The Health Resources and Services Administration's Ryan White HIV/AIDS Program in rural areas of the United States: Geographic distribution, provider characteristics, and clinical outcomes.

BACKGROUND: People living with HIV (PLWH) residing in rural areas experience substantial barriers to HIV care, which may contribute to poor HIV health outcomes, including retention in HIV care and viral suppression. The Health Resources and Services Administration's Ryan White HIV/AIDS Program (HRSA RWHAP) is an important source of HIV medical care and support services in rural areas. The purpose of this analysis was to (1) assess the reach of the RWHAP in rural areas of the United States, (2) compare the characteristics and funded services of RWHAP provider organizations in rural and non-rural areas, and (3) compare the characteristics and clinical outcomes of RWHAP clients accessing medical care and support services in rural and non-rural areas. METHODS AND FINDINGS: Data for this analysis were abstracted from the 2017 RWHAP Services Report (RSR), the primary source of annual, client-level RWHAP data. Organizations funded to deliver RWHAP any service ("RWHAP providers") were categorized as rural or non-rural according to the HRSA FORHP's definition of modified Rural-Urban Commuting Area (RUCA) codes. RWHAP clients were categorized based on their patterns of RWHAP service use as "visited only rural providers," "visited only non-rural providers," or "visited rural and non-rural providers." In 2017, among the 2,113 providers funded by the RWHAP, 6.2% (n = 132) were located in HRSA-designated rural areas. Rural providers were funded to deliver a greater number of service categories per site than non-rural providers (44.7% funded for ≥5 services vs. 34.1% funded for ≥5 services, respectively). Providers in rural areas served fewer clients than providers in non-rural areas; 47.3% of RWHAP providers in rural areas served 1-99 clients, while 29.6% of non-rural providers served 1-99 clients. Retention in care and viral suppression outcomes did not differ on the basis of whether a client accessed services from rural or non-rural providers. CONCLUSIONS: RWHAP providers are a crucial component of HIV care delivery in the rural United States despite evidence of significant barriers to engagement in care for rural PLWH, RWHAP clients who visited rural providers were just as likely to be retained in care and reach viral suppression as their counterparts who visited non-rural providers. The RWHAP, especially in partnership with Rural Health Clinics and federally funded Health Centers, has the infrastructure and expertise necessary to address the HIV epidemic in rural America.

Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.

Microsatellite instability (MSI) analysis of colorectal cancers is clinically useful to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC) caused by germline mutations of mismatch repair genes. MSI status may also predict cancer response/resistance to certain chemotherapies. We evaluated the MSI Analysis System (Promega Corp.; five mononucleotide and two pentanucleotide repeats) and compared the results to the Bethesda panel, which interrogates five microsatellite loci recommended by the 1997 National Cancer Institute-sponsored MSI workshop (three dinucleotide and two mononucleotide repeats). Thirty-four colorectal cancers were analyzed by both assays. The overall concordance between the two assays was 85% (29 of 34). There was complete concordance between the two assays for all of the MSI-high (11 of 11) and microsatellite stable (MSS; 18 of 18) cases. In the 11 MSI-high cases, all 5 of the mononucleotide loci in the MSI Analysis System demonstrated shifted alleles (100% sensitivity), and each shift resulted in products that were smaller in size than the germline alleles. All (5 of 5) of the cases interpreted as MSI-low by the Bethesda assay were interpreted as MSS by the MSI Analysis System. Our results suggest that the MSI Analysis System is generally superior and may help resolve cases of MSI-low into either MSI-high or MSS.

Capillary electrophoresis artifact due to eosin: implications for the interpretation of molecular diagnostic assays.

Capillary electrophoresis (CE) is a commonly used tool in the analysis of fluorescently labeled PCR amplification products. We have identified a CE artifact caused by the tissue stain eosin that can complicate the interpretation of CE data. The artifact was detected during routine analysis of a DNA sample isolated from a formalin-fixed, paraffin-embedded tissue sample considered histologically suspicious for a B-cell neoplasm. A standard clinical PCR and CE assay for immunoglobulin heavy chain (IGH) gene rearrangement revealed a weak polyclonal population of rearranged IGH genes and a 71 base peak suspicious for IGH clonality. The spectral properties of the 71 base peak were unusual in that although the dominant fluorescence of the peak was blue, it also fluoresced in green and yellow (blue>green>yellow), raising the suspicion that the peak might represent an artifact. CE analysis of the genomic DNA sample without PCR amplification demonstrated the presence of the 71 base peak, suggesting that the artifact was caused by a contaminant within the DNA sample itself. We demonstrate that eosin, which was used to stain the formalin-fixed tissue during processing, yields a discrete 71 base peak of similar morphology to the contaminant peak on CE analysis. The data suggest that eosin in the fixed tissue was not completely eliminated during nucleic acid extraction, resulting in the artifact peak. We discuss the implications of this potentially common contaminant on the interpretation of CE data and demonstrate that artifacts caused by eosin can be avoided by using more stringent DNA purification steps. Histological dyes may fluoresce, and artifacts from them should be considered when primary peaks contain additional underlying peaks of other colors.

A single nucleotide primer extension assay to detect the APC I1307K gene variant.

Adenomatous polyposis coli (APC) is a tumor suppressor gene important in colorectal tumorigenesis. A genetic variant of APC, I1307K, results from a T-to-A transversion at nucleotide 3920 which converts the wild-type sequence to a homopolymer tract (A(8)). The I1307K alteration is not itself oncogenic, but creates a hypermutable region (A(8)) that is prone to frame-shift mutations. The APC I1307K variant occurs in approximately 6% of the Ashkenazi Jewish population and is reported to approximately double an individual's risk for colorectal cancer. Here we describe a single nucleotide primer extension assay for the detection of the APC I1307K mutation. Following PCR amplification, nucleotide 3920 of the APC gene is directly sequenced using single nucleotide primer extension technology. The assay is in a multiplex format allowing simultaneous forward and reverse sequencing of the I1307K variant, which provides an internal, independent confirmation of each testing result. The assay was validated against 60 samples previously characterized by an allele-specific oligonucleotide (ASO) hybridization assay, with 100% concordance of results. Compared to the ASO assay, this single nucleotide primer extension assay requires significantly less technical time to perform, and has a greatly increased throughput capacity. The single nucleotide extension assay provides a highly sensitive and specific assay to identify individuals with the APC I1307K gene variant who may benefit from increased colorectal screening.

Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay.

FLT3 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells and plays an important role in stem cell survival and differentiation. Two different types of functionally important FLT3 mutations have been identified. Internal tandem duplication mutations arise from duplications of the juxtamembrane portion of the gene and result in constitutive activation of the FLT3 protein. This alteration has been identified in approximately 20% to 30% of patients with acute myelogenous leukemia and appears to be associated with a worse prognosis. The second type of FLT3 mutation, missense mutations at aspartic acid residue 835, occurs in approximately 7.0% of acute myelogenous leukemia cases. These mutations also appear to be activating and to portend a worse prognosis. Identification of FLT3 mutations is important because it provides prognostic information and may play a pivotal role in determining appropriate treatment options. We have developed an assay to identify both internal tandem duplication and D835 FLT3 mutations in a single multiplex polymerase chain reaction. After amplification, the polymerase chain reaction products are analyzed by capillary electrophoresis for length mutations and resistance to EcoRV digestion. Here we describe the performance characteristics of the assay, assay validation, and our clinical experience using this assay to analyze 147 clinical specimens.