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1.
J Clin Microbiol ; 49(6): 2169-74, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21450965

ABSTRACT

Diagnosis of invasive aspergillosis remains a significant problem. PCR testing may aid diagnosis but is not yet included in disease-defining criteria due to a lack of standardization of assays and methodologies. This study investigated the analytical performance and the clinical sensitivity and specificity of the Myconostica MycAssay Aspergillus PCR (MAP) assay compared to those of a validated in-house Aspergillus PCR (IHP) test when testing serum specimens. Serum specimens spiked with Aspergillus genomic DNA had a limit of detection equivalent to 5 genomes and a linear dynamic range of 5 to >5 × 10(4) genomes for both assays. When testing clinical specimens (n = 170), the MAP assay had a sensitivity of 60 to 70% and a specificity of 90.5 to 100%. The IHP assay had a sensitivity of 50 to 80% and a specificity of 100%. A commercially available Aspergillus PCR assay provides a methodology that is standardized and reagents that are quality controlled. This facilitates multicenter evaluation of the clinical utility of PCR diagnosis. The performance of the MAP assay is comparable to that of the IHP assay and to those in previously reported studies evaluating commercial tests (galactomannan enzyme-linked immunosorbent assay).


Subject(s)
Aspergillosis/diagnosis , Aspergillus/isolation & purification , Clinical Laboratory Techniques/methods , Mycology/methods , Polymerase Chain Reaction/methods , Serum/microbiology , Adult , Aged , Aged, 80 and over , Aspergillus/genetics , DNA, Fungal/blood , Humans , Middle Aged , Sensitivity and Specificity
2.
J Clin Microbiol ; 49(5): 1872-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21367988

ABSTRACT

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤ 3.5 copies/µl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.


Subject(s)
Molecular Diagnostic Techniques/methods , Mycology/methods , Pneumocystis carinii/isolation & purification , Pneumonia, Pneumocystis/diagnosis , Polymerase Chain Reaction/methods , Adult , Aged , Female , Humans , Immunocompromised Host , Male , Middle Aged , Pneumocystis carinii/genetics , Sensitivity and Specificity
3.
Diagn Microbiol Infect Dis ; 67(4): 392-4, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20638611

ABSTRACT

Fungal polymerase chain reaction (PCR)-based diagnostic methods are at risk for contamination. Sample collection containers were investigated for fungal DNA contamination using real-time PCR assays. Up to 18% of blood collection tubes were contaminated with fungal DNA, probably Aspergillus fumigatus. Lower proportions of contamination in other vessels were observed.


Subject(s)
Aspergillus/genetics , Blood/microbiology , DNA, Fungal/isolation & purification , Specimen Handling , DNA, Fungal/genetics , Humans , Polymerase Chain Reaction
4.
Genetics ; 178(2): 777-86, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18287407

ABSTRACT

ASIP is an important pigmentation gene responsible for dorsoventral and hair-cycle-specific melanin-based color patterning in mammals. We report some of the first evidence that the avian ASIP gene has a role in pigmentation. We have characterized the genetic basis of the homozygous lethal Japanese quail yellow mutation as a >90-kb deletion upstream of ASIP. This deletion encompasses almost the entire coding sequence of two upstream loci, RALY and EIF2B, and places ASIP expression under control of the RALY promoter, leading to the presence of a novel transcript. ASIP mRNA expression was upregulated in many tissues in yellow compared to wild type but was not universal, and consistent differences were not observed among skins of yellow and wild-type quail. In a microarray analysis on developing feather buds, the locus with the largest downregulation in yellow quail was SLC24A5, implying that it is regulated by ASIP. Finally, we document the presence of ventral skin-specific isoforms of ASIP mRNA in both wild-type quails and chickens. Overall, there are remarkable similarities between yellow in quail and lethal yellow in mouse, which involve a deletion in a similar genomic position. The presence of ventral-specific ASIP expression in birds shows that this feature is conserved across vertebrates.


Subject(s)
Agouti Signaling Protein/genetics , Coturnix/genetics , Feathers , Pigmentation/genetics , 5' Untranslated Regions/genetics , Animals , Chickens/genetics , Crosses, Genetic , Female , Male , Mammals , Molecular Sequence Data , Mutation , RNA/genetics , RNA/isolation & purification , Sequence Deletion
5.
BMC Genet ; 9: 7, 2008 Jan 15.
Article in English | MEDLINE | ID: mdl-18197963

ABSTRACT

BACKGROUND: The lavender phenotype in the chicken causes the dilution of both black (eumelanin) and red/brown (phaeomelanin) pigments. Defects in three genes involved in intracellular melanosomal transport, previously described in mammals, give rise to similar diluted pigmentation phenotypes as those seen in lavender chickens. RESULTS: We have used a candidate-gene approach based on an expectation of homology with mammals to isolate a gene involved in pigmentation in chicken. Comparative sequence analysis of candidate genes in the chicken identified a strong association between a mutation in the MLPH gene and the diluted pigmentation phenotype. This mutation results in the amino acid change R35W, at a site also associated with similar phenotypes in mice, humans and cats. CONCLUSION: This is the first time that an avian species with a mutation in the MLPH gene has been reported.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Chickens/genetics , Feathers , Melanins/genetics , Pigmentation/genetics , Point Mutation , Animals , Female , Male , Phenotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
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