Amit Phansalkar

Consensus Characterization of 16 FMR1 Reference Materials: A Consortium Study

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Evaluation of the Abbott Investigational Use Only RealTime Hepatitis C Virus (HCV) Assay and Comparison to the Roche TaqMan HCV Analyte-Specific Reagent Assay

ABSTRACT The accurate and sensitive measurement of hepatitis C virus (HCV) RNA is essential for the clinical management and treatment of infected patients and as a research tool for studying the biology of HCV infection. We evaluated the linearity, reproducibility, precision, limit of detection, and concordance of viral genotype quantitation of the Abbott investigational use only RealTime HCV (RealTime) assay using the Abbott m2000 platform and compared the results to those of the Roche TaqMan Analyte-Specific Reagent (TaqMan) and Bayer Versant HCV bDNA 3.0 assay. Comparison of 216 samples analyzed by RealTime and TaqMan assays produced the following Deming regression equation: RealTime = 0.940 (TaqMan) + 0.175 log10 HCV RNA IU/ml. The average difference between the assays was 0.143 log10 RNA IU/ml and was consistent across RealTimes dynamic range of nearly 7 log10 HCV RNA IU/ml. There was no significant difference between genotypes among these samples. The limit of detection using eight replicates of the World Health Organization HCV standard was determined to be 7.74 HCV RNA IU/ml by probit analysis. Replicate measurements of commercial genotype panels were significantly higher than TaqMan measurements for most samples and showed that the RealTime assay is able to detect all genotypes with no bias. Additionally, we showed that the amplicon generated by the widely used Roche COBAS Amplicor Hepatitis C Virus Test, version 2.0, can act as a template in the RealTime assay, but potential cross-contamination could be mitigated by treatment with uracil-N-glycosylase. In conclusion, the RealTime assay accurately measured HCV viral loads over a broad dynamic range, with no significant genotype bias.

Likelihood ratios to assess genetic evidence for clinical significance of uncertain variants: Hereditary hemorrhagic telangiectasia as a model

Clinical laboratories performing gene sequencing discover previously unreported and/or uncharacterized variants. Often these are missense or intronic mutations in which the contribution to disease cannot be predicted, and consequently these mutations are reported as variants of uncertain significance. Follow-up to assess family concordance is recommended by the American College of Medical Genetics to provide genetic evidence for clinical significance. Although family concordance studies show whether a variant segregates with disease in the family, the strength of evidence varies depending on the number and degree of relatedness of family members available for testing. We investigated a statistical model which accounts for the pedigree, inheritance patterns, and penetrance to determine the likelihood of a variant being a causative or deleterious mutation. We used hereditary hemorrhagic telangiectasia (HHT) as a model for an autosomal dominant disease. Pedigree data were transferred to MLINK, and a Bayesian analysis was calculated to determine the likelihood that a variant is causative of disease. In applying this analysis to HHT pedigrees we found Bayes Factors of variants showing odds in favor of causality ranging from approximately 4:1 to over 400:1. These numbers provide an objective measure of the strength of genetic evidence. Other parameters such as amino acid severity predictions, ortholog and paralog comparisons and functional assays can be included in the analysis to increase the evidence of causality.

Evaluation of the Abbott Investigational Use Only RealTime HIV-1 Assay and Comparison to the Roche Amplicor HIV-1 Monitor Test, Version 1.5

Abbott Moleculars m2000 system and RealTime HIV-1 assay (RealTime) were evaluated for sensitivity, reproducibility, linearity, ability to detect diverse HIV-1 subtypes/groups, and correlation to the Roche AMPLICOR HIV-1 MONITOR Test, Version 1.5 (Amplicor). The limit of detection was determined using the second International World Health Organization Standard and Viral Quality Assurance standard material. Serial dilutions of four patient samples were used to determine inter- and intra-assay reproducibility and linearity. Samples representing HIV-1 groups M, N, and O were evaluated in the RealTime, Amplicor, and Siemens Versant HIV-1 branched chain DNA 3.0 (Versant) assays. Archived Amplicor-tested samples were tested with the 1 ml, 0.5 ml, and 0.6 ml versions of the RealTime assay. Probit analysis predicts a limit of detection of 21.94 IU/ml using the World Health Organization Standard and 26.54 copies/ml using Viral Quality Assurance material with the 1 ml assay. Linearity and reproducibility were very good between approximately 1.60 to 6.0 log(10) copies/ml. All three assays produced similar measurements for all Group M subtypes tested; the RealTime assay was the only assay that detected all three Group O samples tested. Correlation with the Amplicor assay was good, although the RealTime assay measured between 0.342 and 0.716 log(10) copies/ml lower on average, depending on the input volume. The automated RealTime assay exhibits excellent sensitivity, dynamic range, reproducibility, and group/subtype detection, albeit with consistently lower values than Amplicor.

Limited Applicability of Direct Fluorescent-Antibody Testing for Bordetella sp. and Legionella sp. Specimens for the Clinical Microbiology Laboratory

ABSTRACT The rapid diagnosis of infections with Bordetella and Legionella species is important for patient management. With observed increases in direct fluorescent-antibody (DFA) testing volumes, we retrospectively compared the performance characteristics of DFA testing to those of culture and PCR. For Bordetella sp., samples were classified as positive by DFA testing (184 [3%] of 6,195 samples) and culture (150 [2%] of 6,251 samples) significantly less often than by PCR (2,557 [10%] of 26,929 samples). Of 360 samples tested by both DFA and PCR methods, 81 (16 by DFA testing and 79 by PCR) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 18% and 99%, respectively. Of 1,426 samples tested by both DFA and culture methods, 48 (44 by DFA testing and 15 by culture) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 73% and 98%, respectively. For Legionella sp., samples were identified as positive by DFA testing (31 [0.25%] of 12,597 samples) and culture (85 [0.6%] of 13,572 samples) significantly less often than by PCR (27 [4%] of 716 samples). Of 62 samples tested by both DFA and PCR methods, none were positive for Legionella sp. by DFA testing and 3 were positive by PCR. Of 3,923 samples tested by both DFA and culture methods, 22 (3 by DFA testing and 21 by culture) were positive for Legionella sp., with a sensitivity and specificity of DFA testing of 9.5% and 100%. Overall, DFA testing for Bordetella sp. and Legionella sp. is an insensitive method, and despite its continued popularity, clinical microbiology laboratories should not offer it when more sensitive tests like PCR are available.