Frequency of the CYP2C19*17 polymorphism in a Chilean population and its effect on voriconazole plasma concentration in immunocompromised children.

Invasive fungal infections (IFIs) are the most frequent cause of morbidity and mortality in immunocompromised children. Voriconazole is the first-line antifungal choice in the treatment of IFIs like aspergillosis. Voriconazole pharmacokinetics vary widely among patients and voriconazole is metabolized mainly in the liver by the CYP2C19 enzyme, which is highly polymorphic. The CYP2C19*17 allele is characterized by the presence of four single nucleotide polymorphisms expressing an ultra-rapid enzyme phenotype with an accelerated voriconazole metabolism, is associated with low (sub-therapeutic) plasma levels in patients treated with the standard dose. Considering that in our center a high percentage of children have sub-therapeutic levels of voriconazole when treated with standard doses, we sought to determine the frequency of the CYP2C19*17 polymorphism (rs12248560) in a Chilean population and determine the association between voriconazole concentrations and the rs12248560 variant in immunocompromised children. First, we evaluated the frequency of the rs12248560 variant in a group of 232 healthy Chilean children, and we found that 180 children (77.6%) were non-carriers of the rs12248560 variant, 49 children (21.1%) were heterozygous carriers for rs12248560 variant and only 3 children (1.3%) were homozygous carriers for rs12248560 variant, obtaining an allelic frequency of 12% for variant in a Chilean population. To determine the association between voriconazole concentrations and the rs12248560 variant, we analyzed voriconazole plasma concentrations in a second group of 33 children treated with voriconazole. In these patients, carriers of the rs12248560 variant presented significantly lower voriconazole plasma concentrations than non-carriers (p = 0,011). In this study, we show the presence of the rs12248560 variant in a Chilean population and its accelerating effect on the pharmacokinetics of voriconazole in pediatric patients. From these data, it would be advisable to consider the variant of the patient prior to calculating the dosage of voriconazole.

Efficacy and safety of withholding antimicrobial treatment in children with cancer, fever and neutropenia, with a demonstrated viral respiratory infection: a randomized clinical trial.

OBJECTIVES: To determine efficacy and safety of withholding antimicrobials in children with cancer, fever and neutropenia (FN) with a demonstrated respiratory viral infection. METHODS: Prospective, multicentre, randomized study in children presenting with FN at five hospitals in Santiago, Chile, evaluated at admission for diagnosis of bacterial and viral pathogens including PCR-microarray for 17 respiratory viruses. Children positive for a respiratory virus, negative for a bacterial pathogen and with a favourable evolution after 48 h of antimicrobial therapy were randomized to either maintain or withhold antimicrobials. Primary endpoint was percentage of episodes with uneventful resolution. Secondary endpoints were days of fever/hospitalization, bacterial infection, sepsis, admission to paediatric intensive care unit (PICU) and death. RESULTS: A total of 319 of 951 children with FN episodes recruited between July 2012 and December 2015 had a respiratory virus as a unique identified microorganism, of which 176 were randomized, 92 to maintain antimicrobials and 84 to withdraw. Median duration of antimicrobial use was 7 days (range 7-9 days) versus 3 days (range 3-4 days), with similar frequency of uneventful resolution (89/92 (97%) and 80/84 (95%), respectively, not significant; OR 1.48; 95% CI 0.32-6.83, p 0.61), and similar number of days of fever (2 versus 1), days of hospitalization (6 versus 6) and bacterial infections throughout the episode (2%-1%), with one case of sepsis requiring admission to PICU in the group that maintained antimicrobials, without any deaths. CONCLUSIONS: The reduction of antimicrobials in children with FN and respiratory viral infections, based on clinical and microbiological/molecular diagnostic criteria, should favour the adoption of evidence-based management strategies in this population.

Euroforgen-NoE collaborative exercise on LRmix to demonstrate standardization of the interpretation of complex DNA profiles.

There has been very little work published on the variation of reporting practices of mixtures between laboratories, but it has been previously demonstrated that there is little consistency. This is because there is no current uniformity of practice, so different laboratories will operate using different rules. The interpretation of mixtures is not solely a matter of using some software to provide 'an answer'. An assessment of a case will usually begin with a consideration of the circumstances of a crime. Assumptions made about the numbers of contributors follow from an examination of the electropherogram(s)--and these may differ between the prosecution and the defence hypotheses. There may be a necessity to evaluate several sets of hypotheses for any given case if the circumstances are uncertain. Once the hypotheses are formulated, the mathematical analysis is complex and can only be accomplished by the use of specialist software. In order to obtain meaningful results, it is essential that scientists are trained, not only in the use of the software, but also in the methodology to understand the likelihood ratio concept that is used. The Euroforgen-NoE initiative has developed a training course that utilizes the LRmix program to carry out the calculations. This software encompasses the recommendations of the ISFG DNA commissions on mixture interpretation and is able to interpret samples that may come from two or more contributors and may also be partial profiles. Recently, eighteen different laboratories were trained in the methodology. Afterwards they were asked to independently analyze two different cases with partial mixture DNA evidence and to write a statement court-report. We show that by introducing a structured training programme, it is possible to demonstrate, for the first time, that a high degree of standardization, leading to uniformity of results can be achieved by participating laboratories.

A new multiplex PCR for differential identification of Shigella flexneri and Shigella sonnei and detection of Shigella virulence determinants.

Most of the multiplex PCR (mPCR) used to identify Shigella do not discriminate between Shigella species or serotypes. We designed a mPCR to differentiate between S. flexneri and S. sonnei strains based on the detection of markers associated with the she pathogenicity island described in Shigella. In addition, specific primers were included to detect the Shigella virulence determinants ShET-1 and ShET-2 enterotoxin genes. The analysis of 304 Shigella strains from Chile and 79 Shigella strains from other geographic locations indicated that the mPCR described here detected all Shigella species and specifically differentiated S. flexneri and S. sonnei. The technique was sensitive, reproducible, specific and simple to perform, providing a new tool with the potential to be employed for epidemiological and diagnostic purposes.

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group.

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

STR data for the AmpFlSTR Profiler Plus and COfiler loci from the Maghreb (North Africa).

Allele frequencies for 13 STRs (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539) included in the AmpFlSTR Profiler Plus and COfiler kits were determined for a population sample from the Maghreb (Northern Africa).

Population data of 13 STRS in southern Spain (Andalusia).

Allele frequencies for the 13 STRs included in the AmpFl/STR Profiler Plus and Cofiler kits were determined for an Andalusian population (southern Spain).

Population data on the D1S1656 and D12S391 STR loci in Andalusia (south Spain) and the maghreb (north Africa).

Allele and genotype frequencies for two tetrameric short tandem repeat (STR) loci were determined in two population samples, from Andalusia (S Spain; n = 127) and the Maghreb (N Africa; n = 40). After denaturing polyacrylamide gel electrophoresis, 14 alleles were identified for D12S391 and 13 alleles for D1S1656. No deviations from the Hardy-Weinberg equilibrium were detected. Some statistical parameters of forensic interest (H, PD, EC) were also calculated, and the data obtained for both populations were compared. Sequencing data of several intermediate D12S391 alleles designated 17.3, 18.3, and 19.3 are also presented.

Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters.

The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, PGAL1, PCHA1, PCYC1-HSE2, and PGPH1. The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.

Effect of gene amplification on threonine production by yeast.

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.

Isolation of a mutant allele that deregulates the threonine biosynthesis in Saccharomyces cerevisiae.

We have cloned the yeast allele HOM3-R2, that codes for a mutant aspartate kinase which is insensitive to feedback inhibition by threonine, by gap-repair. A strain carrying this allele in a multicopy plasmid, or integrated into the genome, accumulates 14-times and 8-times more threonine than the wild-type, respectively. The sequence of the mutant allele differs from that of the wild-type in a single base pair change, namely a G by an A, at position 1355 in the open reading frame. The fact that the presence of this mutant allele in a cell induces threonine overproduction points to aspartate kinase as the key enzyme in the regulation of threonine biosynthesis in yeast.