Application of the Weighted-Incidence Syndromic Combination Antibiogram (WISCA) to guide the empiric antibiotic treatment of febrile neutropenia in oncological paediatric patients: experience from two paediatric hospitals in Northern Italy.

BACKGROUND: Guidelines about febrile neutropenia in paediatric patients are not homogeneous; the best empiric treatment of this condition should be driven by local epidemiology. The Weighted-Incidence Syndromic Combination Antibiogram (WISCA) addresses the need for disease-specific local susceptibility evidence that could guide empiric antibiotic prescriptions based on outcome estimates of treatment regimens obtained as a weighted average of pathogen susceptibilities. This study developed a WISCA model to inform empirical antibiotic regimen selection for febrile neutropenia (FN) episodes in onco-haematological paediatric patients treated at two Italian paediatric tertiary centres. METHODS: We included blood cultures from patients with a bloodstream infection and neutropenia admitted to the Paediatric Haematology-Oncology wards in Padua and Genoa Hospitals from 2016 to 2021. WISCAs were developed by estimating the coverage of 20 antibiotics as monotherapy and of 21 combined regimens with a Bayesian probability distribution. RESULTS: We collected 350 blood cultures, including 196 g-negative and 154 g-positive bacteria. Considering the most used antibiotic combinations, such as piperacillin-tazobactam plus amikacin, the median coverage for the pool of bacteria collected in the study was 78%. When adding a glycopeptide, the median coverage increased to 89%, while the replacement of piperacillin-tazobactam with meropenem did not provide benefits. The developed WISCAs showed that no monotherapy offered an adequate coverage rate for the identified pathogens. CONCLUSIONS: The application of WISCA offers the possibility of maximizing the clinical utility of microbiological surveillance data derived from large hospitals to inform the choice of the best empiric treatment while contributing to spare broad-spectrum antibiotics.

Reduce susceptibility to cefiderocol in gram negative bacteria in children: Is hope already lost before it's even arrived?

BACKGROUND: In last years the diffusion of carbapenem resistance in Gram-negative bacteria (CR-GNB) is increasing worldwide, mainly due to the expression of carbapenemases. Cefiderocol has molecular characteristics that ideally confers activity against all CR-GNB, but resistant strains have already been identified. We describe cefiderocol susceptibility profile among multi-drug resistant Gram-negative isolated from pediatric patients. METHODS: Prospective, single pediatric center study, 1st January 2020-15th June 2023. All GNB carbapenemases producers or phenotypically carbapenem-resistant isolated in the study period were tested for cefiderocol susceptibility. Clinical and microbiological data were collected. A descriptive analysis was performed, comparing the groups of cefiderocol-resistant vs. cefiderocol-susceptible Enterobacterales and non-fermenting Gram-negative bacteria (NF-GNB). RESULTS: Forty-seven GNB were tested for cefiderocol susceptibility; 38% were cefiderocol-resistant: 16/30 (52%) among Enterobacterales and 2/17 (12%) among NF-GNB. None of the patients were previously exposed to cefiderocol. Looking at Enterobacterales, resistance to ceftazidime/avibactam was higher among cefiderocol-resistant vs. cefiderocol-susceptible strains (62% vs 36%, respectively), as MBL expression (67% vs. 36%, respectively). Too few NF-GNB were cefiderocol-resistance to draw any conclusion. No difference in ICU admission and mortality was identified comparing cefiderocol-resistant vs. susceptible strains. Patients colonized/infected by cefiderocol-resistant strains had been previously hospitalized more frequently. CONCLUSION: In our cohort cefiderocol resistance was mostly registered among Enterobacterales, and especially among MBL producers' strains (that were alongside resistant to ceftazidime/avibactam). This could be explained by the known possible cross resistance mechanism among ceftazidime/avibactam and cefiderocol. Also, correlation of cefiderocol-resistance with previous hospitalization could be associated with horizontal resistance transmission. Looking at our data, we believe that cefiderocol should be use cautiously, especially empirically and in monotherapy, due to the high resistance rate.

Changes in the Use of Antibiotics for Methicillin-Resistant Staphylococcus aureus Bloodstream Infections in Children: A 5-Year Retrospective, Single Center Study.

Monitoring antibiotic use in the pediatric population is a challenge, especially when determining a relationship between specific pathogens, infections, and antibiotic use. We retrospectively analyzed the consumption of anti-methicillin-resistant Staphylococcus aureus (MRSA) drugs from 2017 to 2021 at Istituto Giannina Gaslini by means of defined daily dose (DDD) adopted for adults by World Health Organization. We observed a statistically significant increase in the use of daptomycin and ceftaroline, combined with a decrease in the use of vancomycin. In the same period, we observed an increase in the proportion of bloodstream infections due to MRSA with vancomycin minimally inhibitory concentration (MIC mg/L) = 1, that represented the 100% of cases in 2021. This aspect was combined with the observation that in the 59% of cases, where vancomycin plasma concentrations were evaluated, it was not possible to achieve a ratio of the 24-h area under the concentration-time curve and MIC (AUC0-24/MIC) of vancomycin ≥ 400 mg/L. This study confirms that DDD can be used in pediatrics to monitor antibiotic consumption in relationship with infections epidemiology. Moreover, it describes the presence of vancomycin MIC creep for MRSA also in pediatrics and the difficulties in obtaining effective vancomycin plasma concentrations in children.

Early and Late Onset Neonatal Sepsis: Epidemiology and Effectiveness of Empirical Antibacterial Therapy in a III Level Neonatal Intensive Care Unit.

Bloodstream infections play an important role in neonatal morbidity and mortality. In this study, we retrospectively analyzed etiology and antibiotic resistance profiles of bacteria isolated from blood or Cerebro Spinal Fluid (CSF) cultures to evaluate the appropriateness of initial empirical therapy of neonatal sepsis. METHODS: microbiological data from patients admitted to Neonatal Intensive Care Unit (NICU), from January 2005 to October 2018, were anonymously extracted from the Laboratory of Microbiology database. According to the neonatal sepsis definition for patients admitted to NICU, positive cultures obtained within the first 72 h of life were labeled as Early Onset Sepsis (EOS); and Late Onset Sepsis (LOS) for those obtained later. RESULTS: 859 bacterial strains, 846 from blood and 13 from CSF, were detected in 611 neonates. In EOS, 75 blood cultures were found: 61 yielded Gram-positives and 14 Gram-negatives. Coagulase Negative Staphylococci (CoNS) represented the majority (52% n = 39). Streptococcus agalactiae and Escherichia coli were both isolated in 8% (n = 6) of cases. 784 strains were isolated in LOS: 686 (87%) Gram-positives and 98 (13%) Gram-negatives. CoNS represented most pathogens (n = 560, 71.4%) followed by Staphylococcus aureus (n = 57, 7.3%) and Enterococcus faecalis (n = 33, 4.2%). Ampicillin/gentamicin therapy resulted effective in 15/20 (75%) of EOS isolates. Internal protocol for LOS initial empirical therapy, calling for piperacillin/tazobactam and vancomycin resulted effective in 98.5% (734/745) of LOS strains. CONCLUSIONS: knowledge of local epidemiology of resistant pathogens, both in EOS and LOS, is fundamental to set up an effective empirical therapy in NICU. Aminoglycosides were fundamental in EOS. On the other side, LOS empirical therapy with vancomycin is sustained by the observation of 38% of methicillin resistance among S. aureus and about 95% in CoNS.

Pathways and microbiome modifications related to surgery and enterocolitis in Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital anomaly of the enteric nervous system. Abnormal microbiome composition was reported in HSCR patients. In this study, we addressed and analyzed microbiome modifications with relation tosurgery and HSCR associated enterocolitis (HAEC). METHODS: The faecal microbiome of 31 HSCR patients (overall 64 samples) was analyzed. HAEC was diagnosed and classified according to a combination of Pastor's and Elhalabi's criteria. Stool samples were analyzed by 16S sequencing (7 out of 9 polymorphic regions). Compositional and relative abundance profiles, as well as the functional potentials of the microbial community, were analyzed with the marker gene sequencing profiles using PICRUSt. RESULTS: The relative abundance of Bacteroidetes showed a severe decrease with slow recovery after surgery. Conversely, Proteobacteria transiently increased their abundance. Noteworthy, a strong linkage has been found between Proteobacteria descendants and HAEC occurrences. The inferred functional analysis indicated that virulence factors and fimbriae or pili might be associated with HAEC. CONCLUSIONS: Our study, addressing microbiome dynamics, demonstrated relevant changes after surgical manipulation. Alpha-diversity analyses indicated that surgery deeply affects microbiome composition. Proteobacteria and Enterobacteriaceae seem to play a pivotal role in HAEC occurrences. Several virulence factors, such as fimbriae or pili, might explain the HAEC-predisposing potential of selected microbiomes. These results suggest some innovative therapeutic approaches that deserve to be tested in appropriate clinical trials.

Validation of the Giannella Risk Score for the Prediction of Infection by Carbapenemase-producing Enterobacteriaceae in the Pediatric Population.

BACKGROUND: Despite efforts made to prevent the spread of multi-drug-resistant bacteria, carbapenemase-producing Enterobacteriaceae (CPE) has become one of the most dangerous threat worldwide. However, data on the epidemiology of CPE and on the correlation between CPE colonization and infection are scanty. The objectives of this study were first to describe the epidemiologic characteristics of colonizations and invasive CPE infections in the pediatric population, and second, to apply the Giannella Risk Score (GRS) to the pediatric population for the assessment of the risk of invasive CPE infection in patients with already known colonization. METHODS: Pediatric patients with evidence of colonization by CPE were retrospectively enrolled. For each colonized patient, the subsequent development of an infection by CPE was then assessed for a 90-day period after the first CPE isolation; GRSs were compared between patients who had developed any type of CPE infection and those without infection. RESULTS: A total of 215 patients (113 males and 102 females) with at least 1 isolation of CPE during hospitalization were analyzed. Median age was 5.6 years [interquartile range (IQR), 1.89-12.2 years]. Overall, 28 CPE infections (13%) were documented: 23 blood stream infections and 5 complicated urinary tract infections. The 30-day mortality of invasive CPE infections was 34.8%. The GRS values in patients with any CPE infection were statistically higher than in noninfected patients: median GRS 9 (IQR, 4-12.5) versus 4 (IQR, 2-4), respectively; P < 0.0001. The analysis of the receiver operating characteristic curves identified a GRS cut-off value ≥8 as the best predictor of CPE infection. The likelihood ratio of the results was <2 and the informedness of the test had a value <0.50. CONCLUSIONS: Our study confirms that the spread of CPE is an impelling problem also in the pediatric population, with a high mortality rate of invasive infections. However, the application of the GRS appears to be poorly informative in the pediatric setting; it might sometimes help to identify patients at very low-risk of CPE infection, in whom it is reasonable to spare targeted antimicrobial treatments.

Antibiotic susceptibility of Staphylococcus aureus isolated from skin lesions in children. A retrospective analysis from a tertiary care Italian pediatric hospital.

Antibiotic susceptibility of S. aureus was retrospectively assessed in 1833 strains isolated from skin lesions observed in an Italian tertiary care hospital. Methicillin resistance was more frequent in outpatients than in inpatients (18% vs. 14%, p = 0.04) as well as resistance to cotrimoxazole (8% vs. 4.1%, p < 0.001). Resistance to ampicillin was 99% in both groups, while for clindamycin it was 11% and 14%, respectively. Among topical antibiotics fusidic acid showed the better resistance profile (3%). Antibiotic resistance in pediatric skin infection in outpatients could represent a therapeutic problem in Italy.

Gut Microbiota in T1DM-Onset Pediatric Patients: Machine-Learning Algorithms to Classify Microorganisms as Disease Linked.

AIMS: The purpose of this work is to find the gut microbial fingerprinting of pediatric patients with type 1 diabetes. METHODS: The microbiome of 31 children with type 1 diabetes at onset and of 25 healthy children was determined using multiple polymorphic regions of the 16S ribosomal RNA. We performed machine-learning analyses and metagenome functional analysis to identify significant taxa and their metabolic pathways content. RESULTS: Compared with healthy controls, patients showed a significantly higher relative abundance of the following most important taxa: Bacteroides stercoris, Bacteroides fragilis, Bacteroides intestinalis, Bifidobacterium bifidum, Gammaproteobacteria and its descendants, Holdemania, and Synergistetes and its descendants. On the contrary, the relative abundance of Bacteroides vulgatus, Deltaproteobacteria and its descendants, Parasutterella and the Lactobacillus, Turicibacter genera were significantly lower in patients with respect to healthy controls. The predicted metabolic pathway more associated with type 1 diabetes patients concerns "carbon metabolism," sugar and iron metabolisms in particular. Among the clinical variables considered, standardized body mass index, anti-insulin autoantibodies, glycemia, hemoglobin A1c, Tanner stage, and age at onset emerged as most significant positively or negatively correlated with specific clusters of taxa. CONCLUSIONS: The relative abundance and supervised analyses confirmed the importance of B stercoris in type 1 diabetes patients at onset and showed a relevant role of Synergistetes and its descendants in patients with respect to healthy controls. In general the robustness and coherence of the showed results underline the relevance of studying the microbioma using multiple polymorphic regions, different types of analysis, and different approaches within each analysis.

A fast and reliable method for detecting SNP rs67384697 (Hsa-miR-148a binding site) by a single run of allele-specific real-time PCR.

Surface expression of human leukocyte antigen (HLA)-class I molecules is critical for modulating T/natural killer lymphocytes' effector functions. Among HLA molecules, HLA-C, the most recently evolved form of class I antigens, is subjected to both transcriptional and multiple post-transcriptional regulation mechanisms affecting its cell surface expression. Among the latter a region placed in the 3' untranslated region of HLA-C transcript contains the single nucleotide polymorphism (SNP) rs67384697 "G-ins/del" that has been found to be strictly associated with surface levels of HLA-C allomorphs because of the effect on the binding site of a microRNA (Hsa-miR-148a). Higher expression of HLA-C has been proved to influence HIV-1 infection via a better control of viremia and a slower disease progression. More importantly, the analysis of SNP rs67384697 "G-ins/del" combined with the evaluation of the HLA-Bw4/-Bw6 C1/C2 supratype, as well as the killer immunoglobulin-like receptor genetic asset, has proved to be pivotal in defining the status of Elite Controllers in the Caucasian population. Here we describe a new reliable and fast method of allele-specific real-time PCR to monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a in a high-throughput format that can be easily applied to studies involving large cohorts of individuals.

Moyamoya vasculopathy shows a genetic mutational gradient decreasing from East to West.

BACKGROUND: Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease characterized by bilateral steno-occlusive changes at the terminal portion of the internal carotid arteries and an abnormal vascular network at the base of the brain determining stroke in children. Patients with a similar vasculopathy and associated conditions are affected by the moyamoya syndrome (MMS). Most of the studies focused on MMD were carried out on East-Asian population. Ring Finger 213 (RNF213) has been identified as the strongest susceptibility gene for MMD in East-Asian people. Overall, 74.5% of the East-Asian patients carry the founder variant p.Arg4810Lys of RNF213 never reported in Caucasians. A different genetic landscape among the diverse ethnic populations seems to exist. METHODS: We sequenced the coding sequence region of RNF213, TGFB1 and PDGFRB in 21 ethnically homogeneous Italian children with moyamoya; comprehensive sequencing data are available from parents of eight of them. The analyses were carried out by NGS on Thermo-fisher PGM platform. We also performed a comprehensive review of the literature about the variations of these three genes in Caucasian patients. RESULTS: Several new variants of RNF213 gene were detected, in particular, two new pathogenic mutations on RNF213 (p.Trp4677Leu and p.Cys4017Ser) were identified in one MMS case and in one MMD case, respectively. Moreover, in a MMS case a new probably causing disease mutation p.Pro1063Thr of PDGFRB was detected. CONCLUSIONS: The genetic susceptibility of Asian moyamoya vasculopathy seems to differ from the Caucasian disease. No additional differences seem to exist between MMD and MMS.

Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

Activating Killer Immunoglobulin Receptors and HLA-C: a successful combination providing HIV-1 control.

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

HLA-B and HLA-C Supratyping by Pyrosequencing®.

Usually, HLA typing has been performed either by serology-based typing incubating a panel of known anti-HLA antibodies with viable lymphocytes of unknown HLA type or by molecular typing including medium-resolution HLA typing by Sequence Specific Oligonucleotide Probes (SSOP) or high-resolution HLA typing by Sequence Based Typing (SBT). Traditionally, HLA antigens have been defined using serological techniques, but these methods have several disadvantages, such as low resolution, the requirement for viable cells, and cell surface expression of HLA molecules. HLA type screening methods are categorized as low, medium, and high resolution, and only sequencing-based typing methods provide the highest resolution and are considered the gold standard for HLA typing.Among the HLA SBT based-methods, the Pyrosequencing(®) technique is an extremely versatile and accurate real-time sequencing technique with some advantages compared to classic Sanger method.Here, we describe a quick and inexpensive method that allows through the use of Pyrosequencing subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I80, or -Bw4 T80 and HLA-C1, or -C2 groups. In particular, this analysis is focused on the amino acids around residue 80. This method demonstrated good sensitivity, specificity, and reproducibility. Using a quantitative allele acquisition mode, the method provides accurate sequence information required for the definition of heterozygous and/or homozygous samples.

A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

Receptor modulation and functional activation of human CD34+ Lin- -derived immature NK cells in vitro by Mycobacterium bovis Bacillus Calmette-Guerin (BCG).

It is not yet clear whether immature NK (iNK) cells are bystanders to or rather participate in immune responses to pathogens that may colocalize in areas of NK-cell maturation such as bone marrow or lymph nodes. Mycobacteria, including Bacillus Calmette-Guerin (BCG), have been shown to interact with peripheral NK cells and in vivo may colocalize in areas of iNK-cell development. We studied infection with BCG of human cord blood CD34(+) Lin(-)-derived cultures containing myelomonocytes and iNK cells in vitro. Increased iNK-cell DNAM-1 expression, transient natural cytotoxicity receptor modulation, and production of IFN-γ were observed. Transcriptional receptor modulation was associated to BCG challenge, which determined increased iNK-cell cytotoxic activity against tumor cell lines and also increased killing of immature dendritic cells (iDCs). No requirement for cell contact was recorded for BCG-induced iNK-cell activation, while cytokine production including IL-18, IL-10, GM-CSF, and TGF-ß contributed to the observed effects. Thus, iNK cells are affected by mycobacteria in vitro and may contribute to shaping of adaptive mature innate responses through iDC-iNK cross-talk. In addition, iNK-cell activation by BCG may represent a novel additional mechanism contributing to the effects observed upon BCG administration in vivo.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

BACKGROUND AIMS: The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. METHODS: We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. RESULTS: Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. CONCLUSIONS: We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients.

BACKGROUND: Human cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97. OBJECTIVES: Here a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases). STUDY DESIGN: The system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients. RESULTS: The UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations. In particular, the H520Q viral mutation, known to increase GCV resistance (IC50=10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50=2.9) also increased 3 times. CONCLUSIONS: PSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients.

Troubleshooting fine-tuning procedures for qPCR system design.

Quantitative real-time PCR (qPCR) has been improved and optimized over the past decade for a wide range of applications. Design of primers and probes is one of the crucial steps to obtain high system efficiency of qPCR since design pitfalls influence negatively amplification performances. We report the results of some experiments. First, we demonstrate the utility of optimal primer design and concentration in PCR by constructing suboptimal primers, for instance with hairpin and primer-dimers secondarystructures, and quantifying the decrease in efficiency of amplification. Second, we show the adverse effects of the target sequence harboring stable secondary structures on the primer binding sites. Finally, we let see that the mere use of probe-based detection is not enough to ensure robustness of qPCR data, because the eventual detrimental products generated by primers not well designed may influence in any case the PCR efficiency.