Irene Vanni

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

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.

Human leukocyte antigen–B (-Bw6/-Bw4 I80, T80) and human leukocyte antigen–C (-C1/-C2) subgrouping using pyrosequence analysis

Specific combinations of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I molecules characterized by a particular residue 80 are significantly associated with outcomes in different pathologic conditions, such as autoimmunity, pathogenic infection, cancer, and reproductive failure. Thus, a simplified method for HLA typing used in association with the analysis of KIR genotype (Kirotype) is of particular interest to extend the analysis of larger series. Here, we describe a quick and inexpensive method that allows use of pyrosequencing, a helpful subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I(80) or -Bw4 T(80), HLA-C1, or -C2 groups and HLA-A allotypes sharing Bw4+ epitope or the rare HLA-B allotypes displaying the C1 motif. In particular, this analysis is focused on the amino acids around residue 80, known to be relevant in defining the affinity of KIR/HLA interaction and in the functional effects. This method was demonstrated to have good sensitivity, specificity, and reproducibility of detection and it was validated using a panel of HLA-typed International Histocompatibility Workshop (IHW) cell lines and clinical isolates. Using an allele quantitative acquisition mode, the method permitted us to obtain an accurate sequencing as required in heterozygous and/or homozygous sample definition.

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. J. Clin. Lab. Anal. 25:389–394, 2011.

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.

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.

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.