Dry-Reagent-Based PCR as a Novel Tool for Laboratory Confirmation of Clinically Diagnosed Mycobacterium ulcerans-Associated Disease in Areas in the Tropics Where M. ulcerans Is Endemic

ABSTRACT After tuberculosis and leprosy, Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic “gold standard.” Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.

After tuberculosis and leprosy, Buruli ulcer (BU) is the third most common mycobacterial disease in immunocompetent humans. The incidence of BU has been on the rise worldwide. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. The disease mainly affects impoverished inhabitants of remote rural areas, with a preference for children under the age of 15, and is believed to be associated with tropical and subtropical wetlands. Epidemiological studies suggest that swamps and slowly flowing water are the sources of the organism. Recent evidence supports the view that waterborne insects may be involved in the transmission of the infection. However, the precise source of infection is still unknown. The incidence and prevalence of BU worldwide are not precisely defined, as adequate surveillance data based on accurate case confirmation data are lacking. However, prevalences for the disease up to Ͼ20% have been reported from various foci in countries where the disease is highly endemic. In Ghana in 1999, for example, the overall crude national prevalence rate of active lesions was 20.7 per 100,000, but the rate was 150.8 per 100,000 in the district where the disease was most endemic (1,3,6,7,11).
According to World Health Organization standard case definitions, BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema and evolving into a painless ulcer with undermined edges. The lesion may lead to extensive scarring, contractures, and deformations with possible total loss of articulation function. If left untreated, it may even result in loss of limbs or blindness.
Cases that meet these clinical definitions are considered probable cases. Confirmed cases require a positive laboratory diagnosis. Because BU is associated with nonspecific clinical manifestations and an indolent course, every ulcer or nodule in an area of endemicity should be suspected as a Mycobacterium ulcerans infection until proven otherwise. Observations made by various researchers suggest that misclassification of clinically diagnosed BU cases may occur frequently. Early and healed lesions, especially, may be confused with other skin diseases endemic in tropical areas. The differential diagnosis comprises infectious (e.g., abscess, onchoceriasis, leprosy, elephantiasis, yaws, scrofuloderma, mycosis, actinomycosis, herpes, cutaneous leishmaniasis, tropical phagedenic ulcer, venous ulcer, and noma) and other (e.g., insect bites; psoriasis; enlarged lymph nodes; lipoma and other neoplasms; vascular, diabetic, and varicose ulcers; and burns) conditions and poses difficulties, especially in tropical settings with limited access to laboratory facilities (8,12).
Treatment of BU with antibiotics has been widely unsuccessful. Experimental drug treatment trials are under way. The present standard of treatment is surgical removal of the affected tissue, eventually followed by skin grafting. Due to the fundamental lack of understanding of modes of transmission, effective prevention strategies have not yet been developed. At present, disease control in countries of endemicity is limited to early detection through improved active surveillance and surgical treatment to prevent the development of severe ulceration and resulting disabilities (3,6,15).
The laboratory confirmation of BU is complicated by the absence of a diagnostic "gold standard" and a resulting lack of clarity regarding the sensitivities and specificities of different diagnostic laboratory assays. A positive laboratory diagnosis of M. ulcerans-associated disease requires any two of the following findings: detection of acid-fast bacilli in a Ziehl-Neelsenstained smear, positive culture of M. ulcerans (swab or tissue specimen, confirmed by biochemical tests or IS2404 PCR), histopathological confirmation, and positive IS2404 PCR (swab and/or tissue specimen) (6,14,15). Whereas the sensitivities of smear microscopy and culture are relatively low, histopathology and different PCR assays targeting different regions in M. ulcerans IS2404 and 16S rRNA genes provide diagnostic sensitivities of Ͼ90%. PCR can serve as a highly sensitive and reliable tool for presurgical early diagnosis and postsurgical laboratory confirmation of probable cases. Thus, PCR can prevent misclassification and subsequent inadequate treatment of patients in countries of endemicity. In addition, PCR provides reliable laboratory-confirmed incidence and prevalence data (5,9,10,13,16).
However, conventional PCR techniques in most cases are not available in countries of endemicity. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of conventional diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine (BNITM). The method is based on the standard diagnostic IS2404 PCR developed by Stinear et al. (14), and due to the use of lyophilized reagents, is well adapted to tropical conditions and convenient for application in the field. After technical and clinical validation, the method has been successfully installed at the  forms: ulcerative lesions, n ϭ 37; nonulcerative lesions, n ϭ 7 (nodules, n ϭ 4; plaque, n ϭ 1; papule, n ϭ 1; edema, n ϭ 1); clinical data not available, n ϭ 4. The duration of the disease ranged from 1 week to 16 years, with a median duration of 2 months.
To standardize the process of specimen collection, the following criteria were established. Diagnostic swabs (Greiner Bio-One, Essen, Germany) were to be taken from the undermined edges of the lesions before surgery; 10-by 10-mm tissue specimens were to be obtained from surgically excised tissue. In the case of nodules, the tissue specimens should contain a section of the center of the nodule; in the case of ulcers, the specimens should be taken from the edge of the ulcerative lesions containing necrotic tissue sections.
The PCR specimens were stored in 2-ml tubes (Sarstedt, Nümbrecht, Germany) containing 700 l of cell lysis solution (Puregene DNA isolation kit; Gentra Systems, Indianapolis, Ind.) at room temperature until they were processed.
PCR-negative tissue specimens were subjected to histopathological analysis to exclude other infectious and noninfectious conditions considered for differential diagnosis. Ten-by 10-mm tissue sections were stored in 5-ml tubes (Sarstedt) in 10% formaldehyde, and histopathological analysis was carried out according to standardized criteria (4).
DNA preparation. The Puregene DNA isolation kit was used for DNA isolation with minor modifications evaluated for extracting M. ulcerans DNA from tissue samples as described below. All materials mentioned were included in the kit.
Tissue specimens were inactivated at 95°C for 15 min. Subsequently, the specimens were cut, and 5-mm 3 (maximum) pieces were incubated overnight at 55°C in 700 l of cell lysis solution enriched with proteinase K (Sigma-Aldrich, Munich, Germany) to a final concentration of 300 g/ml. The proteinase K was inactivated at 95°C for 15 min. After the specimens were cooled to room temperature, egg white lysozyme was added to a final concentration of 250 g/ml, and the specimens were incubated at 37°C for 1 h.
Processing of swab samples, as well as subsequent procedures of DNA extraction for tissue specimens, were carried out according to the manufacturers' instructions. The DNA pellets were resuspended in 200 l of DNA hydration solution, and extracts were stored at 4°C until they were further processed.
Selection of oligonucleotides and PCR conditions. (i) Standard reference method. Based on the findings in a recent publication (14), amplification of a specific 492-bp-long DNA sequence in M. ulcerans IS2404 (GenBank accession no. AF003002) by PCR was carried out from 2 l of target DNA extract using primers MU5 (5Ј-AGC GAC CCC AGT GGA TTG GT) and MU6 (5Ј-CGG TGA TCA AGC GTT CAC GA). The reaction volume was 20 l, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , 200 M (each) deoxynucleoside triphosphate, and 1 U of Ampli-Taq Gold DNA polymerase. All reagents except the oligonucleotides (TibMolbiol, Berlin, Germany) were purchased from Perkin-Elmer, Weiterstadt, Germany. DNA extracts of diagnostic specimens were tested in 10 0 and 10 Ϫ1 dilutions. Hot-start thermal cycling was conducted in a Primus Thermocycler (MWG Biotech, Ebersberg, Germany) according to the following cycling profile: one initial activation step with polymerase at 94°C for 10 min; 40 cycles, each consisting of denaturation at 94°C for 10 s, primer annealing at 58°C for 10 s, and extension at 72°C for 30 s; and one final extension step at 72°C for 15 min. The amplification products were held at 4°C until they were further processed and detected by agarose gel electrophoresis (1.5%) and ethidium bromide staining (1 g/ml). Because the reaction conditions of the PuReTaq Ready-To-Go PCR bead Technology were fixed as described above and therefore deviated from the standard reference method, the amount of target DNA was extrapolated and adjusted to 2.5 l due to the increased reaction volume.
Technical validation and optimization of PCR. (i) Quantified plasmid DNA standard. The 492-bp target region of the standard reference method was amplified, analyzed by gel electrophoresis, and prepared using the NucleoSpin Extract kit (Macherey Nagel, Düren, Germany) according to the manufacturer's instructions. Subsequently, the amplicon was directly cloned via the T/A sticky end using the AdvanTAge PCR cloning kit (Clontech, Heidelberg, Germany).
Plasmid DNA was prepared using the NucleoSpin Plasmid kit (Macherey Nagel). Afterwards, plasmid concentrations were determined photometrically (260 nm). Because the target region is located within a multiple-copy element and is therefore detectable ϳ50 times per genome (13), the plasmid standard with 1.5 genome equivalents represents ϳ75 copies of the element.
(ii) Validation by testing diagnostic specimens in comparison to the IS2404 reference method. Diagnostic specimens used for validation of the dry-reagentbased PCR method were obtained from 48 clinically diagnosed BU patients. (i) Swabs (n ϭ 19) and tissue specimens (n ϭ 16) from 19 clinically diagnosed BU patients were simultaneously tested at the BNITM by both the standard reference and the dry-reagent-based PCR methods. (ii) After implementation of the method at the KCCR, swabs (n ϭ 22) and tissue specimens (n ϭ 30) from 30 clinically diagnosed BU patients were tested simultaneously at the KCCR (dryreagent-based PCR) and the BNITM (standard reference method).

Internal quality control. (i) Extraction control.
To exclude false-positive PCR results caused by contamination during DNA extraction, an extraction was performed without a specimen and used as an extraction control. The extraction control was required to produce a negative result after PCR.
(ii) PCR run controls. To exclude false-positive and -negative PCR results, the following control reactions were performed.
(a) Negative control reaction. The reaction mixture contained water instead of template DNA, which should not produce a specific band after PCR.
(b) Positive control reaction. PCR was performed using plasmid DNA containing the specific 492-bp DNA sequence of M. ulcerans IS2404 as a template.
(c) Inhibition control reactions. For quality testing of the DNA extract, inhibition control PCRs were run in parallel with the tested samples. These controls consisted of half of the volume of DNA extract, with the IS2404 plasmid DNA as a competitor (1.5 genome equivalents). Possible inhibitory effects of substances in the DNA extract were excluded if a clearly visible PCR amplicon of 492 bp was present in the inhibition control reactions.

Analytic sensitivity.
To establish a dry-reagent-based PCR for the detection of M. ulcerans, IS2404 was selected as the target region. This target had been proven to be appropriate for diagnostic application, and its use is regarded as the standard reference method routinely used for diagnostic purposes (14).
To determine the analytical sensitivity in a quantified DNA standard, we cloned the amplicon of the standard reference method into Escherichia coli.
Replicates of log 10 plasmid serial dilutions were amplified by both the standard reference PCR and the dry-reagent-based PCR. It was shown that the sensitivity was excellent: 1.5 genome equivalents per reaction could be detected with the standard reference PCR in 10 of 10 reactions. When testing arithmetically higher dilutions (ϳ0.15 plasmids per reaction), positive standard reference PCR results were occasionally obtained due to the plasmids randomly distributed among the samples. Therefore, the reaction conditions could be considered already optimized.
The same sensitivity, i.e., 1.5 genome equivalents per reaction, was achieved using the dry-reagent-based PCR formulation, indicating comparable analytical sensitivity.

Sensitivity and specificity. (i) Mycobacterial reference strains.
To confirm the specificity of the dry-reagent-based PCR, 39 M. ulcerans reference strains and 15 different mycobacterial strains (Table 1)  from 19 patients were tested simultaneously with both methods at the BNITM. The accordance (swabs, 94.7%; tissue specimens, 75%) and discordance (swabs, 5.3%; tissue specimens, 25.0%) rates are presented in Table 2.
Histopathological analysis of 27 PCR-negative tissue specimens. Eleven specimens were inappropriate for diagnostic purposes, as the subcutaneous adipose tissue was either missing or not complete. For two specimens, histopathological results were not available. Unspecific dermatitis was found in two specimens, and one specimen was diagnosed as Kaposi's sarcoma. Two specimens showed signs of parasitic infection, one of those being onchocerciasis. One specimen was confirmed as cutaneous tuberculosis.
The histopathological features of eight specimens were compatible with M. ulcerans-associated disease. Six of those could be classified as active disease, one could be classified as inac-tive (healing) stage, and one case could not be definitely confirmed.

DISCUSSION
At present, control strategies for BU in countries of endemicity are limited to early case detection through improved active surveillance and surgical treatment. A diagnostic gold standard for the laboratory confirmation of BU has not yet been established, and sensitive diagnostic techniques like PCR and histopathology are often not available in areas of endemicity (6). Thus, misclassification and delayed diagnosis may occur frequently. According to a retrospective study carried out in Ashanti Region, Ghana, in 1994 to 1996, the average total treatment costs for a BU patient with advanced ulcerative disease requiring prolonged hospitalization were determined to be U.S.$780 as opposed to U.S.$20 to $30 for early cases. Thus, early case detection and subsequent surgical treatment reduce patient-related treatment costs (2).
In order to respond to the urgent need to develop reliable tools for early case detection, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the BNITM.
The implementation of conventional PCR assays in tropical countries is accompanied by various technical difficulties. The transport and storage of reagents and specimens require cold chains and equipment like generators and voltage stabilizers to prevent damage to the reagents and specimens by repeated freezing and thawing due to regularly occurring power cuts. Furthermore, conventional PCR assays require careful han-  dling of reaction components and strict measures by skilled laboratory workers to avoid contamination of reagents. The use of lyophilized, room-temperature-stable PCR reagents and transport buffer for specimens prevents temperature-dependent transport-and storage-related problems. In addition, lyophilized primers and reaction mixtures are easy to handle and less time-consuming for laboratory staff. The chemicals are not sensitive to climatic conditions, like heat and humidity, and thus, the qualities of reagents and reactions are always equal. The risk of contamination is minimized, because instead of at least eight different liquid components, as in conventional PCR assays, only water and template are added to the lyophilized reagents.
The dry-reagent-based PCR is slightly more expensive than the conventional diagnostic PCR (in the range of €2 to €3, depending on the manufacturer); however, the numerous advantages of the assay clearly outweigh the slight financial imbalance. In general, studies of the cost-effectiveness of implementing highly sensitive diagnostic tools like PCR in areas of high endemicity in terms of reducing total treatment costs are required.
With an analytical sensitivity and specificity equal to those of conventional PCR and accordance rates for diagnostic swabs (n ϭ 19) of 95% (Table 2) for both dry-reagent-based and standard reference methods carried out simultaneously at the BNITM, we considered the dry-reagent-based PCR a reliable diagnostic tool. As the discordance rate of 25% (Table 2) for tissue specimens (n ϭ 16) was attributable to technical aspects of specimen collection, i.e., the specimens subjected to the two methods were not obtained from the same location, there was no evidence for a lower sensitivity of the method for tissue samples.
After implementation of the assay at the KCCR, 52 diagnostic specimens from 30 clinically diagnosed patients were simultaneously tested at the KCCR (dry-reagent-based assay) and BNITM (standard reference method). Accordance rates of Ͼ95% (Table 3) for both swabs and tissue specimens suggest that the dry-reagent-based PCR assay is highly reliable and well adapted to application under tropical conditions. The diagnostic specimens tested for technical validation at the BNITM and for validation under tropical conditions simultaneously at the KCCR and the BNITM show relatively low diagnostic sensitivity (31.6 and 27.3%, respectively, for swabs; 18.8 and 36.7%, respectively, for tissue samples [Tables 2 and 3]). As the aim of this study was to establish and validate the dry-reagent-based diagnostic PCR by comparing it with the standard IS2404 reference method, all patients with clinically diagnosed M. ulcerans-associated disease who presented at Dunkwa Government Hospital during the study period were included in the pilot study. Therefore, specific selection criteria, like the type and size of lesion or duration of the disease, were not applied. Preliminary data from an ongoing study at the KCCR aimed at the PCR confirmation of clinically diagnosed early cases suggest high diagnostic sensitivity (Ͼ90%) for early lesions (i.e., where the duration of disease was Ͻ6 months). Of the patients included in the validation pilot study, however, 47.9% had lesions older than 6 months (13 of 48; 27.1%) or data were not available (10 of 48; 20.8%). In those cases, the age of the lesions was likely to affect the diagnostic sensitivity, as older lesions might be in an inactive (healing) stage, and thus, bacilli could not be detected.
In order to explain the relatively high number of PCRnegative tissue specimens (27 of 46; 58.7%), they were subjected to histopathology. Histopathological analysis revealed that 40.7% (11 of 27) of the specimens were not appropriate for diagnostic purposes, as the subcutaneous tissue was either missing or incomplete. In 22.2% (6 of 27) of the specimens, either noninfectious (nonspecific dermatitis and Kaposi's sarcoma) or other infectious (tuberculosis, onchocerciasis, and other parasitic infections) conditions were diagnosed. Of the PCR-negative tissue specimens, 29.6% (8 of 27) showed histopathological features compatible with Buruli ulcer but without detectable acid-fast bacilli. One specimen was in the inactive (healing) stage. Older and inactive lesions tend not to contain detectable amounts of bacilli and thus may not produce positive PCR results. Therefore, only early lesions might be considered suitable for molecular diagnosis of M. ulceransassociated disease. Furthermore, these findings clearly support the need to establish the differential diagnosis before classifying and treating a patient for Buruli ulcer. Thus, diagnostic tools to exclude other conditions that might be mistaken for M. ulcerans-associated disease are required as well. These findings also stress the importance of accurate specimen collection, as only specimens that are taken from necrotic-tissue areas and that include the subcutaneous tissue allow a reliable diagnosis of M. ulcerans-associated disease. Despite the prior definition of criteria for specimen collection, in this pilot study, the technical procedures were not closely monitored, as a close link between the hospital and the laboratory was not yet fully established. The diagnostic sensitivity clearly depends on the quality of specimens, and only a close collaboration between the surgeon and the laboratory guarantees optimized laboratory results.
If these conditions are fulfilled, the dry-reagent-based PCR presented in this study can serve as a reliable and rapid tool for the laboratory confirmation of M. ulcerans-associated disease under tropical conditions. Implementation of the method in major treatment centers in areas of endemicity is under way and will provide diagnostics at the district level.