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1.
J Clin Microbiol ; 49(12): 4273-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22012015

RESUMO

Culture-independent molecular techniques such as real-time PCRs offer the potential for early diagnosis of invasive aspergillosis (IA), thereby reducing the disease-associated mortality rate. PCR-based testing is presently excluded from disease-defining consensus criteria due to lack of standardization and clinical validation. A single-center prospective study was conducted to investigate the performance of the commercially available MycAssay Aspergillus test for detecting Aspergillus DNA in patients with suspicion of IA. To this end, a total of 158 bronchoalveolar lavage (BAL) fluid specimens that were consecutively collected from hematology (n = 68) and intensive care unit (n = 90) patients were examined. Sixteen of 17 (94.1%) specimens from patients with proven/probable IA were MycAssay positive, and 15 of these 16 patients were also positive by an "in-house" PCR assay. A total of 139 of 141 (98.6%) specimens from patients without proven/probable IA were MycAssay negative. Fifteen of 16 (94.1%) MycAssay-positive patients were also positive for BAL fluid galactomannan (GM) at an index cutoff of ≥1.0 (index range, 1.1 to 8.3), as were 3 patients without IA but with pulmonary fusariosis. Interestingly, in seven of the PCR-positive BAL specimens that tested culture positive for Aspergillus species, cycle threshold values were earlier than those of specimens with a culture-negative result. In conclusion, the MycAssay Aspergillus PCR appears to be a sensitive and specific molecular test for the diagnosis of IA, and its performance is comparable to that of the GM assay. However, more large studies are necessary to firmly establish its clinical utility in high-risk settings.


Assuntos
Aspergillus/isolamento & purificação , Técnicas Bacteriológicas/métodos , Líquido da Lavagem Broncoalveolar/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Aspergilose Pulmonar/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Aspergillus/química , Aspergillus/genética , Líquido da Lavagem Broncoalveolar/química , Galactose/análogos & derivados , Humanos , Técnicas Imunoenzimáticas/métodos , Mananas/análise , Estudos Prospectivos , Aspergilose Pulmonar/microbiologia , Sensibilidade e Especificidade
2.
J Clin Microbiol ; 49(6): 2169-74, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21450965

RESUMO

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).


Assuntos
Aspergilose/diagnóstico , Aspergillus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Soro/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspergillus/genética , DNA Fúngico/sangue , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade
3.
J Clin Microbiol ; 49(5): 1872-8, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21367988

RESUMO

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.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Micologia/métodos , Pneumocystis carinii/isolamento & purificação , Pneumonia por Pneumocystis/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Feminino , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Pneumocystis carinii/genética , Sensibilidade e Especificidade
4.
Biochem Pharmacol ; 76(4): 476-81, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18601911

RESUMO

Tachykinin NK(2) receptor antagonists are potentially beneficial in treating various disorders including irritable bowel syndrome, urinary incontinence, depression and anxiety. The current study evaluates the frequency of single nucleotide polymorphisms (SNPs) in the human NK(2) receptor gene (TACR2). In addition, the potency of the endogenous peptide agonist neurokinin A (NKA), and the small molecule antagonists saredutant (NK(2)-selective) and ZD6021 (pan-NK antagonist) at the various NK(2) receptor protein variants were determined. The TACR2 gene was sequenced from 37 individuals. Two amino acid changing SNPs encoding the NK(2) receptor variants Ile23Thr and Arg375His were found. The frequency of the four possible protein variants differed between populations. Site-directed mutagenesis was performed introducing either SNP or both SNPs into the TACR2 gene and the constructs were transfected into CHO cells. NKA-evoked increases in intracellular Ca(2+) were monitored by FLIPR. The potency of saredutant and ZD6021 was evaluated by their ability to inhibit NKA-induced increases in intracellular Ca(2+). NKA evoked increases in intracellular Ca(2+) with a potency ranging between 1 and 5nM in CHO cells expressing the different constructs. Saredutant and ZD6021 blocked NKA-evoked increases in intracellular Ca(2+) with pK(b) values ranging between 8.8-9.3 and 7.9-8.7, respectively. The current study demonstrates that polymorphisms leading to the Ile23Thr and Arg375His amino acid exchanges are highly prevalent in the human TACR2 gene. These polymorphisms however do not appear to affect the potency of the endogenous agonist NKA or the small molecule antagonists saredutant and ZD6021 with respect to intracellular Ca(2+) signalling.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Receptores da Neurocinina-2/genética , Receptores de Taquicininas/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Frequência do Gene , Variação Genética , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Transfecção
5.
Hum Mol Genet ; 16(17): 2114-21, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17597096

RESUMO

The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.


Assuntos
Códon de Terminação/genética , Mutação , Insensibilidade Congênita à Dor/genética , Canais de Sódio/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.7 , Dor/genética , Dor/fisiopatologia , Insensibilidade Congênita à Dor/fisiopatologia , Linhagem , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/metabolismo
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