Understanding false positives and the detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and BD MAX SARS-CoV-2 assays.

Several real-time RT-PCR assays have received Emergency Use Authorization from the United States Food and Drug Administration. The BD MAX™ SARS-CoV-2 assay, run by the BD MAX™ system, is a qualitative test that detects the SARS-CoV-2 specific nucleocapsid phosphoprotein gene regions, N1 and N2. The human RNase P gene is used as the endogenous nucleic acid extraction control. The Cepheid Xpert® Xpress SARS-CoV-2 assay, run by the GeneXpert system, detects the pan-sarbecovirus E gene and the N2 region of the N gene. We evaluated the performance characteristics of the BD and Cepheid assays using matched patient samples. We also analyzed comparative Ct values for both assays using 183 positive samples tested at this facility. In addition, we mitigated reporting false positive results without relying on interpretive software. We found that both systems showed comparable sensitivity. We found an approximately 3.5% false positive rate from the BD MAX™ system results.

Asynchronous Testing of 2 Specimen-Diversion Devices to Reduce Blood Culture Contamination: A Single-Site Product Supply Quality Improvement Project.

OBJECTIVE: Blood culture contamination above the national threshold has been a consistent clinical issue in the ED setting. Two commercially available devices were examined that divert an initial small volume of the specimen before the collection of blood culture to reduce skin contamination. METHODS: Prospectively, 2 different blood culture-diversion devices were made available in the unit supplies to ED clinicians at a single site during 2 different periods of time as a follow-up strategy to an ongoing quality improvement project. Blood samples were collected in the emergency department over a period of 16 months. A retrospective record review study was conducted comparing the use of the 2 specimen-diversion devices with no device (control group) for blood culture contamination rates. The main outcome of monthly blood culture contamination per device was tested using a Bayesian Poisson multilevel regression model. RESULTS: A total of 4030 blood samples were collected and analyzed from November 2017 to February 2019. The model estimated that the mean incidence of contaminated blood draws in the device A group was 0.29 (0.14-0.55) times the incidence of contaminated draws in the control group. The mean incidence of contaminated blood draws in the device B group was 0.23 (0.13-0.37) times the incidence of contaminated draws in the control group, suggesting that initial-diversion methods reduced blood culture contamination. CONCLUSION: Initial specimen-diversion devices supplement present standard phlebotomy protocols to bring down the blood culture contamination rate.

Quimioterapia neoadyuvante como tratamiento para la preservación de la fertilidad en pacientes con cáncer de cérvix en estadio IB: reporte de un caso y revisión de la literatura / Neo-adjuvant chemotherapy as treatment for fertility preservation in patients with stage IB cervical cancer: Case presentation and review of the literature

Presentar el caso de una paciente con cáncer de cérvix en estadio IB1 mayor de dos centímetros, a quien se le administró quimioterapia neoadyuvante seguida de traquelectomía radical con linfadenectomía pélvica por laparoscopia, con el fin de preservar su fertilidad; hacer una revisión de los casos reportados en la literatura con énfasis en los resultados obstétricos y oncológicos.Materiales y métodos: presentación del caso y bésqueda en Medline vía PubMed de los artículos publicados en inglés, francés y español desde enero de 2000 hasta agosto de 2014, sobre la administración de quimioterapia neoadyuvante y cirugía conservadora de la fertilidad en pacientes con cáncer de cérvix en estadio IB.Resultados: se seleccionaron 12 artículos pertinentes, para un total de 55 pacientes. Con un seguimiento entre 14 y 69 meses, se reportaron 4 recaídas y solo una muerte por enfermedad. Resultados obstétricos: 30 embarazos, 24 nacimientos, 3 abortos, 1 embarazo ectópico y 2 embarazos en curso.Conclusión: la quimioterapia neoadyuvante, seguida de cirugía conservadora de la fertilidad, puede considerarse como una alternativa de manejo en pacientes seleccionadas con cáncer de cérvix en estadio IB, con tumores voluminosos, que deseen preservar su fertilidad...

To present the case of a patient with stage IB1 cervical cancer larger than 2 centimetres who received neo-adjuvant chemotherapy followed by laparoscopic radical cervicectomy and pelvic lymphadenectomy for fertility preservation; and to review the literature for case reports emphasizing obstetric and oncologic outcomes.Materials and methods: Case presentation and search of the literature in Medline through PubMed of articles published in English, French and Spanish between January 2000 and August 2014 on the topic of neo-adjuvant chemotherapy and fertility preservation surgery in patients with stage IB cervical cancer.Results: Overall, 12 relevant articles were selected totalling 55 patients. Over follow-up period ranging between 14 and 69 months there were 4 relapses and only one death attributable to the disease. Obstetric outcomes included 30 pregnancies, 24 births, 3 miscarriages, 1 ectopic pregnancy, and 2 on going pregnancies.Conclusion: Neo-adjuvant chemotherapy followed by conservative, fertility preservation surgery may be an option for the management of selected patients with stage IB cervical cancer involving large tumours sizes, who wish to preserve their fertility...

Purine nucleoside phosphorylase deficiency: a mutation update.

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.

Recém-nascidos prematuros e a alta hospitalar: uma revisão integrativa sobre a atuação da enfermagem / Premature newborns and hospital discharge: an integrative review on nursing performance

O objetivo desta pesquisa foi investigar evidências sobre as ações de enfermagem utilizadas junto às mães de prematuros internados em Unidades Neonatais no preparo da alta e apontar dificuldades advindas da prática. As bases Lilacs, Bdenf, Medline e a biblioteca eletrônica SCIELO foram utilizadas para a seleção de sete artigos entre 2004 e 2010. As principais ações de enfermagem citadas foram: apoio ao aleitamento materno; cuidados de higiene; orientações sobre ordenha e vacinação; estímulo ao vínculo afetivo e acompanhamento ambulatorial após a alta. Três artigos relataram dificuldades encontradas sendo elas: intervenção dos pais nos cuidados; problemas de relacionamento entre família e profissionais; e medo dos pais em realizar os cuidados. Concluímos ser necessária a padronização das informações fornecidas pelas equipes de saúde nas Unidades Neonatais, bem como uma sistematização do preparo dos pais para a alta.

Niveles de actividad física, barreras y estados de cambio en una población urbana de un municipio de Colombia / Levels of physical activity, barriers, and stage of change in an urban population from a municipality in Colombia

Objetivo: Evaluar el nivel de actividad física, barreras y estados de cambio, en la población urbana entre 25 y 50 añosde edad del municipio de Santa Rosa de Osos, Antioquia.Materiales y métodos: Se realizó un estudio de prevalencia, con muestreo aleatorio bietápico en 357 personas sinenfermedad cardiovascular conocida; se administró una encuesta prediseñada para evaluar los niveles de actividad física, losestados de cambio y las barreras para la práctica de la actividad física.Resultados: La actividad física baja total fue del 9% y por ámbitos: trabajo 66.9%; transporte 60.8%; hogar 44.8% ytiempo libre 76.2%. Las barreras más relevantes para la práctica de la actividad física fueron la ®carencia de voluntad¼ (70%)y la falta de tiempo (46.2%). Los estados de cambio más prevalentes fueron el ®contemplativo¼ (40.3%) y el de ®preparación¼(17.1%).Conclusión: Se encontraron niveles de actividad física baja en los diferentes ámbitos de la vida diaria, acompañados deuna prevalencia alta de las barreras y de los estados previos a la práctica de la actividad física, principalmente en las mujeresy las personas obesas.

Trinucleotide repeat variants in the promoter of the thiopurine S-methyltransferase gene of patients exhibiting ultra-high enzyme activity.

Thiopurine S-methyl transferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of the thiopurine immunosuppressants. To date, 22 variants have been identified that are predictive of decreased TPMT activity. In contrast, no molecular explanation has been found for the 1-2% of Caucasians who exhibit ultra-high TPMT activity. Here, we report the characterization of polymorphisms within a trinucleotide (GCC) repeat element of the TPMT promoter in two patients with inflammatory bowel disease exhibiting the highest TPMT activity from two testing centres. The first patient was heterozygous for a variant allele carrying seven GCC repeats [(GCC)7], whereas the second patient was heterozygous for a variant allele containing five GCC repeats [(GCC)5]. Fifty patients with inflammatory bowel disease with normal TPMT activity were all homozygous for six GCC repeats [(GCC)6]. Of 200 healthy controls, five were found to be heterozygous for the (GCC)7 variant. Within in vitro reporter gene assays, the mean luciferase activities of the (GCC)6, (GCC)7, and (GCC)5 constructs were 8.0+/-0.26, 13.2+/-0.10 and 12.3+/-0.12, respectively. The significant increase in activity observed for (GCC)5 and (GCC)7 compared with (GCC)6 (P-value

Polymorphisms in folate, pyrimidine, and purine metabolism are associated with efficacy and toxicity of methotrexate in psoriasis.

Methotrexate is the gold standard therapy for moderate to severe psoriasis, but there is marked interpersonal variation in its efficacy and toxicity. We hypothesized that in psoriasis patients, specific common polymorphisms in folate, pyrimidine, and purine metabolic enzymes are associated with methotrexate efficacy and/or toxicity. DNA from 203 retrospectively recruited psoriasis patients treated with methotrexate was collected and genotyped by restriction endonuclease digestion or length polymorphism assays. The reduced folate carrier (RFC) 80A allele and the thymidylate synthase (TS) 3'-untranslated region (3'-UTR) 6 bp deletion were associated with methotrexate-induced toxicity (P=0.025 and P=0.025, respectively). RFC 80A and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) 347G were associated with methotrexate discontinuation (P=0.048 and P=0.038). The TS 5'-UTR 28 bp 3R polymorphism correlated with poor clinical outcome (P=0.029), however, this was not the case when patients with palmoplantar pustular psoriasis were not included in the analysis. Stronger associations between specific polymorphisms and methotrexate-induced toxicity and discontinuation were found in a subanalysis of patients on methotrexate not receiving folic acid supplementation. We have demonstrated preliminary evidence that specific polymorphisms of enzymes involved in folate, pyrimidine, and purine metabolism could be useful in predicting clinical response to methotrexate in patients with psoriasis.

The ITPA c.94C>A and g.IVS2+21A>C sequence variants contribute to missplicing of the ITPA gene.

Inosine triphosphate pyrophosphatase (ITPase) catalyzes the conversion of inosine triphosphate (ITP) to the correspondent monophosphate. The ITPA c.94C>A and g.IVS2+21A>C allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94C>A [P32T] sequence variant is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The aim of this study was to explore the molecular mechanisms of ITPase deficiency. ITPA mRNA was extracted from peripheral blood leukocytes (PBL), Epstein-Barr virus transformed lymphoblast cell cultures, reticulocytes, and cultured fibroblast from patients with known ITPA genotypes. ITPA mRNA was reversed transcribed, sequenced and the relative amounts of misspliced transcripts quantitated from three independent experiments. The ITPA g.IVS2+21A>C sequence variant resulted in missplicing of exon 3. The ITPA c.94C>A allelic variant resulted in missplicing of exons 2 and 3 representing, in PBL samples, 61% of the total mRNA expressed in ITPA c.94C>A homozygotes. We proposed that the ITPA c.94C>A allelic variant destroys an exonic splicing silencing (ESS) element in exon 2, resulting in the activation of two nearby upstream 5' splice sites and missplicing of the exons 2 and 3 cassette causing structural changes to the enzyme and contributing to ITPase deficiency.

Typing TPMT and ITPase to detect azathioprine toxicity.

Genetic variation in thiopurine methyltransferase (TPMT) enzyme activity strongly predicts adverse effects in patients treated with the thiopurine drugs, azathioprine and 6-mercaptopurine, and is one of the classic examples of pharmacogenetics in clinical practice. Recently, polymorphic variation in the gene encoding the enzyme inosine triphosphatase (ITPase) has also been associated with risk of thiopurine-related toxicity. This article reviews the evidence for the role of TPMT and ITPase deficiency as predictors of thiopurine toxicity and makes recommendations for clinical and laboratory practice.

Pharmacogenetic association with adverse drug reactions to azathioprine immunosuppressive therapy following liver transplantation.

Azathioprine (AZA) is a thiopurine prodrug commonly used in triple-immunosuppressive therapy following liver transplantation. Approximately 1 in 10 patients suffers side effects in response to the drug, the most problematic being bone marrow toxicity. There is evidence that polymorphisms in the genes encoding thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPase) predict adverse drug reactions to AZA therapy. Furthermore, common genetic polymorphisms in the gene encoding methylenetetrahydrofolate reductase (MTHFR) may have an indirect impact on thiopurine drug methylation by influencing levels of the methyl donor S-adenosylmethionine (SAM). The aim of this study was to determine whether polymorphisms in these candidate pharmacogenetic loci predict adverse drug reactions to AZA immunosuppressive therapy in liver transplant patients. A series of 65 liver transplant recipients were recruited to the study from the Liver Transplant Out-Patient clinic at The Royal Infirmary of Edinburgh. Clinical response to AZA was retrospectively correlated against TPMT activity, TPMT*2, *3A, and *3A genotypes, inosine triphosphatase (ITPA) 94C>A and IVS2+21A>C genotypes, and MTHFR 677C>T and 1298A>C genotypes. Variant TPMT, ITPA, and MTHFR genotypes were not significantly associated with adverse drug reactions to AZA, including bone marrow suppression. However, the 2 patients who suffered nodular regenerative hyperplasia (NRH) were both heterozygous for the TPMT*3A mutation. In conclusion, our findings suggest that TPMT, ITPA, and MTHFR genotypes do not predict adverse drug reactions, including bone marrow suppression, in liver transplant patients. However, the possible association between NRH and a heterozygous TPMT genotype should be investigated further.

Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase).

Adverse drug reactions to azathioprine (AZA), the pro-drug of 6-mercaptopurine (6-MP), occur in 15% to 28% of patients and the majority are not explained by thiopurine methyltransferase (TPMT) deficiency. Inosine triphosphate pyrophosphatase (ITPase) deficiency results in the benign accumulation of the inosine nucleotide ITP. 6-MP is activated through a 6-thio-IMP intermediate and, in ITPase deficient patients, potentially toxic 6-thio-ITP is predicted to accumulate. The association between polymorphism in the ITPA gene and adverse drug reactions to AZA therapy was studied in patients treated for inflammatory bowel disease. Sixty-two patients with inflammatory bowel disease suffering adverse drug reactions to AZA therapy were genotyped for ITPA 94C>A and IVS2 + 21A>C polymorphisms, and TPMT*3A, *3C, *2 polymorphisms. Genotype frequencies were compared to a consecutive series of 68 controls treated with AZA for a minimum of 3 months without adverse effect. The ITPA 94C>A deficiency-associated allele was significantly associated with adverse drug reactions [odds ratio (OR) 4.2, 95% confidence interval (CI) 1.6-11.5, P = 0.0034]. Significant associations were found for flu-like symptoms (OR 4.7, 95% CI 1.2-18.1, P = 0.0308), rash (OR 10.3, 95% CI 4.7-62.9, P = 0.0213) and pancreatitis (OR 6.2,CI 1.1-32.6, P = 0.0485). Overall, heterozygous TPMT genotypes did not predict adverse drug reactions but were significantly associated with a subgroup of patients experiencing nausea and vomiting as the predominant adverse reaction to AZA therapy (OR 5.5, 95% CI 1.4-21.3, P = 0.0206). Polymorphism in the ITPA gene predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6-thioguanine, should be considered for AZA-intolerant patients with ITPase deficiency.

Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype in British Asians and Caucasians.

OBJECTIVE: Polymorphisms in the TPMT gene open reading frame (ORF) are associated with reduced TPMT activity. Variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the gene consisting of combinations of Type A, B and C repeat units, may modulate TPMT activity. Here we present the allele frequencies of genetic modifiers of TPMT activity in a British Asian population, as well as the concordance between intermediate TPMT activity and ORF and VNTR genotypes in a predominantly Caucasian population. METHODS: VNTR type and ORF mutations were determined in two selected TPMT activity ranges, intermediate activity (4-8 U, 108 patients), normal (12-15 U, 53 patients) and in 85 British Asians. RESULTS: In British Asians, TPMT*3C was the prevalent mutant allele (four heterozygotes). One patient was heterozygous for TPMT*3A. Overall VNTR frequencies did not differ from Caucasians. Three new VNTR alleles were designated VNTR*6c, VNTR*6d, and VNTR*7c. Forty-one percent of patients with intermediate activity were heterozygous for a TPMT ORF mutation (3A, 2B, 1C). Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D' = 1), VNTR*4b - TPMT*3C (D' = 0.67) and VNTR*6a - TPMT*1 (D' = 1) alleles. As a result, significant differences (P < 0.05) in the distribution of Type A, B or the total number of repeats summed for both alleles, were found between the ORF heterozygous intermediate activity group and the wild-type intermediate or normal activity groups. No significant difference was found between the two wild-type groups. CONCLUSION: Our results suggest that TPMT gene VNTRs do not significantly modulate enzyme activity.

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G-->A, 561G-->A, 708G-->A) and two associated with ITPase deficiency (94C-->A, IVS2+21A-->C). Homozygotes for the 94C-->A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C-->A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A-->C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C-->A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A-->C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A-->C heterozygotes and 94C-->A/IVS2+21A-->C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.