Quantitative nested real-time PCR assay for assessing the clinical course of tuberculous meningitis

Takahashi, Teruyuki; Tamura, Masato; Takahashi, Shirushi; Matsumoto, Koichí; Sawada, Shigemasa; Yokoyama, Eise; Nakayama, Tomohiro; Mizutani, Tetsuya; Takasu, Toshiaki; Nagase, Hiroki

doi:10.1016/j.jns.2007.01.071

Cited by 27 publications

(48 citation statements)

References 14 publications

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“…To maintain a linear quantitative relationship between the amount of final amplified product and the amount of target sequence in the nested PCR, the first-round PCR was limited to the exponential phase of the reaction. 29,47,48 The approach used to quantify telomere length on permanent paraffin-embedded tissue sections was validated in our laboratories 19,20 and is based on a PNAbased FISH assay. The probe we use in telomere-specific FISH is a relatively small, directly labeled PNA probe with superior hybridization properties, eg, the ability to hybridize in very low salt conditions, that prevent competitive re-annealing of the target genomic DNA sequences compared with more traditional FISH techniques that use DNA or RNA hybridization probes.…”

Section: Discussionmentioning

confidence: 99%

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Wang

Meeker

Kowalski

et al. 2011

The American Journal of Pathology

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Telomere dysfunction and aberrant telomerase expression play important roles in tumorigenesis. In thyroid tumors, three possibly inhibitory splice variants of the active full-length isoform of human telomerase reverse transcriptase (hTERT) may be expressed. These variants might regulate telomerase activity and telomere length because it is the fraction of the full-length isoform, rather than the total transcript level, that correlates with enzymatic activity. Telomerase reactivation may be critical in the early stages of tumorigenesis, when progressive telomere shortening may be limiting cell viability. The aim of this study was to investigate the relationship between telomere length and hTERT splice variant expression patterns in benign and well-differentiated malignant thyroid tumors. Telomere lengths of 61 thyroid tumors were examined by fluorescence in situ hybridization, comparing tumors with adjacent normal thyroid tissue on the same slide. Expression patterns of hTERT splice variants were evaluated by quantitative and nested RT-PCR. Telomere length was inversely correlated with percentage of full-length hTERT expression rather than with total hTERT expression levels. Short telomeres and high fractions of full-length hTERT transcripts were associated with follicular and papillary thyroid carcinomas, whereas long telomeres and low levels of full-length hTERT were associated with benign thyroid nodules. Intermediate levels of full-length hTERT and telomere length were found in follicular variant of papillary thyroid carcinomas and follicular adenomas.

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Section: Discussionmentioning

confidence: 99%

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Wang

Meeker

Kowalski

et al. 2011

The American Journal of Pathology

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“…A recent example of this method includes the use of an IS6110 uniplex PCR assay that had 98% sensitivity (NPV, 99%) and a specificity of 100% (PPV ϭ 100%) against the "gold standard" of culture (167) and an overall sensitivity of 76.4% (NPV, 59.9%) and specificity of 89.2% (PPV, 94.7%) when clinical TBM was included (166). Several methods of PCR have been evaluated, including quantitative nested real-time PCR (208)(209)(210), the Amplicor Mycobacterium tuberculosis Test (23,24), and a variety of other in-house PCR assays (45,46,51,83,104,111,120,123,138,141,165,189,193). These assays have been evaluated specifically in HIV-coinfected patients (59,180) and also as a means to monitor therapy (180,210).…”

Section: Molecular and Biochemical Analysismentioning

confidence: 99%

“…Several methods of PCR have been evaluated, including quantitative nested real-time PCR (208)(209)(210), the Amplicor Mycobacterium tuberculosis Test (23,24), and a variety of other in-house PCR assays (45,46,51,83,104,111,120,123,138,141,165,189,193). These assays have been evaluated specifically in HIV-coinfected patients (59,180) and also as a means to monitor therapy (180,210). While many of those studies demonstrated improved sensitivity over traditional smear and culture methods, several other studies highlighted the low sensitivities associated with these PCR assays (25,68,112,164,206).…”

Section: Molecular and Biochemical Analysismentioning

confidence: 99%

Central Nervous System Tuberculosis: Pathogenesis and Clinical Aspects

et al. 2008

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SUMMARY Tuberculosis of the central nervous system (CNS) is a highly devastating form of tuberculosis, which, even in the setting of appropriate antitubercular therapy, leads to unacceptable levels of morbidity and mortality. Despite the development of promising molecular diagnostic techniques, diagnosis of CNS tuberculosis relies largely on microbiological methods that are insensitive, and as such, CNS tuberculosis remains a formidable diagnostic challenge. Insights into the basic neuropathogenesis of Mycobacterium tuberculosis and the development of an appropriate animal model are desperately needed. The optimal regimen and length of treatment are largely unknown, and with the rising incidence of multidrug-resistant strains of M. tuberculosis, the development of well-tolerated and effective antibiotics remains a continued need. While the most widely used vaccine in the world largely targets this manifestation of tuberculosis, the BCG vaccine has not fulfilled the promise of eliminating CNS tuberculosis. We put forth this review to highlight the current understanding of the neuropathogenesis of M. tuberculosis, to discuss certain epidemiological, clinical, diagnostic, and therapeutic aspects of CNS tuberculosis, and also to underscore the many unmet needs in this important field.

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“…Studies indicate, however, that nested PCR will retain its utility for quantitation if the first-round PCR is maintained in the exponential phase (35). Indeed, quantitative nested real-time PCR assay has been developed and used in some studies without apparent distortion in the amplified product ratio (36). Further, we also optimized our nested PCR reaction using thyroid cell lines to ensure accurate product ratios (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Differentiating Alternative Splice Variant Patterns of Human Telomerase Reverse Transcriptase in Thyroid Neoplasms

Wang

Kowalski

Tsai

et al. 2008

Thyroid

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Background: Although fine-needle aspiration (FNA) biopsy of thyroid nodules is very sensitive in detecting thyroid malignancy, it remains ambiguous in 20-30% of cases. Current biomarkers for thyroid cancer lack either the sensitivity or specificity to substantially address this clinical problem. The aim of this study was to investigate the gene expression patterns of human telomerase reverse transcriptase (hTERT) alternative splice variants in benign and malignant thyroid tumors in an attempt to find a more reliable biomarker in the differential diagnosis of thyroid nodules. Methods: One hundred and thirty-three thyroid tumors from eight histopathological tumor types were collected from patients undergoing thyroid surgery at Johns Hopkins Hospital. Gene expression patterns of hTERT alternative splice variants were investigated in the tumors by nested reverse transcriptase-PCR. Telomerase enzyme activity was evaluated in a subset of 16 samples associated with the different hTERT patterns. Association of c-myc expression and hTERT patterns was also examined. Results: Malignant thyroid tumors exhibited a greater proportion of the active full-length hTERT transcript (0.57 AE 0.15) than inactive splice variants, a À (0.13 AE 0.02), or b À =a À b À deletion transcripts (0.30 AE 0.11; p < 0.001). The opposite was observed in benign tumors, which exhibited greater proportions of b À =a À b À deletion transcripts (0.64 AE 0.08) than either the full-length (0.19 AE 0.06) or a À deletion transcripts (0.17 AE 0.02; p < 0.001). Similar results were observed among a diagnostically challenging subset of 50 thyroid tumors that were suspicious for malignancy on FNA. Further, increased telomerase enzymatic activity was only associated with expression of the full-length hTERT isoform. In contrast, c-myc expression, which has been implicated in hTERT regulation, correlated with overall hTERT transcription without specificity for expression of the full-length isoform. Conclusions: These differences in gene expression patterns of hTERT alternative splice variants may provide a useful adjunct to FNA diagnosis of suspicious thyroid tumors.

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Quantitative nested real-time PCR assay for assessing the clinical course of tuberculous meningitis

Cited by 27 publications

References 14 publications

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Central Nervous System Tuberculosis: Pathogenesis and Clinical Aspects

Differentiating Alternative Splice Variant Patterns of Human Telomerase Reverse Transcriptase in Thyroid Neoplasms

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