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Journal of Clinical Microbiology, July 2000, p. 2743-2745, Vol. 38, No. 7
Wadsworth Center, New York State Department
of Health,1 and Department of Medicine,
Albany Medical College,4 Albany, New York;
Department of Respiratory Medicine, Semmelweis University,
Budapest, Hungary2; and Gen-Probe
Incorporated, San Diego, California3
Received 22 December 1999/Returned for modification 13 February
2000/Accepted 29 March 2000
Mycobacterium celatum type 1 was found to cross-react
in the AccuProbe Mycobacterium tuberculosis complex assay.
Subsequently, we found a statistically significant increase in the
relative light units with lower temperatures, suggesting that it is
necessary to perform this AccuProbe assay at between 60 and 61°C. We
also recommend the inclusion of M. celatum type 1 as a
negative control.
The introduction and routine
application of nucleic acid probes, such as the AccuProbe
Mycobacterium tuberculosis complex assay (TB AccuProbe;
Gen-Probe Incorporated, San Diego, Calif.), has considerably shortened
the time required for identification of the M. tuberculosis
complex while providing high sensitivity and specificity
(15). However, in a study by Butler et al. (2) cross-reactivity was observed between M. celatum type 1, but
not type 2, in the TB AccuProbe. DNA sequencing showed that M. celatum type 1 differs by a single nucleotide from the probe used
in the assay (6), while type 2 differs by four nucleotides.
Recently, Bull and coworkers identified a novel subtype of the pathogen M. celatum, type 3, that also showed cross-reactivity in the
TB AccuProbe (1); however, the DNA sequence for the probe
region was not reported.
M. celatum is a newly recognized, slow-growing, nonpigmented
species whose biochemical characteristics and colony morphology are
similar to those of M. avium complex, M. malmoense, M. shimoidei, and M. xenopi
(1, 3, 11). The three types of M. celatum can be
distinguished from one another and from other mycobacteria by DNA
sequencing or restriction fragment length polymorphism analysis of
selected genes and by high-performance liquid chromatography (3, 10, 11, 17; J. Baldus-Patel, D. G. Leonard,
X. Pan, J. M. Musser, R. E. Berman, and I. Nachamkin, Abstr.
99th Gen. Meet. Am. Soc. Microbiol., abstr. U-18, p. 637, 1999). In
recent years, a considerable amount of clinical evidence has indicated that M. celatum can lead to fatal disease in both
immunocompetent and immunocompromised patients (4, 8, 14,
18). Therefore, the impact of misidentification in the TB
AccuProbe can be significant (5).
Thus, M. celatum type 1 (ATCC 51131) was submitted for
identification to 137 laboratories participating in New York State's proficiency testing program. Ninety of the 137 laboratories used the TB
AccuProbe, and one-third of those reported false-positive (>30,000)
relative light units (RLU). These results indicate that in spite of
Gen-Probe's previous modification of the selection time from 5 to 10 min (16; 1993 original and 1994 revised package inserts for the AccuProbe cultural identification test, Gen-Probe Incorporated), a considerable number of laboratories still obtained false-positive TB AccuProbe results with M. celatum.
To identify possible reasons for the false-positive results, we
requested procedural details from the 90 laboratories. Unfortunately, this information did not reveal any significant differences in methodology between those with positive and those with negative RLU
values. However, in a previous report on cross-reactivity of M. terrae with the TB AccuProbe (7), it was found that
changes in temperature influenced the specificity of the test results. Thus, we sought to determine the effects of both selection step time
and temperature by testing M. celatum type 1 (ATCC 51131) in
the TB AccuProbe in one of our laboratories. First, the M. celatum isolate was tested to ensure that M. tuberculosis was not also present in the suspension. This test was
negative. We then performed experiments using M. celatum
previously grown on either Löwenstein-Jensen medium or in
Middlebrook 7H9 broth. When broth was used, 1.5 ml of broth was
centrifuged, the supernatant was removed, and the pellet was
resuspended in 500 µl of broth. One-hundred-microliter aliquots of
loopfuls of growth from solid media or resuspended broth were tested
according to the TB AccuProbe protocol (AccuProbe culture
identification test revised package insert). It was noted that the
suspensions from the solid media contained considerably more bacteria
than those prepared from broth-grown cultures. Experiments were carried
out in triplicate with selection times of 9, 10, 11, and 12 min at
60°C (standard temperature) and with temperatures of 58, 59, 60, and
61°C at 10 min (standard selection time). Suspensions of M. tuberculosis and M. avium previously grown on solid
media were included as positive and negative controls. The RLU values
are expressed as means + standard errors of the means (SEM).
Comparisons were performed by the Mann-Whitney U test.
The results on the effect of the selection time are shown in Fig.
1A. There were no statistically
significant differences between the mean RLU values at the different
selection times for suspensions from the same medium. However, the RLU
values from the solid medium suspension were significantly higher than
those from the broth-grown suspension (P < 0.05). As
stated before, the solid medium suspension contained a greater cell
density than the broth-grown suspension. Thus, these results suggest
that the M. celatum cell density could influence the RLU
value. However, the highest RLU values obtained were still under the
cutoff of 30,000 (Fig. 1A).
0095-1137/00/$04.00+0
False-Positive Results for Mycobacterium
celatum with the AccuProbe Mycobacterium tuberculosis
Complex Assay
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FIG. 1.
(A) Effect of selection time on RLU value in the
AccuProbe M. tuberculosis complex assay with M. celatum. There were no statistically significant differences
between the mean RLU values with solid or liquid medium for the
different selection times. The RLU values from the solid medium
suspension were significantly higher than those from the broth-grown
suspension at all selection times (P < 0.05). Data are
expressed as means plus SEM. The RLU cutoff is 30,000. (B) Effect of
temperature on RLU value in the AccuProbe M. tuberculosis
complex assay with M. celatum. The Mann-Whitney U test
revealed significant differences between the mean RLU values with solid
medium for 58°C versus 60 and 61°C, between 59°C versus 60 and
61°C, and 60°C versus 61°C (P < 0.05). The RLU
values from the solid medium suspension were significantly higher than
those from the broth-grown suspension at all selection temperatures
(P < 0.05). Data are expressed as means plus SEM. The
RLU cutoff is indicated by a horizontal line.
As to the effect of the temperature, the RLU value obtained with a suspension from solid media was found to decrease as the temperature was increased, as shown in Fig. 1B. Statistical analysis revealed a significant difference between the RLU values from the solid medium suspension at 58°C versus 60 and 61°C, at 59°C versus 60 and 61°C, and at 60 versus 61° (P < 0.05). There were no significant differences between the RLU values from the broth-grown suspension at the different temperatures. But once again, the RLU values from the solid medium suspension were significantly higher than those from the broth-grown suspension at all temperatures (P < 0.05).
In summary, our experiments revealed that the number of organisms present could strongly influence the RLU value, as there was a statistically significant difference between the RLU values of suspensions derived from broth versus solid medium. These results suggest that the presence of a larger biomass of M. celatum from the solid medium resulted in increased signal. Furthermore, in agreement with the previous findings of Ford et al. (7) for cross-reaction of M. terrae in the TB AccuProbe, we also found that changes in temperature can significantly affect the RLU value when a heavy inoculum of M. celatum is present. We found a statistically significant increase in RLU with lower temperatures; however, varying the selection time did not appear to have an effect on the signal with M. celatum. These results suggest that in order to eliminate cross-reactivity with M. celatum, it is imperative to perform the test with a selection temperature between 60 and 61°C rather than the presently recommended 60 ± 1°C.
Of the 90 participating laboratories that used the TB AccuProbe, only one reported a selection temperature (59.5°C) lower than the 60°C recommended by Gen-Probe. However, only 9 (11%) participants checked the temperature of their heating instruments before each testing, while the majority (51%) checked the temperatures only annually, and some did not check at all (3%). The controlled experimental results of this study strongly suggest that frequent temperature checks on heating instruments are warranted. The guidelines of the National Committee for Clinical Laboratory Standards for molecular diagnostic methods for infectious diseases state the following (13): "Quality control charts that indicate acceptable ranges should be posted on all water baths, incubators, and heating blocks. Temperatures should be checked and recorded on these charts daily."
In order to monitor the performance of the test, we recommend that M. celatum type 1 (ATCC 51131) be used as a negative control either instead of or along with the presently suggested M. avium (ATCC 25291). Furthermore, when identifying members of the M. tuberculosis complex, cellular and colony morphology should be taken into consideration (9, 12). Finally, since M. celatum may be difficult to identify with conventional biochemical tests, we recommend the use of molecular diagnostic techniques and/or high-performance liquid chromatography for the most accurate identification of this organism (3, 10, 11, 13a, 17).
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ACKNOWLEDGMENTS |
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We thank Olivia Montaño for excellent technical assistance.
Á. Somoskövi was supported in part by grant no. 1D43TW00915 from the Fogarty International Center, National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509. Phone: (518) 474-2196. Fax: (518) 474-6964. E-mail: salfinger{at}wadsworth.org.
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