An investigation of fingerstick blood collection for pointof- care HIV-1 viral load monitoring in South Africa

Background: Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution.Objective: To assess the feasibility and ease of collecting 150 μL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture.Methods: Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded.Results: One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 μL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay.Conclusion: The findings of this study support the feasibility of collecting 150 μL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting

Standard reference sequences for submission of HLA genotyping for the 18th International HLA and Immunogenetics Workshop

The International human leukocyte antigen (HLA) and Immunogenetics Workshops (IHIWs) have fostered international collaborations of researchers and experts in the fields of HLA, histocompatibility and immunology. These IHIW collaborations have comprised many projects focused on achieving a variety of specific goals. The international and collaborative nature of these projects necessitates the collection and analysis of complex data generated in multiple laboratories, often using multiple methods of acquisition. Collection and storage of these data in a consistent way adds value to IHIW projects, which can be extended to future work. DNA-based genotyping data, especially HLA genotyping data, can be transmitted in the form of a Histoimmunogenetics Markup Language (HML) document. HML facilitates clear communication of a genotype and supporting metadata, such as, sequencing platform, laboratory assays, consensus sequence, and interpretation. Sequence information can be reported relative to known reference sequences, which add meaning and context to genotypes. Selecting the correct reference sequence for a given allele sequence is nuanced, and guidelines have emerged through collaborative community efforts such as Data Standards Hackathons. Here, we describe the guidelines established for the selection of reference sequences to be used in transmission of HLA (and MICA/MICB) genotyping data for the 18th IHIW.Transplantation and autoimmunit

Clustering and Alignment of Polymorphic Sequences for HLA-DRB1 Genotyping

Located on Chromosome 6p21, classical human leukocyte antigen genes are highly polymorphic. HLA alleles associate with a variety of phenotypes, such as narcolepsy, autoimmunity, as well as immunologic response to infectious disease. Moreover, high resolution genotyping of these loci is critical to achieving long-term survival of allogeneic transplants. Development of methods to obtain high resolution analysis of HLA genotypes will lead to improved understanding of how select alleles contribute to human health and disease risk. Genomic DNAs were obtained from a cohort of n = 383 subjects recruited as part of an Ulcerative Colitis study and analyzed for HLA-DRB1. HLA genotypes were determined using sequence specific oligonucleotide probes and by next-generation sequencing using the Roche/454 GSFLX instrument. The Clustering and Alignment of Polymorphic Sequences (CAPSeq) software application was developed to analyze next-generation sequencing data. The application generates HLA sequence specific 6-digit genotype information from next-generation sequencing data using MUMmer to align sequences and the R package diffusionMap to classify sequences into their respective allelic groups. The incorporation of Bootstrap Aggregating, Bagging to aid in sorting of sequences into allele classes resulted in improved genotyping accuracy. Using Bagging iterations equal to 60, the genotyping results obtained using CAPSeq when compared with sequence specific oligonucleotide probe characterized 4-digit genotypes exhibited high rates of concordance, matching at 759 out of 766 (99.1%) alleles. © 2013 Ringquist et al

Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string

Knowledge of an individual's human leukocyte antigen (HLA) genotype is essential for modern medical genetics, and is crucial for hematopoietic stem cell and solid-organ transplantation. However, the high levels of polymorphism known for the HLA genes make it difficult to generate an HLA genotype that unambiguously identifies the alleles that are present at a given HLA locus in an individual. For the last 20 years, the histocompatibility and immunogenetics community has recorded this HLA genotyping ambiguity using allele codes developed by the National Marrow Donor Program (NMDP). While these allele codes may have been effective for recording an HLA genotyping result when initially developed, their use today results in increased ambiguity in an HLA genotype, and they are no longer suitable in the era of rapid allele discovery and ultra-high allele polymorphism. Here, we present a text string format capable of fully representing HLA genotyping results. This Genotype List (GL) String format is an extension of a proposed standard for reporting killer-cell immunoglobulin-like receptor (KIR) genotype data that can be applied to any genetic data that use a standard nomenclature for identifying variants. The GL String format uses a hierarchical set of operators to describe the relationships between alleles, lists of possible alleles, phased alleles, genotypes, lists of possible genotypes, and multilocus unphased genotypes, without losing typing information or increasing typing ambiguity. When used in concert with appropriate tools to create, exchange, and parse these strings, we anticipate that GL Strings will replace NMDP allele codes for reporting HLA genotypes

Estimating KIR Haplotype Frequencies on a Cohort of 10,000 Individuals: A Comprehensive Study on Population Variations, Typing Resolutions, and Reference Haplotypes

The killer cell immunoglobulin-like receptors (KIR) mediate human natural killer (NK) cell cytotoxicity via activating or inhibiting signals. Although informative and functional haplotype patterns have been reported, most genotyping has been performed at resolutions that are structurally ambiguous. In order to leverage structural information given low-resolution genotypes, we performed experiments to quantify the effects of population variations, reference haplotypes, and genotyping resolutions on population-level haplotype frequency estimations as well as predictions of individual haplotypes. We genotyped 10,157 unrelated individuals in 5 populations (518 African American[AFA], 258 Asian or Pacific Islander [API], 8,245 European[EUR], 1,073 Hispanic[HIS], and 63 Native American[NAM]) for KIR gene presence/absence (PA), and additionally half of the AFA samples for KIR gene copy number variation (CNV). A custom EM algorithm was used to estimate haplotype frequencies for each population by interpretation in the context of three sets of reference haplotypes. The algorithm also assigns each individual the haplotype pairs of maximum likelihood. Generally, our haplotype frequency estimates agree with similar previous publications to within <5% difference for all haplotypes. The exception is that estimates for NAM from the U.S. showed higher frequency association of cB02 with tA01 (+14%) instead of tB01 (-8.5%) compared to a previous study of NAM from south of the U.S. The higher-resolution CNV genotyping on the AFA samples allowed unambiguous haplotype-pair assignments for the majority of individuals, resulting in a 22% higher median typing resolution score (TRS), which measures likelihood of self-match in the context of population-specific haplo- and geno-types. The use of TRS to quantify reduced ambiguity with CNV data clearly revealed the few individuals with ambiguous genotypes as outliers. It is observed that typing resolution and reference haplotype set influence haplotype frequency estimates. For example, PA resolution may be used with reference haplotype sets up to the point where certain haplotypes are gene-content subsets of others. At that point, CNV must be used for all genes.This study was supported by funding from Office of Naval Research grants ONR N00014-12-1-0142, ONR N00014-13-1-0039, ONR N00014-14-1-0028 and ONR N00014-15-1- 0848. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa

Background. Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution.Objective. To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture.Methods. Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded.Results. One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay.Conclusion. The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting