Dear Khaled:

PCR is not restricted by the presence of viable organisms in the sample, thus rendering possible a retrospective diagnosis of tuberculosis from archival material with no cultural examination and whose storage in formalin severely hampers processing by usual bacteriological methods. After formalinized tissue specimen processing you  can  detect  M. tuberculosis by acid-fast stain and can extract DNA , then amplified it by PCR.

As far as microbiological methods are concern; isolation is not possible from formalized tissue. Acid fast staining, histopathology and immunohistochemistry can be performed. Pcr can be uesd to detect the presence of bacterial DNA.

DNA extraction and PCR if, viable cell culture can not be performed.

Diagnosis of tuberculosis (TB) in formalin-fixed, paraffin-embedded tissue specimens depends on microscopic examination (Zeihl-Neelsen stain (ZN-stain), and histopatholgy) but ZN-stain sensitivity is still of primary limitation and histopathological features of chronic granulomatous inflammation can be

found in various diseases other than TB. Accurate and early diagnosis of tuberculosis in formalin-fixed, paraffin-embedded tissue samples is important for effective management. The present study aimed to compare between TB-polymerase chain reaction (PCR), ZN-stain, and histpathological examination for diagnosis of TB in formalin fixed histologic specimens. Out of fifty different tissue specimens clinically diagnosed as TB, ZN-stain was positive in (34%), histological examination was positive in (52%) and TBPCR was positive in (64%). TB-PCR was the most sensitive (100.0%) with specificity of 90.0% while sensitivity of ZN-stain was the lowest (50%). It was concluded that the PCR assay was more rapid, specific and reliable for TB diagnosis. In addition it should be implemented as routine test in histopathology as well as microbiology laboratories for the diagnosis of TB.

Rapid, reliable diagnosis of tuberculosis is essential to

initiate correct treatment, avoid severe complications,

and prevent transmission. Conventional microbiological

methods may not be an option if samples

are formalin-fixed and paraffin-embedded (FFPE) for

histopathological examination. With the demonstration

of necrotizing granulomatous inflammation, tuberculosis

becomes an important differential diagnosis,

although it was not initially suspected. Following

paraffin extraction, BDProbeTec ET strand displacement

amplification for detection of Mycobacterium

tuberculosis complex (MTC) was applied to 47 prospectively

and 19 retrospectively collected FFPE samples

from various sources with granulomatous inflammation

and results were compared to tuberculosis

notification. Of the prospective samples, 20 were from

patients who were notified as having tuberculosis and

the assay was positive in 18 (90%). Specificity was

100%. For 27 of the patients with prospectively collected

FFPE specimens, culture was performed on a

specimen collected at a later date from the same location.

Culture revealed MTC in 14 and nontuberculous

mycobacteria in four. BDProbeTec ET was positive

in 13 (92.8%) of the patients with positive MTC

culture and negative in the remaining. The sensitivity

and specificity in 19 archival samples was 40% and

100%, respectively, compared to notification data.

The assay provided rapid, correct diagnosis on different

sources of FFPE samples collected prospectively

and therefore offers an important supplementary

method for patients where tuberculosis was not initially

suspected. (J Mol Diagn 2004, 6:231–235)

Am J Respir Crit Care Med. 1998 Oct;158(4):1150-5.

Polymerase chain reaction to detect Mycobacterium tuberculosis in histologic specimens.

Salian NV1, Rish JA, Eisenach KD, Cave MD, Bates JH.

Author information

Abstract

There is a need for rapid and sensitive detection of Mycobacterium tuberculosis in tissue specimens. A polymerase chain reaction (PCR)-based assay for the diagnosis of tuberculosis was evaluated in 60 formalin-fixed tissue specimens, the target for the amplification being a segment of IS6110 in the M. tuberculosis chromosome. Of the 60 formalin-fixed, paraffin-embedded tissue specimens studied, 57 showed granulomatous inflammation and 53 had been cultured for mycobacteria; 10 were positive for M. tuberculosis and three were positive for other mycobacteria. Of 60 samples, 15 showed acid-fast bacilli on special staining. When done comparatively on a positive culture for M. tuberculosis, PCR for M. tuberculosis DNA in 60 tissue samples was 100% sensitive and 93% specific, having a positive predictive value of 76.9% and negative predictive value of 100%. PCR for M. tuberculosis DNA done on tissue samples was positive for 14 of 19 patients who had a clinical diagnosis of tuberculosis, negative for all six patients with nontuberculous mycobacterial infections, and negative for all 33 patients who had a diagnosis of a disease other than mycobacterial infection. When compared with the clinical diagnosis of tuberculosis, PCR for M. tuberculosis DNA in these patients' tissues was 73.6% sensitive and 100% specific, having a positive predictive value of 100% and negative predictive value of 88.6%. These data indicate that PCR amplification is useful for detecting M. tuberculosis DNA in formalin-fixed tissue specimens, and that it can be used to increase diagnostic accuracy in patients who have perplexing diagnostic problems associated with a granulomatous tissue response.

PMID:

9769274

DOI:

10.1164/ajrccm.158.4.9802034

Evaluation of PCR in Detection of Mycobacterium tuberculosis from

Formalin-Fixed, Paraffin-Embedded Tissues: Comparison

of Four Amplification Assays

GIULIA MARCHETTI,1

* ANDREA GORI,1 LIDIA CATOZZI,1 LUCA VAGO,2 MANUELA NEBULONI,2

M. CRISTINA ROSSI,1 ANNA DEGLI ESPOSTI,1 ALESSANDRA BANDERA,1

AND FABIO FRANZETTI1

Clinic of Infectious Diseases1 and Pathology Unit,2 “Luigi Sacco” Hospital,

University of Milan, Milan, Italy

Received 24 September 1997/Returned for modification 18 December 1997/Accepted 4 March 1998

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium

tuberculosisfrom 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 the mtp40 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 mg yielded the

highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsenpositive,

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.

SUMMARY

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.

KEYWORDS: Tuberculosis; PCR; Diagnosis; Paraffin-embedded tissue.

thank you very much drVielma  , have come across of TB and lymphoma in the same patient?

PCR may be.

No culture