIs due to their relevant effectiveness and safety. Particularly, their use may be advisable in combination therapy in case of severe infections (including endocarditis or persistent bacteriemia) or in monotherapy in subjects at higher risk of drugs-induced toxicity with older regimens. On the contrary, caution should be taken in case of suspected/ascertained central nervous system infections due to inconsistent data regarding penetration of these drugs in cerebrospinal fluid and brain tissues.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing.

BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.