PCR detection of Mycobacterium tuberculosis in necrotising non-granulomatous lymphadenitis using formalin-fixed paraffin-embedded tissue: a study in Thai patients

AimsTo investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In the absence of material submitted for tuberculosis (TB) culture, MTBC detection in FFPE tissue can be an important aid to diagnosis.MethodsWe collected 144 FFPE tissue blocks (lung and lymph node) for IS6110 real-time PCR. Two DNA extraction methods (QIAamp FFPE tissue kit and NucliSENS easyMAG) were assessed within a general laboratory setting. PCR results were compared with histology and culture.ResultsIn the histological MTBC and culture MTBC (TB-positive) groups, 72.4% were IS6110-positive and 27.6% negative. IS6110-negative results were obtained from 98%, 61.5% and 84% of the histologically MTBC-negative (TB-negative) group, histologically TB/no culture group and sarcoidosis group, respectively. Review of 19 IS6110-positive patients in the latter three groups showed that 15 had clinical TB. Thirteen of 15 (86.7%) IS6110-positive patients in the histological TB/no culture group and 2 of 4 (50%) IS6110-positive patients in the sarcoidosis group were clinically diagnosed with TB which highlights the difficulty of a pathological diagnosis.ConclusionsIS6110 real-time PCR using easyMAG extracted DNA is a moderately sensitive, specific and rapid method for MTBC detection in FFPE material, but must be interpreted in the overall clinical context. PCR results can be available in around 5 h from FFPE specimen receipt, with minimal hands-on time.

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Detection of Mycobacterium Bovis in Formalin-Fixed, Paraffin-Embedded Tissues of Cattle and Elk by PCR Amplification of an IS6110 Sequence Specific for Mycobacterium Tuberculosis Complex Organisms

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879700900304 ◽

1997 ◽

Vol 9 (3) ◽

pp. 244-249 ◽

Cited By ~ 48

Author(s):

Janice Miller ◽

Allen Jenny ◽

Jack Rhyan ◽

Dennis Saari ◽

David Suarez

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Tuberculosis Complex ◽

Rapid Diagnosis ◽

Paraffin Sections ◽

Tuberculosis Complex ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Positive Results ◽

Formalin Fixed ◽

Pcr Test

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk ( Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex ( M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria ( M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

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Evaluation of PCR in Detection of Mycobacterium tuberculosis from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.36.6.1512-1517.1998 ◽

1998 ◽

Vol 36 (6) ◽

pp. 1512-1517 ◽

Cited By ~ 59

Author(s):

Giulia Marchetti ◽

Andrea Gori ◽

Lidia Catozzi ◽

Luca Vago ◽

Manuela Nebuloni ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

False Negative ◽

Pcr Amplification ◽

Positive Culture ◽

Single Copy ◽

High Rate ◽

Antigen Gene ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of themtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 μg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

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RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

Modern Pathology ◽

10.1038/modpathol.3801008 ◽

2008 ◽

Vol 21 (3) ◽

pp. 326-333 ◽

Cited By ~ 11

Author(s):

Eijun Itakura ◽

Rong-Rong Huang ◽

Duan-Ren Wen ◽

Eberhard Paul ◽

Peter H Wünsch ◽

...

Keyword(s):

Pcr Detection ◽

In Situ Pcr ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies

Modern Pathology ◽

10.1038/modpathol.2013.48 ◽

2013 ◽

Vol 26 (9) ◽

pp. 1222-1227 ◽

Cited By ~ 13

Author(s):

Bryan H Schmitt ◽

MaryAnn Regner ◽

Kathy A Mangold ◽

Richard B Thomson Jr ◽

Karen L Kaul

Keyword(s):

Helicobacter Pylori ◽

Pcr Detection ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Gastric Biopsies ◽

Formalin Fixed

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Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652008000600002 ◽

2008 ◽

Vol 50 (6) ◽

pp. 321-326 ◽

Cited By ~ 10

Author(s):

Denise Barcelos ◽

Marcello F. Franco ◽

Sylvia Cardoso Leão

Keyword(s):

Mycobacterium Tuberculosis ◽

Miliary Tuberculosis ◽

Clinical Samples ◽

Actin Gene ◽

Tissue Handling ◽

Beta Actin ◽

Inhibitory Factors ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

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