Tuberculosis caused by Mycobacteriumtuberculosis remains one of the major public health problems worldwide, killing more persons than any other agent (approximately 1.8 million per year).1 Tuberculosis does not affect the developing world alone, but is reemerging rapidly in many industrialized nations where it was previously considered controlled. The dramatic increase in the global incidence of tuberculosis is facilitated by the spread of HIV infection and the appearance of multidrug-resistant strains of mycobacteria.2,3 Cutaneous involvement represents only 2% of extrapulmonary tuberculosis.4,5 In the past, it was more prevalent in temperate countries with cold and humid climates with a few hours of daily sunlight, malnutrition, and low socioeconomic conditions, which are predisposing factors for cutaneous tuberculosis. The prevalence of pulmonary tuberculosis is estimated at 50 cases for every 100,000 inhabitants.6,7 The skin manifestations present secondary to hematogenous spread or from direct tissue extension from a latent or an active underlying focus of infection. Several Mexican studies show the incidence of dermatological symptomatology in cutaneous tuberculosis.3,8,9 One of these studies reveals that up to 3% of 114 Mexican patients with skin problems were diagnosed with cutaneous tuberculosis, with 20% of these cases affecting children younger than 15 years and with higher overall incidence among females.8 At the “Dr. Ladislao de la Pascua” Dermatologic Clinic, 178 of 19,664 biopsies performed between 1985 and 1995 were cutaneous tuberculosis (0.91%). Therefore, this comprises an important point to support the diagnosis of cutaneous tuberculosis in all suspect lesions. Cutaneous tuberculosis is not lethal, but it may be a cause of complications or destruction of the skin.
The classification of cutaneous tuberculosis is complicated by the clinical profile of the polymorphic variables of these diseases.4 Definitive diagnosis requires the demonstration of mycobacteria, which is not always possible in skin lesions. In these latter cases, some patients exhibit strong reactivity to mycobacterial antigens [purified protein derivative (PPD)] and are considered as tuberculids. Rosacea-like tuberculosis, papulonecrotic tuberculosis, and erythema induratum of Bazin may belong to the tuberculids group.8,9 However, because demonstration of the presence of mycobacteria in tuberculids in situ has not been possible to date, it has been suggested that the lesions are caused by a hyperergic host-immune response to a bacterial infection in the lung or lymph nodes.8,9 Given the difficulty associated with demonstrating the presence of M. tuberculosis in skin lesions by routinely available microbiological and histological tests, cutaneous tuberculosis is often diagnosed only after the exclusion of other possibilities.3,8,9 Institution of timely therapy is thus often delayed.
Polymerase chain reaction (PCR) has been used previously to amplify DNA segments from M. tuberculosis and other mycobacteria in different clinical samples.10–13 PCR is at least as sensitive as routine culture techniques14,15 and can be used on formalin-fixed paraffin-embedded tissues.16–20 The usefulness of a PCR test for the diagnosis of cutaneous tuberculosis has been demonstrated in cases of lupus vulgaris tuberculosis and scrofuloderma tuberculosis in an accidental exogenous inoculation, and in erythema induratum of Bazin,21–24 in one patient with coexisting papulonecrotic tuberculosis and erythema induratum of Bazin.25,26 These sensitive and rapid PCR tests constitute a significant improvement in the diagnosis of cutaneous tuberculosis.
We investigated the presence of mycobacterial DNA in its clinical forms that are most frequently observed at the “Dr. Ladislao de la Pascua” Dermatologic Center. The PCR method designed allowed for the amplification of 3 genomic mycobacterial DNA fragments and simultaneous discrimination among M. tuberculosis, M. bovis, and other nontuberculosis mycobacterial DNA.10 In addition, nested PCR was performed to further increase the sensitivity and specificity of the method.9
MATERIALS AND METHODS
Bacterial Strains and DNA Isolation
The mycobacterial strain used as control was M. tuberculosis H37Rv. The strain was grown in Middlebrook 7H9 (Difco, Franklin Lakes, NJ) and DNA was isolated using the phenol–chloroform method. DNA was precipitated with isopropanol and resuspended in 50 μL of distilled water,27 and 100 ng of this solution was used for PCR amplification.
Patients and Samples
In this study, we included 30 paraffin-embedded skin biopsies from randomly selected patients at the “Dr. Ladislao de la Pascua” Dermatologic Center; 10 of these were from men and 20 from women, with a mean age of 30.5 years (range, 3–63 years) (Table 1). Multiple clinical forms of cutaneous tuberculosis were included (Table 1). Previous diagnosis was based on the following criteria: the clinical type and localization of the lesions; histopathological analysis of the samples; intradermal PPD test; and response to administered antituberculous drugs. Negative controls were 4 normal skin biopsies, and the internal PCR control was the mixture of reagents involved in the assay. As a positive control, we used M. tuberculosis DNA isolated from a patient with pulmonary tuberculosis.
Preparation of Biopsies
Biopsies were fixed with 10% formalin and embedded in paraffin. The embedded tissue blocks were cut into 20-mm-wide sections, and these sections were treated according to the procedure described by Volkenandt.28 Briefly, paraffin was removed from the tissues, after which the tissues were heated at 70°C for 30 minutes.15 Genomic DNA was isolated with guanidine isothiocyanate and phenol, using 1.0 mL of TRIzol (Gibco BRL) according to the procedure described by Chomczynski.29 DNA was precipitated with 75% ethanol and resuspended in 8 mM NaOH. Insoluble material was removed by centrifugation at 7500g for 10 minutes at 4°C. The final solution was heated at 55°C for 20 minutes. A total of 10 μL of the DNA sample were used for PCR amplification.30 The samples were randomly analyzed.
We used 100 ng of DNA from the M. tuberculosis strain H37Rv to standardize the test. The sequences of the primers are shown in Table 2. In the initial amplification, primers MT1 and MT2 amplified the gene encoding the 32-kDa alpha antigen present in all described mycobacteria,14 whereas primers IS5 and IS6 amplified the IS6110 insertion element,31–33 and PT1 and PT2 were used to amplify the species-specific gene mtp40, encoding the Mtp40 protein, which is specific for M. tuberculosis.14,15 Nested PCR further amplified an internal region of the mtp40 gene of M. tuberculosis.14 All reactions were performed in a final volume of 50 μL, with 1X reaction buffer, 1.25 U of Taq polymerase, 0.2 mM of each dNTP, 2.5 mM of MgCl2, 10 pM of MT1-MT2, 15 pM of IS5-IS6, and 20 pM of PT1-PT2. The reactions were performed in a thermocycler (RoboCycler 40; Stratagene). Multiple PCR conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 1 minute, alignment at 71°C for 2 minutes, and extension at 72°C for 3 minutes, for 35 cycles, with a final extension at 72°C for 10 minutes. Sensitivity limits for multiple PCRs were established by assaying serial dilutions (1:10) of 10,000-0.000,001 pg/μL of M. tuberculosis genomic DNA. The multiple PCR products were subjected to nested PCR under the following conditions: 94°C for 5 minutes, followed by 30 cycles at 94°C for 1 minute, 74°C for 2 minutes, and 72°C for 2 minutes, followed by a final extension at 72°C for 7 minutes.
We used 10 μL of the PCR products for the analysis by horizontal electrophoresis in 2.0% agarose gels (Horizon 58, Life Technologies, Gibco BRL) containing 0.5 μg/mL of ethidium bromide.34 Electrophoresis bands were visualized in an image analyzer (IS-1000 Digital Imaging System; Alpha Innotech Corporation).
The multiple PCR products were subjected to nested PCR. Ten microliters of the multiple PCR products was subjected to a second round of amplification using the 44–265-bp strand from the mtp40 gene as target,17 using 20 pM of the primers. The mtp40 primer sequences were obtained from Gen Bank NCBI (gibbsq 146622): PT3: 5′ CAC CAC GTT CGG GAT GCA CTG C and PT4: 5′ CTG ATG GTC TCC GAC ACG TCG (223-bp fragment). Nested PCR conditions were as follows: initial denaturalization at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, alignment at 74°C for 2 minutes, and extension at 72°C for 2 minutes, with a final extension at 72°C for 7 minutes. The amplified products were analyzed as described previously. The sensitivity of the nested PCR was determined by amplifying the products from the multiple PCR dilution assay as described previously.
Multiple PCR and Nested PCR Standardization
The following factors were taken into account: (1) the sizes of the amplified fragments were sufficiently different to be distinguishable on agarose gels; (2) the primers had no potential matches with sequences at nonspecific target sites, and (3) the optimal DNA–primer annealing temperatures were nearly the same for all template–primer combinations.31Figure 1a depicts the electrophoretic pattern obtained after multiple PCR, and lane 5 presents the simultaneous amplification of the 506-bp fragment corresponding to the alpha antigen, the 984-bp fragment corresponding to the IS6110 insertion sequence, and the 396-bp fragment corresponding to the species-specific mtp40 gene. Lanes 2, 3, and 4 illustrate the amplification products when the primers were used in separate reactions.
Nested PCR further amplified an internal region of the mtp40 gene of M. tuberculosis; Figure 1b reveals a 223-bp product corresponding to the internal fragment of the mtp40 gene. The sensitivity of the multiple PCR and the nested PCR was 100 pg and 10 fg of DNA, respectively (Fig. 2). A nested PCR was used to increase the sensitivity of the test.
Multiple and Nested PCR Applied to Skin Biopsies
We analyzed 30 paraffin-embedded tissue samples obtained from patients and 4 normal skin controls. Figure 3a shows results obtained from 4 patients and one normal skin control; lanes 1–4 correspond to patients 22–25 and lane 5 corresponds to the normal skin control. None of the 34 samples analyzed (30 from patients and 4 from normal controls) produced visible amplification products after multiple PCR. The positive and negative controls demonstrated the expected results.
All 34 samples from the multiple PCR reaction were subjected to a second round of amplification using internal primers for the mtp40 gene. Figure 3b presents results from 4 of these in which lanes 1, 2, and 4 correspond to patients 22, 23, and 25, respectively, which amplified the expected 223-bp product. However, the DNA of patient 24 could not be amplified (lane 3). In 24 of 30 cases, the M. tuberculosis mtp40-specific band was amplified by nested PCR (Table 3).
Ten patients with a histopathological diagnosis of tuberculids corresponded to erythema induratum of Bazin, 7 to rosacea-like tuberculosis, and one to papulonecrotic tuberculosis. Figure 4 depicts the appearance and histopathology of different cutaneous tuberculosis. Figure 4a shows the appearance of an erythema induratum lesion where recurrent tender subcutaneous nodules occur mainly on the lower legs of a patient with tuberculosis, with the histopathology of the lesion demonstrating suppurative inflammation and foci of necrosis (Fig. 4b). Figure 4c reveals the appearance of a rosacea-like lesion where we may observe erythematosus papules and nodules, as well as pustules. The histopathology demonstrated epithelioid cell granulomas with surrounding mononuclear cell infiltrates and Langhans giant cells in the superficial and deep dermis (Fig. 4d).
Identification of M. tuberculosis DNA in the skin biopsies was achieved in 80% of erythema induratum of Bazin and in all the rosacea-like tuberculosis and papulonecrotic tuberculosis forms of cutaneous tuberculosis (Table 3). It was possible to demonstrate M. tuberculosis DNA in some of the other forms of cutaneous tuberculosis studied, including in 4 of the 5 cases (80%) of verrucosa tuberculosis, in 2 of the 2 cases (100%) of lupus vulgaris tuberculosis, and in 2 of the 5 (40%) cases of scrofuloderma tuberculosis (Table 2).
Cutaneous manifestations of tuberculosis have been found in less than 0.1% of individuals seen at dermatology clinics.35 An acid-fast bacillus smear is useful if lesions have a high bacterial load, as observed in lichen scrofulosorum, miliary tuberculosis, and tuberculosis gumma. Currently, PCR appears to be the most useful technique in multibacillary forms of cutaneous tuberculosis.12,13 In one report of acid-fast bacilli–negative specimens, the overall sensitivity of PCR was found to be 50%–72%.20 The PCR method used in our study allowed for simultaneous amplification of the M. tuberculosis species–specific mtp40, IS6110 insertion sequence, and alpha antigen-encoding gene. Thus, in a single step, M. tuberculosis could be identified and differentiated from M. bovis and other nontuberculosis mycobacteria. This method ensures the detection of M. tuberculosis lacking the IS611016 insertion sequence because the species-specific mtp40 gene and the alpha antigen-encoding gene are amplified.
Although the multiple PCR method was designed to detect any mycobacterial DNA present in biopsy samples, none of the patients with cutaneous tuberculosis exhibited positive results, and it was necessary to perform a species-specific nested PCR to increase the sensitivity of detection. It is known that only a small number of mycobacteria are present in this type of clinical samples.8,18,19,21,22 For this reason, we improved sensitivity with nested PCR to detect the mycobacterial DNA. Amplification from paraffin-embedded tissues is often a method with low efficiency for obtaining DNA because it is affected by tissue-fixing solutions, storage time, and conservation conditions.17 Importantly, the nested PCR allowed for the successful amplification of M. tuberculosis DNA in 80% of the samples.
The detection limit of the multiple PCR was 100 pg of DNA. Using a similar assay, Del Portillo et al10 reported a detection limit of 1 pg of DNA. Nested PCR increased the sensitivity of our method to 10 fg of M. tuberculosis DNA, making it a very powerful method for the detection of mycobacteria.
Some authors support that cutaneous lesions of the group called tuberculids are related to M. tuberculosis.36 In fact, the role of mycobacteria in the pathogenesis of tuberculids has been controversial because the isolation and culture of the bacteria is not easily achieved by routine techniques. It has been suggested that, even if the bacillus is present in the lesions, it is removed by a strong host-immune response and can therefore not be directly implicated in the pathology.21,22,37–42 However, the results presented herein support a direct association between M. tuberculosis and the chronic nodular lesions denominated tuberculids. In this study, M. tuberculosis DNA was identified in 16 of the 18 tuberculid cases (88.9%). These findings imply that the mycobacterium is present in the lesions. We suggest that the mycobacteria cannot be cultured from tuberculid lesions because this type of samples contains a small number of bacteria. Notwithstanding this, it was possible to detect them using nested PCR. In agreement with other authors,35 we have demonstrated that this method comprises a promising tool for the diagnosis of cutaneous tuberculosis. At the present time in Mexico, diagnosis is based on exclusion criteria and is supported by the clinical features of the lesions, histopathological studies, the reaction of the skin to PPD, and the response to treatment with antituberculous drugs.
M. tuberculosis DNA was amplified in all samples from patients with rosacea-like tuberculosis or papulonecrotic tuberculosis. DNA was not amplified by nested PCR in only 2 of the 10 patients with erythema induratum of Bazin (patients 10 and 28). This failure can be attributed to the presence of undetected PCR inhibitors or to other technical problems; the recovery rate of intact DNA is influenced by variables such as the tissue type, autolysis or necrosis, the presence of nucleases, and the stromal density.16,17 Such variables most probably prevented the detection of DNA from these 2 patients. Erythema induratum of Bazin or erythema induratum is the most frequent lesion observed in Mexico and other countries,21,22 but diagnosis is difficult in that it is easy to confuse it with other similar diseases. It has been designated as nodular vasculitis of the legs.41 Other authors consider PCR as a powerful technique for establishing or confirming the diagnosis of cutaneous tuberculosis and for the determination of mycobacteria in tuberculid lesions.37
The results of this study contrast with the findings of Margall et al,34 where the M. tuberculosis complex was detected by PCR in 77% of the samples with different histopathological varieties of cutaneous tuberculosis; only 10% of the total cases studied corresponded to tuberculids. In this study, M. tuberculosis DNA was amplified in 80% of the total samples, the majority of which (60%) corresponded to tuberculids. These cases have a difficult diagnosis and usually it is not found to be the causal agent. However, Degitz et al43,44 were able to demonstrate M. tuberculosis complex DNA in 5 of the 7 patients with erythema induratum of Bazin and in 4 of the 6 patients with papulonecrotic tuberculosis. In that study, the authors performed PCR from paraffin-embedded skin biopsies and, with further Southern blot analysis, were able to differentiate between the M. tuberculosis complex and atypical mycobacteria. These results are in agreement with our study. Schneider et al38,39 identified M. tuberculosis complex DNA by PCR in 5 of the 20 patients with erythema induratum of Bazin. Four of their samples required a second amplification, as was the case with all our samples. In another study reported by Yen et al,41 the detection of M. tuberculosis from paraffin-embedded and formalin-fixed biopsies was achieved in 5 of the 9 samples (56%) using PCR and Southern blot.
In 3 of the 5 cases of scrofuloderma tuberculosis reported here, diagnosis was not confirmed by implanted detection methods, although this type of tuberculosis is considered typical cutaneous tuberculosis.4,6 Other groups have reported similar observations. Margall et al34 reported only one of the 3 cases of clinically histologically diagnosed scrofuloderma tuberculosis as positive for M. tuberculosis DNA amplified by PCR, and from one cultured sample, it was not possible to isolate the bacterium. Such difficulties can be attributed most probably to necrosis, which is a frequent characteristic of the skin tissue of these patients.
One patient (No. 12) exhibited an ulcerated nodule in the mandible, which simulated an odontogenic fistula. The final diagnosis, however, was scrofuloderma tuberculosis. Of the other 2 cases, patients 7 and 13 exhibited typical scrofuloderma tuberculosis lesions; nested PCR, however, did not detect mycobacterial DNA in these lesions. In patient 24, despite a clinical and pathological diagnosis of warty lupus tuberculosis, the presence of mycobacterial DNA was not demonstrated. The recovery rate of intact DNA is believed to be influenced by variables such as the tissue type, autolysis or necrosis, the nuclease content, and the stromal density.16,17 Such variables, in all likelihood, exerted some effect on the results of our study: Thus, M. tuberculosis DNA was detected in 66.7% of typical cutaneous tuberculosis patients. The latter is in agreement with the report of Degitz et al,43,44 where 53% of paraffin-embedded biopsies from patients with lupus vulgaris tuberculosis were positive for the M. tuberculosis complex DNA detected by PCR.
An important finding from our study is the successful amplification of mycobacterial DNA from rosacea-like tuberculosis, that on a routine diagnostic test, had not been accepted as tuberculosis previously. Rosacea-like tuberculosis can now be considered as cutaneous tuberculosis. Hoon et al12,45 used PCR to detect M. tuberculosis on skin samples. Their test was unable to detect mycobacterial DNA in tuberculids, lupus vulgaris tuberculosis, or warty lupus tuberculosis samples. It was, however, efficient for skin samples from immunocompromised patients. Mycobacteria develop better in such patients, and it is therefore easily detected by conventional microbiological culture or PCR techniques of lower sensitivity. These results support the notion that, before our study, lack of success in detecting M. tuberculosis DNA in the skin biopsies from cutaneous tuberculosis patients can be attributed to the absence of a sufficiently sensitive method for amplifying DNA from a very small number of bacteria.
In summary, multiple PCR followed by nested PCR have demonstrated that erythema induratum of bazin, papulonecrotic tuberculosis, and rosacea-like tuberculosis are closely associated with M. tuberculosis infection. The PCR methodology developed in this study is a rapid, highly sensitive, and specific method for the diagnosis of the aforementioned group of cases and for differentiation among mycobacterial species, which will contribute significantly to the timely diagnosis and appropriate therapy in these cases.
The authors are grateful to Dr. Omar Sangüeza for critical review of the manuscript and to Alberto Castillo for excellent technical support.
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