Sensitivity of PCR Targeting Mycobacterium ulcerans by Use of Fine-Needle Aspirates for Diagnosis of Buruli Ulcer

Abstract

In a previous study, we reported that the sensitivity of PCR targeting the IS2404 insertion sequence of Mycobacterium ulcerans was 98% when it was applied to 4-mm punch biopsy samples of Buruli lesions. Fine-needle aspiration (FNA) is a less traumatic sampling technique for nonulcerated lesions, and we have studied the sensitivity of PCR using FNA samples. Fine-needle aspirates were taken with a 21-gauge needle from 43 patients diagnosed clinically with M. ulcerans disease. Four-millimeter punch biopsies were obtained for microscopy, culture, and PCR targeting the IS2404 insertion sequence. The sensitivity of PCR using samples obtained by FNA was 86% (95% confidence interval [95% CI], 72 to 94%) compared with that for PCR using punch biopsy samples. In this study, the sensitivities of culture and microscopy for punch biopsy samples were 44% (95% CI, 29 to 60%) and 26% (95% CI, 14 to 41%), respectively. This demonstrates that PCR on an FNA sample is a viable minimally invasive technique to diagnose M. ulcerans lesions.

Buruli ulcer is a skin disease caused by infection with Mycobacterium ulcerans and was classified recently as one of 13 neglected tropical diseases (8). It has been reported in over 30 countries worldwide, predominantly in humid tropical areas in West Africa. The burden of disease falls mainly on children aged 15 years or younger living in rural areas who have little or no access to health services (2). The disease manifests as a painless nodule, a firm plaque, or an edematous lesion which soon ulcerates. Clinical diagnosis of early nonulcerative lesions is about 70% accurate in experienced hands. Laboratory confirmation requires swabs from undermined edges of the ulcer or punch biopsy for nonulcerative lesions. Currently used methods are histopathology, detection of acid-fast bacilli (AFB) by smear, PCR, or culture. In the past, surgery was widely regarded as the only treatment, but recurrence rates between 6 and 17% have been reported (1, 5, 14). In 2004, the World Health Organization recommended a combination of rifampin and an aminoglycoside (streptomycin or amikacin) for treatment, and early results of treating ambulant patients with this combination for 8 weeks have been promising (4). However, it is desirable to establish the diagnosis early if this treatment is planned.

We developed a modified PCR method targeting the insertion sequence IS2404 of M. ulcerans, which had a sensitivity of 98 to 100% compared with the combination of microscopy (42%), culture (49%), and histology (82%) when applied to 4-mm punch biopsy samples and gave results within 24 h. This was the most useful technique for reaching a treatment decision provided that it was done in a laboratory with high standards to avoid false-positive results (10). Fine-needle aspiration (FNA) is a technique that yields a small sample of cells from tissues suspected to be abnormal. It has become the predominant initial diagnostic technique for lumps at sites including the head, neck, and breast. FNA has achieved this level of application because it is relatively inexpensive, rapid to perform, well accepted by patients, and associated with low morbidity and has a relatively high diagnostic accuracy (7). In the present study, we have investigated for the first time the use of FNA to obtain tissue for detection of M. ulcerans by PCR targeting IS2404.

MATERIALS AND METHODS

Clinical specimen collection.

Patients with a clinical diagnosis of M. ulcerans disease were recruited from Ahafo Ano North District in the Ashanti region of Ghana from September 2005 to June 2007 after informed consent and were screened at Tepa Government Hospital. Ethical approval was obtained from the ethical review committee at the School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Lesions were grouped as nonulcerative (nodules, plaques, or edematous lesions) or ulcers, as described in WHO documents (2, 3, 16). FNA was done by inserting a 21-gauge needle into the center of nonulcerated lesions or into viable inflamed skin immediately adjacent to ulcers and moving it back and forth within the subcutaneous tissue. The needle tip was flushed into 100 μl of TE buffer (1 mM EDTA in 10 mM Tris-HCl, pH 8.0), using a 5-ml syringe. In order to establish the diagnosis of M. ulcerans infection, a 4-mm punch biopsy (Stiefel Laboratories, United Kingdom) was obtained from the same site as the FNA for microscopy, culture, and PCR targeting the IS2404 insertion sequence (10). Tissues were placed in sterile 2-ml skirted tubes with an O ring (Sarstedt, United Kingdom). One biopsy was used for microscopy and culture, and one was used for PCR. Each patient sample for microscopy and culture was processed within 24 h; samples for PCR were stored at −20°C. Microscopy, culture, and PCR were performed at Komfo Anokye Teaching Hospital, Kumasi, Ghana. Samples were coded, and all laboratory tests were performed in a blinded fashion.

Microscopy and culture.

As previously described (10), punch biopsies were cut into pieces with a scalpel blade and homogenized with a sterile mortar and pestle in 2 ml of normal saline. Ziehl-Neelsen (ZN)-stained smears of supernatants were examined under a microscope. Supernatant was removed, and tissue homogenate was decontaminated using the sodium hydroxide (modified Petroff) method (15). Briefly, an equal volume of 1 M NaOH was added to 1 ml of specimen and left to stand for 10 min with occasional shaking and then neutralized with 1 ml of 1 M HCl. The mixture was centrifuged at 3,000 × g for 20 min, and the supernatant was removed. A 0.1-ml sample of sediment was inoculated onto each of two Löwenstein-Jensen (LJ) slopes. LJ slopes were incubated at 31°C, and cultures were examined weekly until visible growth occurred. Slopes were discarded after 6 months of incubation. Positive cultures for M. ulcerans were subcultured on LJ slopes. ZN staining was done to confirm the presence of AFB in positive cultures, and specificity was confirmed by PCR targeting the insertion sequence IS2404.

PCR for M. ulcerans.

Twenty to 40 mg of punch biopsy tissue was minced with a sterile scalpel blade, and a solution containing 0.1 mg proteinase K in digestion buffer was added to the tissue or FNA samples to give final concentrations of 0.5% Triton X-100, 20 mM Tris-HCl, pH 8.3, and 1 mM EDTA. The suspension was incubated at 50 to 55°C for 18 h. DNA extraction was done by the guanidinium thiocyanate-diatom method as described previously (10). Ten microliters of extracted DNA was used in the PCR mix. The PCR was based on the IS2404 insertion element; PU4F (5′-GCGCAGATCAACTTCGCGGT-3′) and PU7Rbio (5′-GCCCGATTGGTGCTCGGTCA-3′) were used as primers. The standard PCR mixture was described previously (10). Electrophoresis of PCR products was performed on a 2% agarose gel stained with ethidium bromide and visualized with UV light.

Each patient's sample was run in triplicate, with two undiluted replicates and one spiked with 5 fg of M. ulcerans DNA to assess inhibition. Cross-contamination was checked by including TE-only negative control vials with each PCR run. The PCR was considered to be positive if all three PCR samples for each patient were positive, positive controls were positive, and negative controls with TE were negative. For PCR-negative specimens, positive controls were positive and negative controls with TE were negative. The gold standard for comparison was the PCR result obtained with a 4-mm punch biopsy sample.

RESULTS

FNA samples were obtained from 45 patients with a clinical diagnosis of M. ulcerans disease. Forty-three of 45 patients for whom the diagnosis of M. ulcerans disease was confirmed with punch biopsies by culture or PCR were included. Of these, 28 had preulcerative lesions and 15 had ulcerative lesions. The median age of patients was 13 (range, 5 to 42) years, and there were 17 (40%) males and 26 (60%) females. The median duration of disease before presentation was 1.0 (range, 0.3 to 8) month. There were 14 nodules (32%), 5 plaques (12%), 9 edematous lesions (21%), and 15 ulcers (35%).

PCR using FNA samples detected 37 of the 43 that were confirmed as having M. ulcerans disease by punch biopsy (Table 1). Therefore, the sensitivity of PCR on FNA samples was 86% (95% confidence interval [95% CI], 58 to 95%) for a single sample, and it rose to 90% (95% CI, 69 to 98%) when a second sample was analyzed. The sensitivities of culture and microscopy obtained with punch biopsy samples were 44% (95% CI, 29 to 60%) and 26% (95% CI, 14 to 41%), respectively, compared to PCR on punch biopsy samples.

TABLE 1.

Comparison of PCRs targeting IS2404, using samples obtained by punch biopsy and FNA, of culture, and of microscopy for AFB for 43 patients with suspected M. ulcerans disease

Test and result	No. of samples with punch biopsy PCR result		Sensitivity (% [95% CI])
	Positive	Negative
FNA-PCR
Positive	37	0	86 (72-94)
Negative	6	0
Culture
Positive	19	0	44 (29-60)
Negative	24	0
Microscopy
Positive	11	0	26 (14-41)
Negative	32	0

FNA samples from all patients with nonulcerated plaque (5) and edematous (9) forms of M. ulcerans disease gave positive PCR results (Table 2). Ten (70%) of 14 FNA samples from those with nodules were positive by PCR, but punch biopsy gave a positive PCR result for all 14. For nonulcerated lesions overall, the sensitivity of processing a first FNA sample was 85.7%, and if a second sample was tested, the sensitivity rose to 89.2% (Table 3). Two (one lower limb and one trunk) of 17 FNA samples obtained from ulcerated lesions gave negative PCR results.

TABLE 2.

Comparison of PCRs targeting IS2404, using samples obtained by punch biopsy and FNA from different forms of M. ulcerans disease lesions

Sample type	No. (%) of PCR-positive samples		Total no. of positive samples
	FNA	Punch biopsy
Nodule (n = 14)	10 (71.4)	14 (100)	12
Plaque (n = 5)	5 (100)	5 (100)	5
Edema (n = 9)	9 (100)	9 (100)	9
Ulcer (n = 15)	13 (86.7)	15 (100)	17

TABLE 3.

Increase in sensitivity after processing a second FNA sample

Sample type	n	No. (%) of PCR-positive samples
		First FNA sample	Second FNA sample	Punch biopsy
Nonulcerative lesions	28	24 (85.7)	25 (89.2)	28 (100)
Ulcers	15	13 (86.7)	14 (93.3)	15 (100)

DISCUSSION

Nucleic acid amplification techniques, including PCR, have had a considerable impact on disease diagnosis on account of their speed, specificity, and enhanced sensitivity. The application of PCR to the diagnosis of Buruli ulcer has the potential to resolve one of the foremost challenges regarding making a treatment decision. Surgery was the mainstay of management of M. ulcerans disease until the WHO issued guidelines in 2004 for the use of streptomycin (15 mg/kg of body weight) and rifampin (10 mg/kg) for 8 weeks. Since then, there has been a compelling need for a cheap and minimally invasive way of obtaining clinical samples, especially for preulcerative lesions of Buruli ulcer, in order to reach a treatment decision. This study has shown in a field setting that FNA can be used to diagnose M. ulcerans disease. Overall, there was a good sensitivity of PCR on FNA samples, and although there was a slight loss in sensitivity compared to that with punch biopsy samples, the FNA procedure was easy, equipment (21-gauge needles) was cheap and easy to obtain, and there were no recorded complications. In contrast, sterile disposable punch biopsy equipment is not easy to obtain in rural health facilities and is relatively expensive. For research studies, it is essential to have the highest possible sensitivity, and punch biopsy would be the first choice, but for routine diagnosis where PCR is available FNA gives clinically useful results.

Fine-needle aspirates from preulcerative nodules, plaques, and edema and from ulcerative forms, obtained using 21-gauge needles, gave a sensitivity of 86% compared with punch biopsy samples, while culture was 44% sensitive and microscopy was 26% sensitive. Diagnosis was confirmed for all 5 nonulcerative plaques, all 9 edematous lesions, and 10 of 14 nodular forms. The loss in sensitivity compared to punch biopsy testing may be due to the small volume of FNA samples; this is supported by the finding that processing a second FNA sample increased the sensitivity to 90%.

The results for AFB smear and culture were consistent with those obtained in the past and suggest that these procedures are too insensitive to be useful for making a treatment decision. In the presence of a strong clinical suspicion when the patient lived in an area of endemicity, ZN staining was only 40 to 43% sensitive (10, 11) and culture of M. ulcerans had a similar sensitivity (35 to 50% [11] and going up to 60% [13]), particularly if culture was performed locally (10). Culture will remain important for establishing the antibiotic sensitivity of M. ulcerans. Histology is the most sensitive of the conventional diagnostic methods when it is available, with 83% sensitivity in our studies in Ghana (10), but a histopathology service is often limited in areas of endemicity. Perhaps the use of more sensitive microbiological techniques, such as a liquid culture system, would result in higher culture positivity. However, because of equipment constraints, this could not be investigated in our study. Currently, the WHO has designated central laboratories where samples for PCR may be transported. For example, in Ghana, there are three such laboratories. One of the six cases not confirmed by FNA was confirmed as Buruli ulcer by culture, and another was confirmed by microscopy. This could be the result of aspiration from an area not containing bacilli and might justify performing multiple aspirations from different parts of the lesion, as reported elsewhere (6).

Results of microscopy for AFB and of PCR using either punch biopsy or FNA samples were available in 24 to 48 h, whereas culture results were available after 6 weeks. M. ulcerans grows slowly in the laboratory on LJ slopes, usually taking 6 to 8 weeks for a positive culture, but it can take up to 6 months (9, 12), so culture could not be used to make a treatment decision.

In conclusion, this is the first study to demonstrate that PCR targeting the insertion sequence IS2404 of M. ulcerans, using FNA samples, can be a useful alternative to punch biopsy in making the diagnosis of M. ulcerans disease when punch biopsy equipment is not available. From cost and sustainability perspectives, a punch biopsy needle costs about $30, while an ordinary 21-gauge needle and a 5-ml syringe cost 20 cents. In addition, 21-gauge needles are routinely available in all health facilities, thus making it easier to use this technique. However, training of key health workers on how to perform FNA will be required to improve the diagnostic accuracy of this technique. Further large-scale studies are needed to validate these preliminary findings.