Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

The impact of anti-mould prophylaxis on Aspergillus PCR blood testing for the diagnosis of invasive aspergillosis.

BACKGROUND: The performance of the galactomannan enzyme immunoassay (GM-EIA) is impaired in patients receiving mould-active antifungal therapy. The impact of mould-active antifungal therapy on Aspergillus PCR testing needs to be determined. OBJECTIVES: To determine the influence of anti-mould prophylaxis (AMP) on the performance of PCR blood testing to aid the diagnosis of proven/probable invasive aspergillosis (IA). METHODS: As part of the systematic review and meta-analysis of 22 cohort studies investigating Aspergillus PCR blood testing in 2912 patients at risk of IA, subgroup analysis was performed to determine the impact of AMP on the accuracy of Aspergillus PCR. The incidence of IA was calculated in patients receiving and not receiving AMP. The impact of two different positivity thresholds (requiring either a single PCR positive test result or ≥2 consecutive PCR positive test results) on accuracy was evaluated. Meta-analytical pooling of sensitivity and specificity was performed by logistic mixed-model regression. RESULTS: In total, 1661 (57%) patients received prophylaxis. The incidence of IA was 14.2%, significantly lower in the prophylaxis group (11%-12%) compared with the non-prophylaxis group (18%-19%) (P < 0.001). The use of AMP did not affect sensitivity, but significantly decreased specificity [single PCR positive result threshold: 26% reduction (P = 0.005); ≥2 consecutive PCR positive results threshold: 12% reduction (P = 0.019)]. CONCLUSIONS: Contrary to its influence on GM-EIA, AMP significantly decreases Aspergillus PCR specificity, without affecting sensitivity, possibly as a consequence of AMP limiting the clinical progression of IA and/or leading to false-negative GM-EIA results, preventing the classification of probable IA using the EORTC/MSGERC definitions.

Gait analysis in children with haemophilia: first Italian experience at the Turin Haemophilia Centre.

AIM: To investigate the functional status in haemophilia patients referred to an Italian paediatric haemophilia centre using gait analysis, verifying any differences between mild, moderate or severe haemophilia at a functional level. METHODS: Forty-two patients (age 4-18) presenting to the Turin Paediatric Haemophilia Centre who could walk independently were included. Therapy included prophylaxis (n = 21), on-demand (n = 17) or immune tolerance induction + inhibitor (n = 4). Patients performed a test of gait analysis. Temporal, spatial and kinematic parameters were calculated for patient subgroups by disease severity and background treatment, and compared with normal values. RESULTS: Moderate (35.7%) or severe (64.3%) haemophilia patients showed obvious variations from normal across a variety of temporal and spatial gait analysis parameters, including step speed and length, double support, swing phase, load asymmetry, stance phase, swing phase and speed. Kinematic parameters were characterized by frequent foot external rotation with deficient plantar flexion during the stance phase, retropelvic tilt, impaired power generation distally and reduced ground reaction forces. Both Gait Deviation Index and Gait Profile Score values for severe haemophilia patients indicated abnormal gait parameters, which were worst in patients with a history of past or current use of inhibitors and those receiving on-demand therapy. CONCLUSION: Functional evaluation identified changes in gait pattern in patients with severe and moderate haemophilia, compared with normal values. Gait analysis may be a useful tool to facilitate early diagnosis of joint damage, prevent haemophilic arthropathy, design a personalized rehabilitative treatment and monitor functional status over time.

Immune response to Hepatitis B vaccine in patients with celiac disease: A systematic review and meta-analysis.

It is debated whether patients with celiac disease (CD) have non-protective antibody responses to HBV vaccination more frequently than non-affected subjects. To perform a literature review and meta-analysis on protective response to HBV vaccination in CD patients. RCTs and observational controlled studies were eligible. Outcome of interest was an anti-HBs (HBsAb) titer ≥ 10 IU/L after last vaccine dose. Comparative index was rate ratio (RR). Heterogeneity between studies was addressed and funnel plots were analyzed. Meta-regression models were applied to investigate effect size due to study-specific variables. Twelve retrospective studies on a total of 1,447 participants and 4 prospective studies on 184 subjects were selected. The RR was 0.732 (95% C.I.: 0.664-0.808) and 0.777 (95% C.I.: 0.629-0.960) in the prospective and retrospective studies, respectively. The I(2), indicating heterogeneity, was 51.1% in retrospective, 39.8% in prospective studies. Non-protective antibody responses occurred more frequently in patients than controls. Due to limitations in the available studies, additional trials to evaluate post-vaccination HBsAb titer in CD patients are needed.

Strong and persistent correlation between baseline and follow-up HIV-DNA levels and residual viremia in a population of naïve patients with more than 4 years of effective antiretroviral therapy.

In a longitudinal study on 181 naïve patients who responded to therapy (mean follow-up 4 years), high baseline human immunodeficiency virus (HIV)-RNA values correlated with high levels of cellular HIV-DNA at all time points (p < 0.0001, p 0.045, p 0.0055, and p 0.0025, respectively) and negatively correlated with undetectable residual viremia (URV; <2.5 copies/mL) at T1, T2, and T3 (p 0.026, p 0.0149, and p 0.0002, respectively). Baseline high HIV-DNA levels predicted the persistence of high values (p 0.0001) and negatively correlated with URV (p 0.0254, p 0.0481, and p 0.0085). These results suggest that baseline viral load, cellular HIV-DNA, and URV were strongly correlated over long-term follow-up of antiretroviral therapy responders.

Hydrogen breath test in patients with severe constipation: the interference of the mixing of intestinal content.

BACKGROUND: The diagnostic accuracy of the hydrogen (H2 ) breath test might be reduced by the release of preformed H2 , trapped in hard stools. Test solution ingestion might induce the mixing of colonic content and a false positive result. We studied severely constipated patients, at diagnosis and after the normalization of bowel function, to clarify whether this mechanism affects test results. METHODS: Twenty functional constipated patients, 10 consecutive patients with functional diarrhea and 10 healthy volunteers underwent (i) a H2 breath test after lactulose, to exclude differences among the groups in fermenting capacity; (ii) breath H2 excretion monitoring after non-absorbable, non-fermentable PEG-electrolyte solution, to exclude the role of the delivery to the colon of preexisting fermentable substrates or of the release of preformed H2 entrapped in the feces; (iii) H2 measurement during a 7-h fasting period, to exclude the role of spontaneous variations of breath gas excretion; and (iv) breath H2 excretion monitoring after PEG, after normalization of bowel function. KEY RESULTS: All the subjects excreted similar amounts of H2 after lactulose. After PEG, only severely constipated patients showed significant breath H2 excretion, theoretically able to induce a false positivity of the lactose breath test in 70% of patients and a false positivity of glucose breath tests in 50% of patients. Breath H2 excretion after PEG disappeared if fecal consistency improved after therapy. CONCLUSIONS & INFERENCES: Severely constipated patients may harbor preformed gas in hard stools which can be released when mixing of the intestinal content is induced. This mechanism may interfere with breath test results.

Effects on bleeding complications of pharmacogenetic testing for initial dosing of vitamin K antagonists: a systematic review and meta-analysis.

BACKGROUND: Although warfarin and other vitamin K antagonists (VKAs) are the most widely used oral anticoagulants for the prevention and treatment of thromboembolic events, a number of factors hamper their manageability, the most important being the inter-individual variability of the therapeutic dose requirement. Following the discovery of the influence of CYP2C9 and VKORC1 polymorphisms on VKA dose requirements, there has been interest in genotype-guided VKA dosing in order to reduce the risk of over-anticoagulation at the time of therapy initiation and hence the risk of bleeding, particularly prominent during the early days of treatment. To assess the impact on clinical outcomes of pharmacogenetic testing for initial VKA dosing, we have performed a systematic review and meta-analysis of the literature. METHODS: MEDLINE, EMBASE and Cochrane databases were searched up to March 2014. Only randomized controlled trials comparing genotype-guided vs. clinically-guided warfarin dosing were included. RESULTS: Nine trials including 2812 patients met the inclusion criteria and were pooled for meta-analytical evaluation. Risk of bias, assessed according to the Cochrane methodology, showed a low risk for the majority of domains analyzed in the included trials. A statistically significant reduction in the risk ratio (RR) for developing major bleeding events was observed in the pharmacogenetic-guided group compared with the control group (RR = 0.47; 95% CI, 0.23-0.96; P = 0.040). CONCLUSIONS: The results of this meta-analysis show that genotype-guided initial VKA dosing is able to reduce serious bleeding events by approximately 50% compared with clinically-guided dosing approaches.

Hydrogen breath test and intestinal gas production.

Despite extensive use in clinical practice, difficulties regarding interpretation of hydrogen breath test are still very frequent, even on research grounds. After the administration of a non-absorbable sugar, such as lactulose, an increase of breath hydrogen and methane is evident; this phenomenon is considered an index of colonic fermentation. It is not clear, however, if the levels of these compounds correlate with the presence and severity of functional symptoms, nor if they accurately reflect gas production at colonic level. So far, apart from flatulence, we have no indications regarding the ability of hydrogen or methane to act as biomarkers of intraluminal events. On the other hand, it has been shown that in functional bowel disease a colonic dysbiosis exists, and that the modification of bacterial flora might result in a reduction of symptom severity. Consequently, it is not clear if hydrogen and methane colonic production could have a role in the pathophysiology of functional complaints, but it is possible that other fermentation products should be taken into consideration, such as acetate, propionate, and alcohol.

HIV coreceptor tropism in paired plasma, peripheral blood mononuclear cell, and cerebrospinal fluid isolates from antiretroviral-naïve subjects.

A survey of HIV coreceptor usage in cerebrospinal fluid (CSF) samples, peripheral blood mononuclear cells (PBMCs), and plasma samples from naïve seropositive patients was conducted. One hundred patients were enrolled in this study. Of the 100 patients, 36 had a primary or recent infection (P-RI), 31 had an early chronic infection (>350 CD4 cells) (ECI), and 33 had a late chronic infection (LCI). All 3 compartments were sampled in a subset of 33 participants, while the remaining 67 patients provided plasma samples and PBMCs only. Seventy-seven patients harbored the R5 virus in plasma samples and had a significantly higher median and percentage of CD4(+) T cells than patients with X4 virus (437 and 281 cells/µl, respectively; P = 0.0086; 20.6% and 18.6%, respectively). The X4 strain was detected more frequently in patients with LCI than in patients with P-RI or ECI (39.3%, 19.4%, and 9.6%, respectively; P = 0.0063). PBMC and plasma tropism was concordant in 90 patients, and 73 had the R5 strain. Among patients with discordant results, 4 had the R5 virus in their plasma and the X4 virus in PBMCs; 6 showed the opposite profile. Plasma, PBMC, and CSF tropism determinations were concordant in 26/33 patients (21 patients had R5, and 5 had X4). The tropism was discordant in 5/33 patients, with the X4 virus in plasma and R5 in CSF; the HIV tropism in PBMCs was X4 in 3 patients. The remaining 2/33 patients had the R5 virus in plasma and PBMCs and the X4 virus in CSF; one of these patients had a P-RI. The discordant tropism in CSF and blood may have implications for chemokine (C-C motif) receptor 5 (CCR5) antagonist use in patients with limited response to antiretroviral therapy (ART) or in responding patients evaluated for simplification of treatment.

[Cryptococcus neoformans meningoencephalitis among apparently immunocompetent patients: description of two cases]. / Meningoencefalite da Cryptococcus neoformans in pazienti apparentement immunocompetenti: descrizione di due casi.

Cryptococcus neoformans is a ubiquitous fungal pathogen which causes human disease ranging from asymptomatic colonization of the lungs, to severe pneumonia, mediastinitis, meningitis or generalized infection. Although cryptococcal infection shows notably opportunistic features, it is sometimes also found among apparently immunocompetent individuals, with an extremely adverse outcome in the case of SNC involvement. Therefore, when faced with a presumed healthy person with anamnestic, clinical, CSF and instrumental findings consistent with chronic meningitis/meningoencephalitis, we must also consider cryptococcosis as a possible cause of disease. This may be rapidly achieved by resorting to quite a simple serological test, namely cryptococcal antigen detection. We describe two cases of cryptococcal meningoencephalitis occurring among apparently immunocompetent subjects (both HIV-negative, not under corticosteroid or immunosuppressive regimen, nor undergoing chemotherapy or radiotherapy. Laboratory diagnostics revealed the existence of reasonable immunological deficit for both subjects. Unfortunately, we were unable to establish whether the alterations in question were preexisting or concomitant with fungal infection. Our patients' course was somewhat problematic, according to findings observed in broader-based studies: this could mostly be explained by the considerable diagnostic delay which often marks cryptococcal infections of immunocompetent individuals. Nevertheless, neither of these two cases were complicated by intracranial pressure increase, leading us to speculate whether this disease may occur less frequently under conditions of substantial immunological integrity.

Immune tolerance with rituximab in congenital haemophilia with inhibitors: a systematic literature review based on individual patients' analysis.

Rituximab, a monoclonal antibody against the pan B-cell antigen CD20, has been successfully used in both adults and children for the management of malignant and non-malignant immune-mediated disorders including acquired haemophilia. On the basis of this positive experience, a number of investigators have recently used this agent in patients with congenital haemophilia and inhibitors refractory to first-line treatments. After a careful electronic and hand search, we have collected 29 studies that included 49 cases. A durable complete remission was obtained in 53% of the cases and no severe adverse events related to rituximab were recorded. A multivariate analysis applied to individual patients' data identified the diagnosis of a mild/moderate haemophilia and the concomitant treatment with factor VIII concentrates and immunosuppression agents as covariates associated with an increased response to rituximab. Large prospective randomized studies with an adequate follow-up are needed to confirm these preliminary findings.

[Epidemiology of reproductive tract infections in a symptomatic population of North-East of Italy]. / Prevalenza di agenti d'infezioni del tratto riproduttivo in una popolazione sintomatica del Nord-Est.

AIM: Reproductive tract infections (RTIs) are one of the main causes of morbidity in the world and sexually transmitted infections (STIs) can give rise to severe sequels (inflammatory pelvic disease, etc.). The epidemiology of these infections is changeable and depends on geographical and economical factors, migratory flows and social and sexual habits. This variability, along with the lack of sufficient data in literature, is a serious problem in the development of screening, prevention and therapy strategies centered on local needs. The aim of our study was to evaluate the epidemiology of reproductive tract infections in a symptomatic population of the Nord-East of Italy. METHODS: In the period January-June 2006, we investigated 207 subjects at the Microbiology and Virology Service of Padua's Hospital, 18-65 years old, males and women, Italian and foreigners. All had symptoms or personal history pointing to a possible reproductive tract infection. For female we collected vaginal and cervical swabs, and for male urethral swabs, for microscopy, the culture for Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma spp., other bacteria and yeasts, and for molecular assay for Chlamydia trachomatis. RESULTS: Among our population, the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis were respectively 6.28%, 1.93% and 3.86%. STIs were more frequent in males, among foreigners, and in patients aged 18-30 years. CONCLUSION: From the results obtained, ideas have emerged in order to arrange a qualitative and quantitative optimization of the diagnosis of RTIs, implementing diagnostic paths based on the different typologies of patients and on the local epidemiology.

A prospective study of BK-virus-associated haemorrhagic cystitis in paediatric patients undergoing allogeneic haematopoietic stem cell transplantation.

We investigated the incidence, risk factors and outcome of haemorrhagic cystitis (HC) in paediatric patients undergoing HSCT and the predictive value of BK viruria and viraemia for developing HC. Over a period of 54 months, 74 patients were recruited. The cumulative incidence of HC was 22%. Among 15 patients prospectively monitored for BK viruria and viraemia, four patients developed HC of grade > or =II. This group, which had two consecutive BK positive samples, showed a sensitivity of 100%, a specificity of 82%, a positive predictive value of 67%, and negative predictive value of 100% for developing HC. Analysed by a receiver-operator characteristic curve (ROC), a urine BK load >9 x 10(6) genomic copies/ml had a sensitivity of 95% and specificity of 90%; while a blood BK load >1 x 10(3) genomic copies/ml had a sensitivity of 40% and a specificity of 93% for HC, respectively. In univariate analysis, BK positivity was the only factor significantly associated with HC. After a median follow-up of 1.8 years, patients with HC showed a lower overall survival, 40 vs 65%, P 0.01, and a lower event-free survival, 42 vs 62%, P 0.03, compared to patients without HC. We conclude that BK detection in urine and/or plasma is a specific predictor for developing HC.

Antimicrobial use and resistance among Gram-negative bacilli in an Italian intensive care unit (ICU).

Gram-negative bacilli antimicrobial resistance remains a significant problem for patients in the intensive care unit (ICU). We performed a retrospective analysis of microbiological data and antibiotic consumption over a 4-year period (2000-2003) in an Italian ICU. Pseudomonas aeruginosa and Klebsiella pneumoniae represented approximately 40% of all isolates. The most significant trend in antimicrobial use was an increase in use of 3(rd )generation cephalosporins, imipenem, and ciprofloxacin. A significant trend toward an increase in resistance rates to piperacillin, 3( rd )generation cephalosporins and ciprofloxacin was observed for K. pneumoniae and a positive correlation between resistance and drug-usage was evident for K. pneumoniae and piperacillin, cefotaxime, ceftazidime, cefepime, and ciprofloxacin, but not for piperacillin/tazobactam. No statistically significant correlations were evidenced for P. aeruginosa. Trends in resistances were studied also for Serratia spp and Proteus spp. Isolation rates of extended-spectrum beta-lactamase (ESBL)-producing strains in pathogens studied were high, especially for K. pneumoniae (72%, 160/222) and Proteus spp (41%, 18/43). In conclusion, the study showed high resistance among Gram-negative organisms isolated in the ICU and significant ESBL production. A significant correlation between antibiotic consumption and increasing resistance was evident for K. pneumoniae.