Abstract
This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.
TEXT
Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, involves the skin, subcutaneous fatty tissue, and bones and predominantly affects children <15 years of age. If left untreated, contractures may cause severe functional limitation. Standardized antimycobacterial treatment consists of rifampin (RMP) and streptomycin administered for 8 weeks. An oral regimen combining RMP and clarithromycin is currently under clinical evaluation (1–3). Notwithstanding the efficiency of chemotherapy, treatment failures and various types of secondary lesions have been reported, suggesting the need for customized clinical management strategies (4–6).
Tuberculosis and leprosy studies have shown that RMP treatment is prone to the development of drug resistance due to missense mutations within the RMP resistance-determining region (RRDR) of the mycobacterial rpoB gene (7, 8). RRDR mutations in M. ulcerans have been described in a mouse model (9); data on drug resistance among clinical isolates, however, are scarce. A pilot study on molecular drug resistance testing conducted by our group from 2004 through 2007 in Ghana revealed a low level (0.9%) of RMP resistance. However, the overall test efficiency of the assay applied in the pilot study (referred to here as assay A) was low (35%) (10). Therefore, the aim of this study was to develop an improved sequencing assay (referred to here as assay B).
The study was approved by the National Togolese Ethics Committee (14/2010/CRBS) and the Ghanaian Kwame Nkrumah University of Science and Technology Ethics Committee (CHRPE/91/10).
The primers MuB-F and MuB-R were designed to amplify a 606-bp region encompassing the RRDR by alignment of (myco)bacterial rpoB genes as retrieved from GenBank (PubMed, NCBI) using DNASIS Max (MiraiBio, San Francisco, CA) (see Table S1 in the supplemental material). MuB-F specifically binds a polymorphic region of the mycobacterial rpoB gene (11); the sequencing primer Bseek-F binds downstream of primer MuB-F (Table 1). Amplification was conducted using the Thermococcus kodakaraensis-derived KOD Hot Start polymerase (Merck, Darmstadt, Germany) followed by performing agarose gel electrophoresis, purification of PCR products, cycle sequencing, and sequence analysis, as previously described (10) (Table 1; see also PCR Protocol S2 and S3 in the supplemental material).
TABLE 1.
Primer sequences and nucleotide positions of rpoB PCR and sequencing primersa
| Assay | Primerb | Sequence (5′ to 3′)c | Nucleotide positiond | Amplicon size, bpe |
|---|---|---|---|---|
| Af | MF | CGA CCA CTT CGG CAA CCG | 785041–785058 | 351 |
| MR | TCG ATC GGG CAC ATC CGG | 785374–785391 | ||
| Bg | MuB-F | CAT CAC CAG CTC GAC GCT | 784912–784929 | 606 |
| MuB-R | TGG ATC TCG TCG GAA ACG | 785500–785517 | ||
| Bseek-F | AAT ACC TGG TCC GCT TGC | 784959–784976 |
Shown are the primer sequences, nucleotide positions within the M. ulcerans genome, and corresponding amplicon sizes. The rpoB gene encodes the beta subunit of (myco)bacterial RNA polymerases. Significant sequence concordances of primers with human or other (myco)bacterial DNA were excluded by Primer BLAST (PubMed, NCBI).
F, forward primer; R, reverse primer. Primers MF and MR were used in assay A for the amplification of a 351-bp fragment of the M. ulcerans rpoB gene (including the RRDR) encompassing the region sequenced by primer MF. Primers MuB-F and MuB-R were used in assay B for the amplification of a 606-bp fragment of the M. ulcerans rpoB gene (including the RRDR) encompassing the region sequenced by primer Bseek-F.
Primer sequence from the 5′ to the 3′ end.
Nucleotide positions are provided for the respective amplicon in M. ulcerans strain Agy99 (GenBank accession no. CP000325 [PubMed, NCBI]).
Amplicon sizes for rpoB PCR of assay A or B, respectively.
For assay A, final concentrations of PCR reagents per 20-μl reaction: 0.5 μM each primer (TIB-Molbiol, Berlin, Germany); 2.5 mM MgCl2, 0.8 mM deoxynucleoside triphosphates (dNTPs), 0.05 U/μl AmpliTaq Gold DNA polymerase, 1× PCR buffer II (Applied Biosystems, Foster City, CA); template DNA, 2 μl; run protocol, 95°C for 5 min, 37 cycles at 95°C for 15 s, 56°C for 15 s, and 72°C for 30 s, and final extension at 72°C for 5 min.
For assay B, final concentrations of PCR reagents per 20-μl reaction: 0.3 μM each primer (TIB-Molbiol); 1.5 mM MgSO4, 0.8 mM dNTPs, 0.02 U/μl KOD Hot Start polymerase, 1× PCR buffer for KOD (Merck, Darmstadt, Germany); template DNA, 2 μl; run protocol, 95°C for 2 min and 39 cycles at 95°C for 20 s, 63°C for 10 s, and 70°C for 15 s.
PCR standards were generated by exact quantification of whole-genome DNA from two M. ulcerans cultures from Ghana by IS2404 quantitative real-time PCR (12, 13). The limits of detection for the two assays were determined by testing 10-fold serial dilutions of PCR standards. The analytical sensitivity of assay B was 10 times higher than that of assay A (100 to 200 and 1,000 to 2,000 copies of the rpoB gene, respectively).
The specificity of assay B was assessed with DNA extracts of 18 closely related human-pathogenic mycobacterial species and five bacterial species frequently colonizing human skin (12, 14–16) (Table 2). Besides M. ulcerans, only M. marinum was amplified. As rpoB wild-type sequences of these two species can be differentiated in two nucleotides by sequencing, assay B was considered M. ulcerans specific.
TABLE 2.
Specificity of assay Ba
| Bacterial species | Sourceb | Isolate origin | Resultc |
|---|---|---|---|
| Mycobacterium abscessus | NRZ | Humand | − |
| Mycobacterium africanum | NRZ | Humand | − |
| Mycobacterium avium | NRZ | Humand | − |
| Mycobacterium bovis | NRZ | Cattled | − |
| Mycobacterium chelonae | NRZ | Humand | − |
| Mycobacterium fortuitum | NRZ | Humane | − |
| Mycobacterium gordonae | NRZ | Humane | − |
| Mycobacterium gordonae | DITM | Humane | − |
| Mycobacterium kansasii | NRZ | Humand | − |
| Mycobacterium leprae | DITM | Humand | − |
| Mycobacterium malmoense | NRZ | Humane | − |
| Mycobacterium marinum | NRZ | Humand | + |
| Mycobacterium microti | NRZ | Moused | − |
| Mycobacterium scrofulaceum | NRZ | Humand | − |
| Mycobacterium smegmatis | NRZ | Humand | − |
| Mycobacterium szulgai | NRZ | Humand | − |
| Mycobacterium tuberculosis | NRZ | Humand | − |
| Mycobacterium ulcerans | DITM | Humand | + |
| Mycobacterium xenopi | NRZ | Humane | − |
| Escherichia coli | MVP | Humane | − |
| Propionibacterium acnes | MVP | Humand | − |
| Staphylococcus aureus | MVP | Humane | − |
| Staphylococcus epidermidis | MVP | Humane | − |
| Streptococcus pyogenes | MVP | Humand | − |
Shown are (myco)bacterial species commonly contaminating human skin and the results of the specificity testing of sequencing assay B.
DNA extracts that were not available at the Department of Infectious Diseases and Tropical Medicine, University Hospital, Ludwig-Maximilians-University, Munich, Germany (DITM) were provided by the National Reference Center for Mycobacteria, Borstel, Germany (NRZ), and the Max von Pettenkofer-Institut, Ludwig-Maximilians-University, Munich, Germany (MVP).
Results of DNA extracts subjected to assay B: +, positive PCR result; −, negative PCR result.
The primary patient isolates were considered pathogenic bacteria.
The primary patient isolates were considered commensals/contaminants of clinical samples.
To determine the performances of the two assays on clinical specimens, 133 whole-genome extracts from IS2404-positive samples collected before the onset of treatment (swab samples, n = 63; fine-needle aspirates [FNA], n = 40; 3-mm punch biopsy specimens, n = 30) from 91 BUD patients from Togo (17, 18) were assessed. P values of <0.05 were considered significant.
Due to initial rpoB PCR inhibition, significantly more DNA extracts had to be diluted when subjected to assay B (54.1%, 72/133) than when subjected to assay A (7.5%, 10/133) (P < 0.01). With a P value of 0.39, the overall rpoB PCR positivity rate (i.e., the proportion of positive rpoB PCR results among all samples tested) was not significantly different in assay A (56.4%, 75/133) and assay B (51.1%, 68/133). However, the rpoB PCR positivity rate of swab samples was significantly higher in assay A (50.8%, 32/63) than in assay B (30.2%, 19/63) (P = 0.02). Among all of the samples with a positive rpoB PCR result in both assays, the proportion of samples yielding a definite sequencing result (overall rpoB sequencing positivity rate) was significantly higher for assay B (98.0%, 48/49) than for assay A (85.7%, 42/49) (P = 0.03). Among all the samples tested, the proportion of samples yielding a definite rpoB sequencing result (overall test efficiency) was 39.8% (53/133) for assay A and 48.9% (65/133) for assay B (P = 0.14). Following stratification of the overall test efficiencies into different sample types, the test efficiency for FNA samples was significantly higher in assay B (70.0%, 28/40) than in assay A (35.0%, 14/40) (P < 0.01) (Table 3).
TABLE 3.
Comparison of the results from clinical samples of Togolese BUD patients subjected to assays Aa and Bb,c
| Test, parameter, and sample collection type | % (no. of samples with a positive result/no. of samples tested) (95% confidence interval) |
|||
|---|---|---|---|---|
| PCR A | PCR B | PCR A and PCR B (concordance rate)d | McNemar's Pe | |
| rpoB PCR | ||||
| Positivity ratef | ||||
| All | 56.4 (75/133) (48.0–64.8) | 51.1 (68/133) (42.6–59.6) | 66.2 (88/133) (58.1–74.2) | 0.39 |
| Swabs | 50.8 (32/63) (38.4–63.1) | 30.2 (19/63) (18.8–41.5) | 57.1 (36/63) (44.9–69.4) | 0.02g |
| Punch biopsy | 73.3 (22/30) (57.5–89.2) | 66.7 (20/30) (49.8–83.5) | 86.7 (26/30) (74.5–98.8) | 0.58 |
| FNA | 52.5 (21/40) (37.0–68.0) | 72.5 (29/40) (58.7–86.3) | 65.0 (26/40) (50.2–79.8) | 0.06 |
| Initial inhibitionh | ||||
| All | 07.5 (10/133) (03.0–12.0) | 54.1 (72/133) (45.7–62.6) | 48.9 (65/133) (40.4–57.4) | <0.01g |
| Swabs | 09.5 (6/63) (02.3–16.8) | 65.1 (41/63) (53.3–76.9) | 41.3 (26/63) (29.1–53.4) | <0.01g |
| Punch biopsy | 06.7 (2/30) (00.0–15.6) | 30.0 (9/30) (13.6–46.4) | 76.7 (23/30) (61.5–91.8) | 0.02g |
| FNA | 05.0 (2/40) (00.0–11.8) | 55.0 (22/40) (39.6–70.4) | 40.0 (16/40) (24.8–55.2) | <0.01g |
| rpoB-Sequencing | ||||
| Positivity ratei | ||||
| All | 85.7 (42/49) (75.9–95.5) | 98.0 (48/49) (94.0–100) | 87.8 (43/49) (78.6–96.9) | 0.03g,j |
| Swabs | 75.0 (9/12) (50.5–99.5) | 100 (12/12) (NAk) | 75.0 (9/12) (50.5–99.5) | 0.07j |
| Punch biopsy | 100 (19/19) (NAk) | 100 (19/19) (NAk) | 100 (19/19) (NAk) | NAk |
| FNA | 77.8 (14/18) (58.6–97.0) | 94.4 (17/18) (83.9–100) | 77.8 (14/18) (58.6–97.0) | 0.15j |
| Efficiencyl | ||||
| All | 39.8 (53/133) (31.5–48.2) | 48.9 (65/133) (40.4–57.4) | 74.4 (99/133) (67.0–81.8) | 0.14 |
| Swabs | 28.6 (18/63) (17.4–39.7) | 27.0 (17/63) (16.0–37.9) | 73.0 (46/63) (62.1–84.0) | 0.84 |
| Punch biopsy | 70.0 (21/30) (53.6–86.4) | 66.7 (20/30) (49.8–83.5) | 90.0 (27/30) (79.3–100) | 0.78 |
| FNA | 35.0 (14/40) (20.2–49.8) | 70.0 (28/40) (55.8–84.2) | 65.0 (26/40) (50.2–79.8) | <0.01g |
Assay A as applied in the pilot study.
The newly developed assay B.
Shown are PCR and sequencing results of all clinical samples subjected to assays A and B.
Proportion of samples with concordant results in assays A and B out of all samples tested by both assays.
P value of McNemar chi-square test for matched pairs of samples with categorical test results.
Overall rpoB PCR positivity rate: the proportion of positive rpoB PCR results among all samples tested (assay A or B, respectively).
P values of <0.05 were considered significant.
Proportion of samples which led to PCR inhibition if tested undiluted out of all samples tested by PCR in assay A or B, respectively.
Overall rpoB-sequencing positivity rate: proportion of samples yielding a definite sequencing result among all samples with a positive rpoB PCR result in both assays.
Fisher's exact test (at least one cell, n < 5).
NA, not applicable.
Overall test efficiency: the proportion of samples yielding a definite sequencing result (assay A and/or B) among all samples tested.
The Mycobacterium genus-specific primers applied in assay A resulted in 30.7% (23/75) coamplification of DNA from other bacterial species (e.g., Corynebacterium species), resulting in nonanalyzable mixed rpoB sequences in these cases. In contrast, assay B did not detect any species other than M. ulcerans strain Agy99 (data not shown).
Furthermore, 12 IS2404 PCR-confirmed whole-genome extracts from the pretreatment samples of 10 BUD patients from Ghana which had yielded contaminated sequences (n = 9) or no sequencing results (n = 3) in assay A (10) were reexamined with assay B. Out of these, 11 (91.7%) rendered definite M. ulcerans rpoB wild-type sequences, and one sample remained negative in assay B.
The current level of RMP resistance among clinical M. ulcerans isolates in countries where BUD is endemic is unknown. Although there is no evidence for, and therefore no general concern about, person-to-person transmission of drug-resistant M. ulcerans strains, individual treatment outcomes of BUD patients may be compromised by drug resistance evolving under chemotherapy. Slow-healing lesions related to drug resistance may negatively influence the manifestation of disabilities; nonhealers and recurrences caused by resistant strains may benefit from timely surgical intervention in default of alternative antimycobacterial drugs. Therefore, rapid molecular assays for the detection of drug resistance constitute valuable tools for supporting individual clinical management decisions and prerequisites for new drug trials.
Although the overall rpoB PCR positivity rates and overall test efficiencies did not differ significantly in the two assays we compared, 20% more rpoB amplicons were generated from swab samples in assay A. However, due to the high specificity of sequencing assay B, out of those swab samples with a positive rpoB PCR in both assays, 100% yielded analyzable sequences in assay B, in contrast to 75% in assay A. In general, assay A resulted in 30% mixed sequences, while assay B yielded a significantly higher proportion of definite sequencing results with 98% analyzable sequences. However, the overall susceptibility of assay B to the presence of PCR inhibitors was considerable, as 50% of all clinical samples had to be diluted, compared to <10% of them in assay A. In turn, dilution leads to decreased M. ulcerans DNA yields, certainly affecting the rpoB PCR positivity rate of assay B. Furthermore, for FNA samples, the overall test efficiency was significantly higher in assay B (70%), suggesting FNAs as the most appropriate sample type for molecular drug resistance testing, which is compatible with current WHO recommendations for sample collection (19).
In conclusion, assay B constitutes a sensitive and 100% M. ulcerans-specific molecular tool for determining rifampin resistance with the highest efficiency in FNA samples. In combination with a recently described 16S rRNA-based viability assay (12), molecular drug resistance testing would also allow a reliable differentiation of individuals harboring viable drug-resistant organisms opposed to mycobacterial DNA residues detectable in secondary lesions (4). Furthermore, novel real-time PCR high-resolution melt analysis assays without allele-specific primers or probes may constitute a promising tool for screening clinical isolates in future studies (20).
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement 241500.
We thank Kerstin Helfrich and Carolin Mengele (DITM) for laboratory assistance, Florian Battke (Dr. Battke Scientia GmbH) for technical advice, Sabine Rüsch-Gerdes, Elvira Richter (Research Center Borstel), and Soeren Schubert (Max von Pettenkofer-Institut, LMU) for providing (myco)bacterial strains, and Basile Kobara (“Programme National de Lutte contre l'Ulcère de Buruli, Lèpre et Pian”) for excellent collaboration.
This article contains parts of the doctoral thesis of Moritz Jansson.
Footnotes
Published ahead of print 29 January 2014
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.03119-13.
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