Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

Identification of HLA-C alleles using PCR—single-strand-conformation polymorphism and direct sequencing

Alleles encoding HLA-C antigens in Japanese were identified by polymerase chain reaction followed by single strand conformation polymorphism (PCR-SSCP) and nucleotide sequencing analyses. The results showed that at least sixteen different alleles code for eight serologically detectable antigen groups and undetectable blanks. Cwl was mainly encoded by Cw*0102, whereas two split antigens of Cw3, Cw9 and Cw10, were encoded by Cw*0303 and Cw*0304, respectively. Cw4 and Cw6 were encoded by Cw*0401 and Cw*0602, respectively. Seven alleles, Cw*0801, Cw*0803, Cw*1202, Cw*1203, Cw*1402, Cw*1403 and Cw*1502, were found to encode serological HLA-C "blanks" in Japanese. Moreover, errors in the published nucleotide sequences of Cw*0501 and Cw*1201 were corrected. Twenty-one HLA-C alleles were distinguished from each other by means of group-specific PCR amplification followed by the SSCP method developed in the present study. The system using genomic DNAs can be used effectively for identification of new HLA-C alleles.     

For-profit hospitals for Europe: the case of Britain and France

The paper first describes the structural characteristics of the for-profit private sector in Britain and in France. In Britain, the recent growth of the for-profit private hospital's sector, although still a small sector, is related to the growth of private insurance. Cost-containment however coupled to rapid increases in premiums is slowing down the momentum. Moreover, the NHS starts charging private hospitals for such services like blood banks. In France the private sector has always been strong, although living in a highly regulated and dependent symbiosis with the public sector. In a second part, speculations are made on the crucial question whether for-profit hospitals are a legitimate alternative for Europe. In the end, an in-between solution is opted for by which the public sector would sub-contract certain functions like data processing, management and others, from the commercial sector.     

2018—2021年成都市非EV - A71感染手足口病患者住院费用及其影响因素研究

目的 了解成都市非EV - A71手足口病患者住院费用及其影响因素。方法 通过成都市公共卫生临床中心病案管理系统,收集2018—2021年手足口病住院病人信息。采用秩和检验、多元线性回归分析不同血清型患者费用的影响因素。结果 共纳入2 310例手足口病患者,直接医疗费用中位数为4 187元。秩和检验结果显示,CV - A10患者的住院天数、重症的费用差异有统计学意义（U = - 4.40, - 3.59; P＜0.05）;CV - A16患者的住院天数、高热的费用差异有统计学意义（U = - 4.90, - 2.51; P＜0.05）;CV - A6患者的性别、住院天数、并发症、重症、支付方式的费用差异有统计学意义（U = - 2.81, - 13.54, - 4.00, - 4.28;H = 13.65; P＜0.05）;其他肠道病毒患者的住院天数、并发症、重症、支付方式的费用差异有统计学意义（U = - 7.46, - 3.25, - 2.45;H = 13.14;P＜0.05）;混合感染患者高热的费用差异有统计学意义（U = - 2.40, P＜0.05）。多元线性回归结果显示,除混合感染外,住院天数是所有患者费用的影响因素。在CV - A10、CV - A6和其他肠道病毒中重症患者的费用更高;在CV - A6和其他肠道病毒中有并发症的患者费用更高。高热是混合感染患者费用的影响因素。结论 成都市非EV - A71感染手足口病患者的病毒血清型不同,费用的影响因素也有所不同。     

毕节市敏感肺结核患者家庭灾难性支出情况分析

目的 了解毕节市敏感肺结核患者家庭疾病灾难性支出（CTC）及影响因素,为降低患者家庭负担提供政策建议。方法 以2020年5—6月期间毕节市登记并在调查时已成功治疗的敏感患者为调查对象,通过概率比例抽样对其进行问卷访谈。灾难性支出采用率描述,用χ2检验和二分类logistic回归分析其原因。结果 毕节市309例肺结核患者家庭总自付费用 M （ P 25, P 75）为5 169.31（2 098.52,14 778.49）元;直接医疗费用、直接非医疗费用和间接费用 M （ P ? P ?分别为1 688.15（987.31,3 337.50）、720.00（200.00,1 425.00）和1 200.00（0.00,8 390.00）,差异具有统计学意义（χ2=63.545, P ＜0.001）。309例患者家庭CTC发生率为52.43%;其中,家庭年收入较低（ OR =25.740,95% CI :9.676～68.473）、住院（ OR =3.515,95% CI :1.782～6.933）、确诊延迟（ OR =2.492,95% CI :1.297～4.789）是导致其发生CTC的危险因素（ P 均＜0.05）。结论 毕节市肺结核患者经济负担以间接费用为主,CTC发生率较为普遍。其中,家庭年收入低、住院和确诊延迟患者是其重要危险因素,可针对性地加强患者健康教育、落实医疗机构诊疗规范以及通过政府和社会给予患者补偿,共同降低其疾病负担减少CTC情况。     

Burden of Providing Informal Care for Patients with Atrial Fibrillation

ObjectivesPatients with atrial fibrillation (AF) have rapid and irregular heart rates, increasing the risk of comorbidities and mortality. Next to formal medical care, many patients receive informal care from their social environment. The objective of this study was to examine the well-being and economic burden of providing informal care to patients with AF in the UK, Italy, and Germany.MethodsCaregivers of patients with AF completed an online survey based on the iMTA Valuation of Informal Care Questionnaire, with questions about their caregiving situation, perceived burden of caregiving, and absence from work due to health problems resulting from caregiving. Care-related quality-of-life utilities were calculated using the Care-related Quality of Life instrument and associated tariffs. Societal costs of caregiving were calculated based on the proxy good method.ResultsA total of 585 caregivers participated in this study. On average, caregivers provided 33 hours of informal care per week to patients (SD 29 hours). On a scale from 0 to 10, their self-rated burden was 5.4. The average Care-related Quality of Life utility was 72. Caregivers primarily indicated problems with daily activities, mental health, and physical health. Still, the vast majority of caregivers (87%) derived fulfillment from providing care. Weekly societal costs of caregiving were on average €636. Comorbidities contributed substantially to the caregiver time and burden.ConclusionsCaring for a patient with AF is associated with substantial objective and subjective burden, but also provides fulfillment from being able to care for a loved one.     

A Multidisciplinary,Community-Based Program to Reduce Unplanned Hospital Admissions

ObjectivesTo evaluate the effect of Hospital Admission Risk Program (HARP) on unplanned hospitalization, bed days, and mortality of enrolled individuals and to evaluate the cost-effectiveness of HARP.DesignA retrospective longitudinal analysis of hospital administrative data.InterventionIndividuals at risk of hospitalization were provided with multidisciplinary, community-based care support managed by care coordinators including integrated care planning, education, monitoring, service linkages, and general practitioner liaison over 6-9 months.Setting and ParticipantsIndividuals who were enrolled into 1 of 8 HARP chronic disease management programs between July 1, 2017, and June 30, 2018, at the Royal Melbourne Hospital, Australia.MethodsHospital admissions between 18 months before and 18 months after HARP enrollment were analyzed. Total hospital costs were compared between 18 months before and 12 months after HARP enrollment.ResultsA total of 1553 individuals with a median age of 71 years (interquartile range 60-81), 63.4% males, were admitted to HARP. Both unplanned hospitalizations and bed days were reduced during the HARP intervention compared to within 3 months before enrollment in each of the HARP management programs. After the HARP intervention, cardiac coach, cardiac heart failure, chronic respiratory, diabetes comanagement, and medication management programs had higher hospitalizations and bed days than individuals’ baseline of at least 3 months before HARP enrollment. Individuals in cardiac heart failure and chronic respiratory management programs had a higher mortality rate than other HARP chronic disease management programs. Individuals in cardiac coach, diabetes comanagement, and medication management programs had lower hospital costs during the HARP intervention compared to within 3 months before HARP enrollment.Conclusions and ImplicationsHARP reduced unplanned hospitalization and bed days but did not return individuals’ hospital use to baseline before the intervention. The variations in mortality between HARP chronic disease management programs implies that condition-specific goals between programs is preferable.     

Formal and Informal Costs of Care for People With Dementia Who Experience a Transition to Hospital at the End of Life: A Secondary Data Analysis

ObjectivesTo explore formal and informal care costs in the last 3 months of life for people with dementia, and to evaluate the association between transitions to hospital and usual place of care with costs.DesignCross-sectional study using pooled data from 3 mortality follow-back surveys.Setting and ParticipantsPeople who died with dementia.MethodsThe Client Service Receipt Inventory survey was used to derive formal (health, social) and informal care costs in the last 3 months of life. Generalized linear models were used to explore the association between transitions to hospital and usual place of care with formal and informal care costs.ResultsA total of 146 people who died with dementia were included. The mean age was 88.1 years (SD 6.0), and 98 (67.1%) were female. The usual place of care was care home for 85 (58.2%). Sixty-five individuals (44.5%) died in a care home, and 85 (58.2%) experienced a transition to hospital in the last 3 months. The mean total costs of care in the last 3 months of life were £31,224.7 (SD 23,536.6). People with a transition to hospital had higher total costs (£33,239.2, 95% CI 28,301.8-39,037.8) than people without transition (£21,522.0, 95% CI 17,784.0-26,045.8), mainly explained by hospital costs. People whose usual place of care was care homes had lower total costs (£23,801.3, 95% CI 20,172.0-28,083.6) compared to home (£34,331.4, 95% CI 27,824.7-42,359.5), mainly explained by lower informal care costs.Conclusions and ImplicationsTotal care costs are high among people dying with dementia, and informal care costs represent an important component of end-of-life care costs. Transitions to hospital have a large impact on total costs; preventing these transitions might reduce costs from the health care perspective, but not from patients' and families' perspectives. Access to care homes could help reduce transitions to hospital as well as reduce formal and informal care costs.