Diagnostic Molecular Pathology最新文献

Dematiaceous fungi are a diverse group of "darkly" pigmented fungi, which contain melanin in their cell walls and are commonly found in soil worldwide. Although morphology and histochemical stains may aid identification in tissue sections, these means for species identification are not specific. In-situ hybridization (ISH) for abundant fungal rRNA sequences may provide a means for detecting dematiaceous fungi. In this study, a 24-base synthetic biotin-labeled oligonucleotide probe targeting rRNA sequences of a variety of dematiaceous fungi was developed. This probe was tested on a cohort of 29 patients with culture-proven cases of dematiaceous fungal-associated rhinosinusitis (26 allergic fungal sinusitis, 2 fungal ball, and 1 acute invasive fungal sinusitis). Fungal cultures were positive for Alternaria species (10), Bipolaris species (5), Curvularia species (10), Cladosporium species (1), Scedosporium prolificans (1), Scopulariopsis species (1), and dematiaceous species, not otherwise specific (1). ISH showed positivity in fungal organisms in 24 of 29 specimens. ISH was negative in culture-proven examples of Rhizopus species, Aspergillus species, Fusarium species, Paecilomyces species, Histoplasmosis capsulatum, Candida species, and Blastomyces dermatitidis. ISH with a dematiaceous-specific fungal probe may be useful for differentiating dematiaceous fungi from other filamentous fungi in tissues, particularly those responsible for fungal rhinosinusitis.

Renal cell carcinomas (RCCs) with an Xp11.2 translocation predominantly affect young patients, and can present at an advanced stage. However, more cases in older patients and incidentally detected cancers at earlier stages are also being identified. As the histology of Xp11.2 RCCs overlaps with clear cell and papillary RCCs, it is not infrequent that Xp11.2 RCCs are overlooked and misdiagnosed. The objective of this study was to validate the use of fluorescence in-situ hybridization (FISH) for identifying Xp11.2 RCCs. One hundred fifty-eight consecutive, unselected renal tumors were evaluated in tissue microarrays, including 109 clear cell RCCs, 20 papillary RCCs, 3 RCCs with mixed papillary and clear cell features, 1 Xp11.2 translocation RCC, 8 chromophobe RCCs, 10 oncocytomas, and 7 angiomyolipomas. FISH evaluation was performed blinded to karyotype data, available in about two-thirds of cases. Furthermore, conventional sections of 4 Xp11.2 RCCs, 4 RCCs with mixed papillary and clear cell features, and 4 cases of alveolar soft part sarcoma (the latter for control purposes) were also assessed by FISH. Break-apart signals were homogeneously identified throughout tumor cells in 2 cases from the tissue microarrays including 1 known Xp11.2 RCC and 1 misdiagnosed Xp11.2 RCC. All conventional sections from the Xp11.2 RCC and alveolar soft part sarcoma cases were positive for the TFE3 rearrangement by FISH. All remaining cases were negative. Our study shows the clinical application of FISH in formalin-fixed, paraffin-embedded tissue for detection of Xp11.2 translocation RCCs and other tumors with this genetic aberration.

Background: The association of vancomycin treatment failure with minimum inhibitory concentration (MIC) creep is concerning, as most isolates are still considered to be in the susceptible range. Several studies have suggested that the accessory gene regulator (agr) group II polymorphism is predictive of vancomycin treatment failure. We assessed the associations between increased vancomycin MIC, agr group II locus, and vancomycin treatment failure in Methicillin-resistant Staphylococcus aureus (MRSA) bacteremias.

Methods: MRSA isolates from 99 inpatient bacteremias were studied. Susceptibility testing was conducted by an automated method (MicroScan) and by the gradient diffusion method (E-test). Vancomycin MICs were stratified into 3 groups for analysis: MIC ≤ 1, MIC > 1 but ≤ 2, and MIC >2 μg/mL. Strains were typed by repetitive-polymerase chain reaction analysis and the agr locus was identified by multiplex polymerase chain reaction. Failure of vancomycin treatment was defined as persistent bacteremia after 72 hours, death at 30 days, or treatment change due to clinical failure.

Results: Among 99 bacteremic patients, there were 82 agr group II and 15 agr group I isolates. There was no relationship between higher vancomycin MICs and isolate agr II loci (42 of 82) (P=0.59). Earlier vancomycin exposure was significantly associated with increased MIC (P=0.03). Vancomycin treatment failure was observed in 12 patients: 3 required an alternate regimen, 4 had persistent positive cultures, and 5 whose deaths were attributable to MRSA infection. Survival in agr group II was 57 of 82 (69%) versus agr group I in which it was 14 of 15 (93%), (P=0.06).

Conclusions: We did not identify any significant association between MIC creep and vancomycin failure or between higher vancomycin MICs and agr group II. However, a higher mortality was seen in agr group II than agr group I.

The American College of Medical Genetics recommends that each laboratory should confirm abnormal or ambiguous results detected by array comparative genomic hybridization (aCGH). At present, the gold standard method for aCGH confirmation is fluorescent in situ hybridization (FISH). However, FISH is not well suited for small tandem duplications or very small deletions that are detectable by oligonucleotide arrays. Therefore, we developed and validated multiplex ligation-dependent probe amplification (MLPA) for aCGH confirmation. The method performance validation showed linearity through the expected analytical measurement range (0.05 to 2 genome equivalents). The interassay normalized coefficient of variation averaged 3.7% across 12 control and target probes. This low imprecision allowed detection of 20% mosaicism with exceptional confidence (P<0.006). Comparison with a combined gold standard of phenotype, aCGH, karyotype, and/ or FISH showed 100% concordance for 218 samples using an X/Y chromosome-specific probe set (95% confidence interval, 98.3%-100.0%). Patient-specific probe sets also showed 100% concordance to the gold standard for 18 genomic targets. In conclusion, we have developed and validated an MLPA assay using a novel approach to accommodate the fact that positive controls would not be available at the time of testing. We initially validated the MLPA method using X/Y chromosome-specific probes and well-characterized samples and then validated new probe sets by comparision with reference populations. We have successfully incorporated aCGH confirmation using custom-designed MLPA into our normal workflow, and used it for confirmation of all abnormal or ambiguous results.

The presence of myxovirus resistance protein A (MxA) RNA was studied in 55 febrile children with primary immunodeficiency, 27 of whom underwent hematopoietic cell transplantation, and in 28 age-matched controls. The level of MxA RNA was above the cutoff, established as the 95th percentile found in controls, with primary immunodeficiency either undergoing transplantation or not in febrile patients, and with a documented diagnosis of infection by adenovirus, cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, and rotavirus. The presence of rare viral infections, unrecognized among those that more frequently occur in patients with primary immunodeficiency and in patients undergoing transplantation, may explain the high MxA RNA levels observed in some patients with fever but undetectable genomes or antibodies for the more common viruses. The level of MxA in febrile patients with acute graft versus host disease was below the cutoff, with a median level comparable with that observed in patients with primary immunodeficiency, who did not undergo transplantation and were without fever and infections, but significantly lower compared with controls. The level of MxA was well correlated with viral infections in follow-up samples. These data indicate that the measurement of MxA RNA is simple and useful to detect viral infections and in distinguishing them from acute graft versus host disease after allogeneic cell transplantation.

Type-specific surveillance of human papillomavirus (HPV) has been proposed as an early indicator of vaccine impact. Longitudinal comparison of HPV typing results requires stable assays with high type-specific reproducibility. Assays are evolving and the impact of even minor changes in the assay format may be difficult to anticipate. We initiated a population-based study of HPV with the prototype line blot (PLB) assay. These reagents were replaced by the research use only Linear Array (LA) HPV Genotyping kit. The assays are similar in principle and earlier comparisons found increased sensitivity and detection of more types per sample with LA; however, in samples from women with cervical abnormalities, the overall concordance was good. Slight changes in sensitivity may be more significant in samples from a general population with lower viral loads in the samples. Residual extracts from 3001 self-collected vaginal swabs from women in the general US population originally tested with PLB were retested with LA. With LA, all the samples were hybridized. PLB hybridization was restricted to samples with probable amplicon in gel electrophoresis. For HPV detection, the agreement between the 2 assays was 78.6% (κ=0.55) with a positive concordance of 52.8%. However, this masks the observation that repeat testing with LA led to the detection of HPV in nearly twice as many samples. Agreement improves if comparison was restricted to the samples hybridized. These results emphasize that assay comparisons should consider the clinical-epidemiologic context of sample collection. Studies designed to examine temporal trends in type-specific prevalence should archive residual material to permit retesting if assays change.

Earlier we had reported a large prevalence of the Q318X mutation in the CYP21A2 gene with 35.3% in Tunisian patients with a classical form of 21-hydroxylase deficiency, in contrast with 0.5% to 13.8% as described in other populations. Here we present the analysis of the Q318X mutation in a healthy Tunisian population. We screened 136 individuals by the polymerase chain reaction (PCR)/random fragment length polymorphism method, which was confirmed by direct sequencing. Surprisingly, 17 Q318X carriers were identified, for a carrier frequency of 12.5% (95% confidence interval: 7.86-19.20). To explain this unexpectedly high rate we suggest that the haplotype with Q318X mutation and duplicated CYP21A2 gene could be very frequent in the Tunisian population. To test our hypothesis, we used 2 different quantitative PCR methods, that is, multiplex ligation-dependent probe amplification and real-time PCR. The molecular studies showed the presence of a duplicated CYP21A2 gene in all 17 heterozygous Q318X mutation carriers. In addition, both quantitative PCR methods used in this study represent a sensitive and useful approach to detecting copy number variations of the CYP21A2 gene. We have identified a very high frequency of carriers with duplicated CYP21A2 gene haplotype in a healthy Tunisian population. This finding complicates the molecular diagnosis of 21-hydroxylase deficiency and we recommend that, whenever a Q318X is identified, the structure of the CYP21A2 region should be determined to discriminate between the severe Q318X mutation and the normal Q318X variant.

KRAS mutations exhibit significant predictive and prognostic value in cancer. Efficient, sensitive, and accurate molecular approaches are required to evaluate KRAS mutation status, even when mutant alleles are restricted to a small portion of a clinical sample, which otherwise contains wild-type alleles. We describe a highly sensitive method to detect KRAS mutations by high-resolution melting (HRM) analysis after mutation enrichment by fast-COLDpolymerase chain reaction (PCR). Using 10 ng of starting DNA and after fast-COLD-PCR of a 76-bp region containing KRAS codons 12 and 13; the amplicons undergo a nested conventional PCR reaction followed by HRM analysis. Samples exhibiting aberrant melting profiles are sequenced to identify mutation type and position. Serial dilution experiments indicate a sensitivity of approximately 0.3% mutant-to-wild type for HRM-based mutation detection and the ability to directly sequence mutation-containing samples. A number of lung adenocarcinoma specimens earlier characterized were screened. Fast-COLD-PCR-HRM analysis correctly identified KRAS mutations and also showed a previously undetected, low-level missense GGT > TTT complex mutation. On account of the short target regions and low requirement of starting DNA, this rapid, cost-effective, and sensitive fast-COLD-PCR-HRM approach is expected to find broad application for detecting low-abundance KRAS mutations in a wide range of clinical specimens.

BK virus nephropathy is not an infrequent complication of renal transplantation associated with high rates of graft loss. Although antibodies against SV40 antigen detect different viruses of the polyomavirus family, immunohistochemistry is widely used to confirm the diagnosis of BK virus nephropathy in renal biopsies. Here we aimed to validate the novel silver-enhanced in situ hybridization (SISH) technique for the automated detection of BK virus in renal transplant biopsies. Two different patient cohorts were included. Twenty-nine consecutive patients suspicious for BK virus infection were investigated by SISH and chromogenic in situ hybridization. An additional 26 renal biopsies positive by SV40 immunohistochemistry from 19 patients were analyzed by SISH. Polyomavirus DNA serum levels, as determined by nested PCR analysis, were available for all of these patients. The presence of BK virus DNA in renal tubular cells was identified in 5 of the suspicious cases by both, SISH and chromogenic in situ hybridization . One additional patient was only positive in the SISH. In the second cohort, SISH was positive in all SV40 positive biopsies, but SISH signals were less extensive than SV40 immunohistochemistry. Our results show that the BK virus SISH is an ancillary tool for the detection of polyomavirus DNA in renal biopsies using bright-field microscopy. However, its diagnostic value in comparison with standard immunohistochemistry seems to be limited.

Activating mutations of K-ras have been described in approximately 40% of patients with colorectal cancer, and are associated with resistance to epidermal growth factor receptor-targeted antibodies, such as cetuximab and panitumumab. Cost-effective and easy methods to determine K-ras mutations are urgently needed. Samples from 31 patients were tested. In laboratory 1, a real-time polymerase chain reaction (PCR)-based technique was used. All samples (n=31) were additionally tested using a restriction fragment length polymorphism (RFLP) analysis in laboratory 2. All results were confirmed by direct sequencing. In the first run, a concordance of real-time PCR and RFLP was observed in 77.4% (24 of 31) of samples. After resampling and reevaluation, a concordance of 93.5% (30 of 31) could be achieved. One of 7 (6.5%) initial discordant cases showed a mutation using real-time PCR and no mutation using RFLP, but the mutation was confirmed by direct sequencing. Real-time PCR and RFLP can be considered as valid K-ras mutation detection techniques. However, in patient probes with lower amounts of tumor cells and wild-type K-ras, reanalysis of further tumor tissue is recommended.