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Background and objectives: Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load

Laparoscopic Palomo varicocelectomy is one the most common approaches adopted to treat pediatric varicocele, but postoperative hydrocele still remains a potential problem with this procedure. This study aimed to evaluate the outcome of a new technique of lymphography using indocyanine green (ICG)-enhanced fluorescence to perform lymphatic sparing laparoscopic Palomo varicocelectomy.The records of 25 patients who underwent laparoscopic left varicocelectomy in our unit from March 2017 to March 2018 were retrospectively evaluated. The average patients' age was 13.7 years (range 12-16). All patients had a high degree varicocele associated with left testicular hypotrophy and symptoms. All procedures were performed in laparoscopy using three trocars. After trocars' positioning, 2 mL of ICG solution was directly injected into the left testicle. Using ICG fluorescence, the lymphatic vessels were clearly identified and spared, and then the entire spermatic bundle was clipped and divided according to Palomo's principle.The average operative time was 18 minutes (range 10-25). No conversions to open surgery and no allergy or other adverse reactions induced by ICG were reported. At a maximum follow-up of 18 months, no recurrence of varicocele or postoperative hydrocele was recorded.Our preliminary experience showed that ICG fluorescence lymphography is a safe and effective option to perform lymphatic sparing laparoscopic Palomo varicocelectomy in children and adolescents with high degree varicocele. The intratesticular injection of ICG and use of fluorescence vision allowed identification of lymphatic vessels in 100% of cases. No allergy to ICG or postoperative hydrocele was reported in our experience.

Purpose We reported our preliminary experience using ICG fluorescence in pediatric minimally invasive surgery (MIS) with the aim to standardize indications, dose, timing, and modality of administration of ICG according to different organs. Methods ICG technology was adopted in 46 MIS procedures performed in our unit over the last 18 months: 30 left varicocele repairs; 5 cholecystectomies in obese adolescents; 3 tumor excisions; 3 nephrectomies; 2 partial nephrectomies; 3 lymphoma excisions. ICG solution was injected intravenously in all cases except for varicocelectomy in which it was injected into the testis. The ICG injection was performed intra-operatively in all cases except for cholecystectomy in which it was injected 18 h prior to the procedure. Results All procedures were completed laparoscopically without conversions or intra-operative complications. No adverse or allergic reactions to ICG were reported. Conclusion Our preliminary experience showed that ICG fluorescence is a safe, useful, and versatile technique to adopt in pediatric MIS to achieve a better identification of anatomy and an easier surgical dissection or resection in challenging cases. Currently, the main indications are varicocelectomy, difficult cholecystectomy, tumor excision, nephrectomy, and partial nephrectomy. The main limitation is the needing of a special equipment to use ICG technology.

Background: The traditional open excision of pilonidal sinus disease (PSD) is associated with a painful postoperative course and high recurrence rates. We recently published our technique of pediatric endoscopic pilonidal sinus treatment (PEPSiT). We aimed to report our long-term outcome including using PEPSiT for recurrent PSD after failed open repair. Methods: All patients with recurrent PSD after open excision who underwent PEPSiT in our unit over the past 2 years were included in the study. During surgery a fistuloscope was introduced through the fistula's orifice. All identifiable hairs were removed using endoscopic forceps. Thereafter, the cavity was debrided with endobrush and ablated with monopolar electrode. External openings were not closed. Results: In the past 2 years, 40 patients with PSD underwent PEPSiT. Ten of 40 patients (6 boys and 4 girls with an average age of 16.8 years [range = 14-18]) had recurrent PSD after open surgery and were included in the study. The average operative time was 27.7 minutes (range = 24-43). No perioperative complications occurred. The average analgesic requirement was 20 hours (range = 16-26) and the average hospitalization was 22.4 hours (range = 18-36). The average time to return to full daily activities was 2.3 days (range = 1-5) and all patients were highly satisfied of postoperative course. At 1 month postoperatively, the external openings were completely healed. No recurrence was recorded at a mean follow-up of 18 months (range = 6-24). Conclusions: Our results demonstrated that PEPSiT is an excellent technique for surgical treatment of PSD in children and teenagers. In fact, it is technically easy and fast to perform, with a short and painless hospital stay and it allows to the operated patients an early return to full daily activities without any physical limitations compared with open repair. In addition, it is also effective for treatment of recurrent PSD after failed open repair.

Sliding indirect inguinal hernias containing ovary are not uncommon in girls. We reported our experience with laparoscopic treatment of inguinal ovarian hernias in female infants and children with the aim to standardize the surgical technique. METHODS: The medical records of all girls who underwent laparoscopic inguinal hernia repair in our unit over the past 5 years were retrospectively reviewed. Only patients with an ovary found intraoperatively in the hernia sac were included in the study. All patients younger than 1 year received preoperatively a bowel preparation with simethicone and enemas. RESULTS: A total of 289 girls (median age 3.2 ± 0.5 years) underwent laparoscopic inguinal hernia repair during the study period. Thirty-seven patients (12.8%) had an ovarian hernia and were included in the study. Of these 37 girls, 9 (28.1%) were younger than 1 month, 20 (62.5%) ranged in age from 2 months to 1 year, and 3 (9.4%) were from 1 to 7 years. The average operative time was 23.7 minutes (range 18-43 minutes). No necrotic ovary was found intraoperatively, and all the procedures were accomplished laparoscopically. Neither intraoperative nor postoperative complications were reported. A patency of the contralateral canal of Nuck was found in 16 of the 37 patients (43.2%) and repaired during the same procedure. The average length of hospitalization was 21.8 hours (range 18-36 hours). No hernia recurrence or ovarian atrophy was recorded at a mean follow-up of 36 months (range 1-60 months). CONCLUSIONS: On the basis of our experience, laparoscopy should be considered the gold standard for the treatment of inguinal ovarian hernias in girls. Key points for standardization of the technique are as follows: bowel preparation in children younger than 1 year, use of 5-mm umbilical balloon trocar, correct positioning of 3-mm working screw trocars, section of the abnormal attachment of ovarian suspensory ligament, section of the periorificial peritoneum, and use of nonresorbable sutures.

SUMMARYThis study assessed changes in prevalence and distribution of HIV-1 non-subtype B viruses in Italian and immigrant patients over two decades in a province in Italy. All HIV-positive patients who underwent genotypic resistance testing were selected. Prevalence of non-subtype B viruses in 3-year periods was calculated. All sequences of non-subtype B and those provided by REGA as unassigned were analysed for phylogenetic relationships. In total, 250/1563 (16%) individuals were infected with a non-subtype B virus. Prevalence increased over time, reaching a peak (31·5%) in 2004–2006. In Italian patients, the most frequent subtypes were B (92·5%) and F1 (4%). F1 subtype was also prevalent in patients from South America (13·6%); in patients of African origin, CRF02_AG (54·9%) and G (12·3%) were the most frequent. HIV-1 non-subtype B infections in Italians were mostly found in patients who acquired HIV sexually. A phylogenetic relationship between F subtypes in Italian and representative HIV-1 sequences from Brazil was found. C subtypes in Italians were phylogenetically related to subtypes circulating in Brazil. Inter-subtype recombinants were also found in the latest years. The HIV-1 epidemic in Brescia province evolved to the point where about 1/3 patients recently diagnosed harboured non-B HIV subtypes. The distribution of HIV-1 non-B subtypes in Italian patients resembled that in South American patients and phylogenetic relatedness between some Italian and South American HIV-1 strains was found. The possible epidemiological link between these two populations would have been missed by looking only at risk factors for HIV acquisition declared by patients. The evidence of inter-subtype recombinants points to significant genetic assortment. Overall our results support phylogenetic analysis as a tool for epidemiological investigation in order to guide targeted prevention strategies.

Background Prevalence and factors associated with etravirine (ETV) resistance mutations among patients failing on first-generation non-nucleoside reverse transcriptase inhibitors (NNRTI) merit investigation. Methods The study comprised an analysis of all sequential patients attending the Institute of Infectious Diseases (Brescia, northern Italy) who performed a genotypic resistance testing (GRT) after ≥3 months of a stable NNRTI-based regimen between 2001 and 2006. Multivariable ordinal logistic regression analysis was performed to assess predictors of ETV resistance mutations. Results Out of 248 strains, 153 (61.7%) harboured ≥1 ETV resistance mutations. In particular, 88 (35.5%), 53 (21.4%) and 12 (4.8%) harboured one, two and three mutations, respectively. The most frequent mutations were G190A (23%), Y181C (23%) and K101E (14.1%). Use of nevirapine (odds ratio [OR] 2.73; 95% confidence level [CI] 1.62–4.62; P500 copies/ml and GRT (per month, OR 1.05; 95% CI 1.01–1.09; P=0.012) were associated with a greater number of ETV resistance mutations. Conversely, higher CD4+ T-cell counts at nadir (per 100 cells/mm3, OR 0.81; 95% CI 0.67–0.98; P=0.029) and use of lamivudine/emtricitabine (OR 0.57; 95% CI 0.37–0.87; P=0.009) were protective. Accumulation of ETV resistance-associated mutations was demonstrated by sequential GRT in 4/35 patients (all treated with nevirapine). Conclusions Mutations associated with ETV resistance were common among patients failing on NNRTI, but prevalence of viral strains harbouring three mutations was low. Use of efavirenz and co-administration of lamivudine reduced the risk of ETV resistance. The continued use of the current NNRTI in a failing regimen may select for additional resistant variants.

Continuous surveillance of HIV primary resistance mutations is highly important due to their potential clinical impact. All patients naive to antiretrovirals who had ≥1 genotypic resistance testing at the Institute of Infectious Diseases (Brescia, Northern Italy) between 2001 and 2006 were analyzed. Primary resistance mutations were defined using epidemiological and clinical criteria. Mutations were interpreted using the Stanford University Algorithm. Logistic regression analysis was used to assess possible predictors of primary resistance mutations. Among 569 patients, 11% presented ≥1 mutation. Prevalence of primary resistance mutations to nucleoside reverse-transcriptase inhibitors (NRTI), non-nucleoside reverse-transcriptase inhibitors (NNRTI), and protease inhibitors (PI) was 6.3%, 6%, and 1.6%, respectively. The most frequent mutations to NRTI were substitutions at position 215 (215Y in 3 patients, and 215 revertants in 16), 41L (13), 219Q (12), and 210W (10). Among mutations to NNRTI, 103N was found in 21 patients, while 181C, 188L, and 190A/S in 8, 3, and 4 patients, respectively. Fifty-one patients (9%) had high-to-intermediate resistance to ≥1 antiretroviral drug before starting the treatment. Regarding the new generation drugs, nine patients had intermediate resistance to etravirine, five patients had intermediate resistance to tipranavir, while five, one, and seven patients had low resistance to etravirine, tipranavir, and darunavir. Homosexuals were more likely to harbor a virus with primary resistance mutations (OR:2.68; 95% CI:1.44-5.00; P=0.002) while non-Italian nationality was protective (OR:0.38; 95% CI:0.17-0.86; P=0.020). Prevalence of primary resistance mutations suggests that genotypic resistance testing should be performed before starting treatment in naïve patients in Italy, particularly when NNRTI are prescribed. © 2008 Wiley-Liss, Inc.

"Objective: The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory Methods: Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). Results: Quantification of HIV-1 DNA was available for 35\/45 (77.8%) samples, and gave a median value of 598 (IQR:252-1,203) copies\/10(6) PBMCs. A total of 56\/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54\/56 (96.4%). Results of tropism prediction by local centers were: 33\/54 (61.1%) R5 and 21\/54 (38.9%) X4\/DM. Conclusion: There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment."

Objective: To determine the impact of genotypic inhibitory quotient (GIQ) for lopinavir (LPV) in patients failing HAART with limited antiretroviral exposure. Design: Retrospective analysis of a prospective trial. Methods: Lopinavir GIQ was calculated as the ratio between the mean trough concentration (Ctrough) and the number of protease mutations using eight different HIV drug resistance mutation lists or algorithms. Early (by week 12) and confirmed (up to week 24) virological response (HIV-RNA < 400 copies/mL, ECVR) was used as dependent variable in logistic regression model. Results: Seventy-one of 109 (65%) patients achieved ECVR. At multivariable logistic regression analysis, each μg/mL increase of GIQ was correlated with increasing probability of ECVR as far as the following mutations were computed: multi-protease inhibitor (PI) associated mutations listed by IAS (OR = 1.17; 95% CI = 0.99-1.39; P = 0.058), mutations associated with LPV resistance by ANRS algorithm (OR = 1.21; 95% CI = 1.02-1.44; P = 0.03), major mutations associated with LPV resistance by Stanford database (OR = 1.16; 95% CI = 1-1.35; P = 0.05), and the whole set of mutations associated with LPV resistance in the same database (OR = 1.22; 95% CI = 1.02-1.46; P = 0.03). Using ROC curve method, a specific threshold GIQ was assessed, above which this parameter could predict ECVR with the highest sensitivity (74.6% with GIQ obtained through Stanford LPV mutations) or specificity (89.5% with GIQ obtained through ANRS LPV mutations). Conclusions: Our results suggest that increasing GIQ can improve virological outcome even in patients with limited exposure to PIs. Further studies are necessary to understand what HIV protease mutations should be considered and whether such mutations should be weighted differently to improve LPV GIQ predictive value. © 2005 Elsevier B.V. All rights reserved.

To assess the prevalence of human T cell leukemia virus type I (HTLV-I) and 2 (HTLV-II) infection and the associated risk factors among immigrants living in Northern Italy, we surveyed 3017 open-population subjects from three geographical areas and 371 prisoners. In the open population, the overall prevalence was 0.3% for HTLV-I and 0.1% for HTLV-II, while among prisoners, HTLV-I and HTLV-II infection were detected in 1.4 and 0.8% of subjects, respectively. HTLV-I prevalence was higher in subjects with multiple sexual partners or sexually transmitted diseases. This association was significant in the open-population group and close to significance in prisoners. Multivariate analysis showed that human immunodeficiency virus (HIV) seropositivity remained significantly associated with HTLV-I infection in both targeted populations (OR: 11.2 in the open population; OR: 9.9 among prisoners), whereas sexual exposure was associated with HTLV-I seropositivity only for prisoners (OR: 14.3). No independent variable was related to HTLV-II infection.

(i) To compare early decrease of HIV plasma viral load (pVL) after two standard-combinations of highly active antiretroviral therapy (HAART) (ii) To evaluate variations of proviral HIV-DNA load on conditions of sustained pVL undetectability. Two different sub-studies of a multicentre prospective randomized controlled trial which compared two first-line HAART (i.e., zidovudine+lamivudine+lopinavir/ritonavir versus tenofovir+lamivudine+ efavirenz). Only patients enrolled at the coordinating centre (University of Brescia) were included in the two sub-studies. In the first sub-study, we calculated pVL decrease with respect to baseline at any of the following time-point: days 1, 3, 7, 14 and 28. Decreases of the pVL were compared between the two treatment groups. In the second sub-study, we'analyzed, variation of proviral HIV-DNA load in CD4+ T-cells from baseline to week 52 only in patients who maintained the same treatment regimen and had sustained undetectable pVL. In either studies, linear regression analysis was used to investigate what factors could influence variations of pVL and of proviral HIV-DNA load. (i) 64 patients were studied. A significant decrease of pVL was found from day 3 on, without statistically significant differences between the two study groups. However, after adjusting for possible confounders, tenofovir+lamivudine+efavirenz resulted to be associated with greater pVL decreases. (ii) 45 patients were studied. Mean proviral HIV-DNA load decreased from 1,610 (95%CI: 879-2,341) to 896 (95% CI 499-1,293) copies/106 cells (P=0.05). Linear regression analysis showed that the decrease of proviral DNA load during follow-up was independently and inversely correlated with age. Further studies are needed to compare pVL decay between antiretroviral regimens and assess whether proviral HIV-DNA load is a surrogate marker of treatment effectiveness. © 2008 Bentham Science Publishers Ltd.

Data from 197 patients for whom highly active antiretroviral therapy (HAART) failed, who started a new regimen chosen under the guide of resistance testing results interpreted by experts, were retrospectively studied, provided that at least 2 determinations of adherence and plasma drug concentrations were performed during the follow-up. Univariate and multivariable logistic regression analyses were conducted, using confirmed virologie response at week 24 as outcome measure (i.e., achievement of undetectable HIV plasma viral load at any time point before week 24 and its maintenance up to week 24). Suboptimal drug concentrations (odds ratio [OR]: 0.3; 95% confidence interval [CI] 0.2-0.7; p = 0.006) and suboptimal adherence (OR: 0.4; 95% CI 0.2-0.8; p = 0.014) were both negative independent predictors of sustained virologic response, while the use of boosted protease inhibitor-containing regimens resulted to be protective (OR: 2.4; 95% CI 1.1-5.3; p = 0.032). © Mary Ann Liebert, Inc.