| Abstract|| |
Background and Aim: Cutaneous tuberculosis (CTB) is still difficult to diagnose due to its varied clinical presentation and limitations of diagnostic methods. The aim of this study was to evaluate the results of diagnostic laboratory tests available for CTB. Materials and Methods: Twenty-six skin biopsy specimens belonging to clinically suspected cases of CTB were studied retrospectively. The specimens were divided into two portions, one part processed for histopathological evaluation and the other was used for microscopy and inoculation for the isolation of mycobacteria. Polymerase chain reaction (PCR) technique was applied to 14 of 26 specimens to detect Mycobacterium tuberculosis complex (MTBC) DNA. Results: Of the 26 biopsy specimens, 11 were confirmed as CTB by identification of MTBC in culture and/or histopathologic affirmation. Of these, four were lupus vulgaris, four were TB verrucosa cutis, one was scrofuloderma, one was primary inoculation TB, and one was periorifical CTB. Culture for mycobacteria was positive for five (45.45%) specimens, while histopathologic affirmation was obtained in ten (90.90%) specimens. Acid-fast Bacilli were not demonstrated in any of the specimens on microscopic examination. The PCR was found to be applied to six of the 11 specimens diagnosed as CTB and was positive in two specimens (33.3%), which were positive for growth in culture and histopathological correlation. Conclusion: The recovery rate of MTBC from biopsy specimens was found to be satisfactory for CTB with histopathological correlation, but the combination of culture with a rapid method, PCR, may improve the diagnostic rate.
Keywords: Acid-fast staining, culture, cutaneous tuberculosis, diagnosis, polymerase chain reaction
|How to cite this article:|
Afsar I, Afsar FS. Evaluation of laboratory diagnosis for cutaneous tuberculosis. Indian J Pathol Microbiol 2016;59:274-8
| Introduction|| |
Cutaneous tuberculosis (CTB) is the rarest case of extrapulmonary TB (EPTB) comprising 2% of the total cases of EPTB. Mycobacterium tuberculosis and Mycobacterium bovis are the causative agents of CTB., The major challenge in the diagnosis of EPTB is the frequently atypical clinical presentation simulating other inflammatory and neoplastic conditions, which frequently results in a delay or deprivation of treatment. Therefore, a high degree of suspicion is required for an early diagnosis and mostly, more than one technique is necessary for the diagnosis.
Although culturing the etiologic agent is the accepted “gold standard” for the diagnosis of TB, various other methods are also employed for the diagnosis of EPTB such as smear microscopy, histopathology, tuberculin skin test, serological assays, interferon-gamma release assays (IGRAs), and nucleic acid amplification (NAA) tests.,, The aim of this study was to evaluate the laboratory results of the skin biopsy specimens, which were finally diagnosed as CTB.
| Materials and Methods|| |
Twenty-six skin biopsy specimens which were admitted by the Ataturk Research and Training Hospital, Department of Medical Microbiology, for evaluation of mycobacteriology from September 2010 to June 2014 were studied retrospectively for acid-fast Bacilli (AFB) staining, growth of mycobacteria in culture, polymerase chain reaction (PCR) if available, and histopathological diagnosis as well as clinical records for clinical presentation.
Skin biopsy specimens
Skin punch biopsies were performed from the active part of the lesions clinically suspected for CTB and divided into two portions, one part processed for histopathological evaluation and the other was used for microscopic examination of AFB and inoculation for the isolation of mycobacteria. One more skin biopsy was performed for PCR technique when available.
Acid-fast staining and culture
Skin biopsy specimens sampled under aseptic conditions were transported to the laboratory in serum physiologic solutions. The samples were broken into pieces by sterile lancets and decontaminated and homogenized with standard sodium hydroxide-N-acetyl-L cysteine method. The sediment was used for making smears to be stained by Ehrlich–Ziehl–Neelsen method for demonstration of Bacilli and inoculating to Löwenstein–Jensen culture tube (Salubris, Turkey) and in Bactec MGIT 960 liquid media (Becton Dickinson). The Löwenstein–Jensen slants were incubated at 37°C and 25°C and examined for growth every week. If no growth was observed after 8 weeks of incubation, the specimen was reported as culture-negative. The Bactec MGIT 960 system is fully automated and provides continuous monitoring. Once a recovery was established in solid media or a positive signal was given by the Bactec system, an Ehrlich–Ziehl–Neelsen staining was performed on the specimens, and the specimens positive for AFB were evaluated by the MGIT TBC ID (Becton Dickinson) test for identification of M. tuberculosis complex (MTBC).
Polymerase chain reaction
The GeneXpert MTB/RIF test (Cepheid, Sunnyvale, CA, USA), which is based on NAA and detection of an MTB-specific region of the rpoB gene, uses real time-PCR (RT-PCR) with molecular beacons. The primers in this assay amplify a portion of the rpoB gene containing the 81 base pair “core” region. The probes can differentiate between the conserved wild-type sequence and mutations in the core region that are associated with rifampicin resistance, and the test also detects mutations associated with rifampin resistance. An amount of 200 µl sediment prepared from skin biopsy specimens after decontamination and homogenization processes was resuspended in phosphate-buffered saline to a 500 µl volume. The sample reagent supplied with the test (1.5 ml) was then added. The mixture was vortexed for 30 s to ensure all bacteria were resuspended. The sample was left to stand for 15 min with intermittent manual shaking. The solution was then transferred to the Xpert cartridge using a Pasteur pipette, and the cartridge was loaded onto the Xpert machine for analysis. Results are interpreted as positive or negative for M. tuberculosis. Positive results are ranked in four categories: Very low, low, medium, or high. Rifampicin resistance results were reported as susceptible or resistant.
| Results|| |
The study group consisted of 26 skin biopsy specimens admitted by the Medical Microbiology Department for Mycobacterial Evaluation. Of the 26 biopsy specimens, 11 were found to be confirmed as CTB by the recovery of M. tuberculosis in culture and/or histopathological affirmation.
Of the 11 cases, four (36.6%) were lupus vulgaris, four (36.6%) were TB verrucosa cutis (TVC), one (9.09%) was scrofuloderma, and one (9.09%) was primary inoculation TB clinically. AFB was not demonstrated in any of the smears prepared by the Ehrlich–Ziehl–Neelsen method. Recovery of MTBC in culture media was established in five (45.45%) cases. Of those five cases, two (40.0%) were TVC, one (20.0%) was scrofuloderma, one (20.0%) was lupus vulgaris, and one (20.0%) was periorificial TB morphologically. The identification test for culture confirmation of MTBC strain yielded M. tuberculosis in all five specimens which were positive in culture. Detection of M. tuberculosis by the PCR method was able to be performed in six (54.54%) of the 11 cases. Of those six cases, the PCR gave positive results in two (33.33%) specimens. The specimens of these two patients in whom PCR positivity was established were also positive in culture and their morphological variants were TVC and periorificial TB. Among the 11 patients diagnosed as CTB by bacteriological and/or histopathological confirmation, both were positive for 4 (36.36%) patients. Histopathological confirmation was observed in (90.90%) of the 11 cases. The only case whose histopathological findings were not compatible with CTB was lupus vulgaris morphologically, but recovery of MTBC in culture confirmed the diagnosis of CTB [Table 1].
|Table 1: Laboratory data and morphological variants of patients diagnosed as cutaneous tuberculosis|
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| Discussion|| |
CTB is currently classified as presentations with high inoculum that are more frequent in endemic countries, and as presentations with low inoculum that are more frequent in countries with a weak endemicity of TB. The great clinical polymorphism, the multiple macroscopic and histological aspects, and the weak efficacy of microbiological examinations make the diagnosis difficult. On occasions, the diagnosis is only established retrospectively, after response to a therapeutic trial. Our study group consisted of the biopsy specimens diagnosed as CTB after microbiological evaluation or the ones microbiologically negative, but confirmed as CTB with histopathologic evaluation.
Diagnosis of TB is confirmed by the presence of AFB and/or isolation of M. tuberculosis in culture from biopsy specimens or fine-needle aspirates., Smear microscopy involves the direct microscopic examination of the clinical sample for the presence of AFB. It is widely used in the diagnosis of EPTB on account of its simplicity, speed, low cost, and minimal requirement for equipment and technical skill, but has drawbacks owing to low and variable sensitivity values and could not differentiate between M. tuberculosis and nontuberculous bacteria. Staining for AFB has low sensitivity, as its detection limit is >104Bacilli per slide or 104Bacilli per ml of specimen., Demonstration of AFB in tissue smears by Ehrlich–Ziehl–Neelsen staining was possible in 9.52% and 9.8% of the CTB cases in two different studies., In another study, no AFB could be demonstrated on routine examination of 100 CTB cases. When multiple sections from each case were examined, AFB could be seen in two cases, one with scrofuloderma with nonspecific histopathology and the other with TVC. The failure of microscopic observation of AFB in all smears was not extraordinary when we particularly consider the small sample size in our study.
The sensitivity of culture is greater than that of the AFB smear and culturing the etiologic agent is the accepted “gold standard” for the diagnosis of TB., The most commonly used culture media for isolation of M. tuberculosis are egg-based media (Löwenstein–Jensen) and semisynthetic media with agar (Middlebrook 7H10 and/H11). However, its drawbacks are that isolation takes 4–6 weeks, due to the slow growth rate of the organism. Possible reasons for nonisolation of M. tuberculosis are explained as the Bacilli are destroyed, nonviable, or highly attenuated, and Bacilli might exist in different forms. AFB was recovered from 7% to 60% of the cases of CTB by various workers.,, In a study of CTB, no difference was found in the yield of culture positivity in clinical types with a satisfactory rate of 55%. This rate was suggested to be due to the use of multiple media for culture and the use of Kirchner's liquid medium for preserving the biopsy material till they are processed. Although the culture was positive in two TVC cases but only in one case of scrofuloderma, lupus vulgaris, and periorificial TB, it is not acceptable to make a comment for the yield of culture positivity in clinical types because of the small sample size of our study.
Even though the conventional culture media requires a long time for the appearance of growth of M. tuberculosis, more rapid methods based on liquid culturing have been developed. The most widely used rapid-culture techniques are radiometric BACTEC (BACTEC 460) and nonradiometric (Bactec MGIT 960), which can detect growth as short as 3–7 days., These techniques are based on the release of a radioactive or fluorochrome marker from a metabolite in the culture medium that is used by the mycobacteria. The release of the marker can be detected by a special device even before the mycobacterial colonies are visible. In addition to identification of mycobacteria, drug susceptibility tests can also be performed. A meta-analysis of ten studies of the Bactec MGIT 960 and BACTEC 460 systems performed on pulmonary or extrapulmonary specimens revealed a sensitivity and specificity for detection of mycobacteria of 81.5% and 99.6% and 85.8% and 99.9%, respectively. The radiometric BACTEC 460 system has the disadvantage that it requires radiolabeling, which renders it difficult and complicated to use. On the other hand, the Bactec MGIT 960 shows a shorter time to the detection of AFB and more convenient nonradiometric technology. These features make the Bactec MGIT 960 system in combination with solid media a valuable alternative.
Conventional microbiological techniques for the diagnosis of CTB have limitations. This prompted the use of molecular techniques for diagnosis. DNA-PCR has been evaluated as a tool in the diagnosis of various forms of CTB, but it has a variable sensitivity. PCR is a reliable, rapid method that has become available in the recent years in the diagnosis of CTB, and this method can be used in the routine diagnostic algorithm when conventional methods fail to identify MTBC. In PCR reactions, discrete fragments of DNA are specifically amplified from specimens in CTB. The PCR gives satisfactory results as few as 100 bacteria per sample in a matter of hours. It was applied to fresh skin biopsy specimens in our laboratory. Although it can also be performed on paraffin-embedded tissue, the possibility of false-negatives increases due to degradation of DNA during the embedding process. If the tissue used for PCR is more than 5-year-old, degradation leads to significant reduction in the amplification of DNA.
A considerable number of regions/sequences of the mycobacterial genome (IS6110, IS986, 65 kDa, and 38 kDa) have been identified as target antigens of PCR. The overall sensitivity of PCR for smear-negative specimens has ranged from about 50% to 72% in different studies using IS110., Suthar et al. found the sensitivity and specificity of DNA-PCR for Mycobacterium tuberculosis as 25% and 73.68%, respectively, and explained the reason for low sensitivity as the predominantly paucibacillary nature of CTB and suggested that the low specificity could be due to the contamination at the laboratory level.
On the other hand, PCR has several limitations. Its sensitivity is reduced when used with smear-negative specimens or paucibacillary samples. This may be explained by the loss of DNA during extraction, failure to sample target DNA during sectioning, or the presence of inhibitory substances. Fixative has also been reported to diminish the PCR signal, particularly when the fixation time is prolonged.,
The recently developed Xpert test based on nested RT-PCR and molecular beacon technology targeting rpoB gene of wild type M. tuberculosis strains has been demonstrated as a rapid test with results for both TB identification and rifampicin resistance in <2 h in a single tube., The performance of Xpert assay has been compared with IS6110-based RT-PCR assay for TB identification in EPTB specimens, and it was found that the Xpert assay exhibited better sensitivity. Tortoli et al. evaluated the utility of Xpert assay in 1476 EPTB specimens and reported 81.3% sensitivity and 99.8% specificity considering culture and clinical diagnosis as the gold standard. A systematic review reported that Xpert reliably detected the vast majority of nonrespiratory samples testing smear-positive and culture-positive for M. tuberculosis, but approximately only two-thirds of smear-negative samples. The two PCR-positive skin biopsy specimens in our study were also culture-positive and compatible with CTB histopathology.
The application of these tests to the reliable diagnosis of TB depends on the careful selection of appropriate gene targets and is challenged by issues peculiar to mycobacteria such as bacterial clumping, low bacterial load, a large variety in the nature of specimen provided for diagnosis, and the difficulty in isolating pure mycobacterial DNA suitable for amplification. The limited shelf-life of the diagnostic cartridges, some operating temperature and humidity restrictions, requirement for electricity supply, unknown long-term robustness, and the need for annual servicing and calibration of each machine are the limitations reported for the Xpert test.
Histopathology testing and isolation of M. tuberculosis in the culture of skin specimens or by PCR have been considered the best diagnostic tool for the detection and diagnosis of CTB. In a study of 193 CTB patients from whom both histopathology and bacteriology results were available, both were positive for 96 (50%) patients, histopathology alone was positive for 78 (41%) patients, and bacteriology alone for 16 (8%) patients. In our study, the 11 patients from whom both histopathology and bacteriology results were available, both were positive for four (36.36%) patients, histopathology alone was positive for six (54.54%) patients, and bacteriology alone for one (9.09%) patient. The PCR method was able to be performed on six (54.54%) specimens and of those six specimens, two (33.33%) were positive for histopathology, bacteriology, and PCR. Correlation of histopathology with bacteriology results indicates that these two indices are complementary to each other, and performing these two diagnostic procedures increases the establishment of the diagnosis of CTB by 8%. However, a negative smear for AFB, lack of granulomas on histopathology, and failure to culture M. tuberculosis do not exclude the diagnosis of EPTB.
An increasing number of new methods with suitable sensitivity and specificity that attempt to speed up diagnosis are being developed such as serological assays and IGRAs.,, However, still the definitive criterion for CTB is isolation of the bacterium in culture or identification of mycobacterial DNA by PCR.
| Conclusion|| |
We evaluated the laboratory tests performed for CTB and found that the results were satisfactory with histopathological correlation, although the study sample size was small. We believe that more encouraging results will be obtained for culture and PCR for CTB if the number of skin biopsy specimens suspected for CTB increases, and the culture should be combined with the most rapid method, PCR.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Ilhan Afsar
Department of Medical Microbiology, Ataturk Research and Training Hospital, Basin Sitesi 35360 Izmir
Source of Support: None, Conflict of Interest: None