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Previous Article | Next Article 
Journal of Clinical Microbiology, February 1999, p. 471-474, Vol. 37, No. 2
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Retrospective Analysis of the Beijing Family of
Mycobacterium tuberculosis in Preserved Lung
Tissues
Lishi
Qian,1
Jan D. A.
Van Embden,2
Adri G. M.
Van
Der Zanden,3
Evert F.
Weltevreden,4
Hongjin
Duanmu,5 and
James T.
Douglas1,*
Department of Microbiology, University of Hawaii at Manoa,
Honolulu, Hawaii 968221;
Laboratory of
Infectious Disease, National Institute of Public Health and
Environmental Protection, 3720 BA, Bilthoven,2
and Department of Microbiology and Infectious Diseases,
Medical Laboratories,3 and
Laboratory of
Clinical Pathology,4 Hospital Center Apeldoorn,
7300 DS Apeldoorn, The Netherlands; and
National
Tuberculosis Control Center, Beijing 101149, People's Republic of
China5
Received 16 July 1998/Returned for modification 23 July
1998/Accepted 9 November 1998
 |
ABSTRACT |
Direct repeat spoligotyping of 85 paraffin-embedded lung biopsies
was used to investigated the occurrence around Beijing of the Beijing
family of Mycobacterium tuberculosis. Samples ranged in
time from 1956 to 1990. Hybridization patterns were found with 49 (58%) samples, and 45 (92%) produced typical Beijing family patterns
extending over the 34-year period.
| |
TEXT |
Within the past few years, new
molecular fingerprinting techniques have made it possible to study
mycobacteria and the epidemiology of tuberculosis with greater
sensitivity and precision than previously possible. Strains can now be
typed according to the polymorphism of DNA fragments. Their spread
between and within communities can be monitored (5, 11), and
common source outbreaks can be identified and investigated (1-3,
7, 10).
Our previous discovery of a predominant clone of Mycobacterium
tuberculosis isolates in China, the Beijing family, which has radiated to neighbor countries (8, 11), raises the question of how long this family of strains was present in the Beijing area. In
cultures collected from 1994 to 1996 from patients living in the
Beijing area, we found more than 80% of isolates were members of the
Beijing family. Not knowing if this represented a recent spread of the
Beijing family, we looked at stored samples collected from individuals
residing in different districts around the Beijing area. Since there
was no access to old cultures from the past, we obtained
paraffin-embedded lung tissues of tuberculosis patients, which had been
stored at the Beijing Tuberculosis Research Institute (The National
Tuberculosis Control Center). The dates of these samples ranged from
1956 to 1990. PCR-based spoligotyping (6) was chosen because
of its sensitivity, specificity, and easy recognition of the
nine-spacer spoligotyping pattern of Beijing family isolates (11).
Eighty-five formalin-fixed paraffin-embedded lung biopsy specimens were
kindly provided by the Department of Pathology, Beijing Tuberculosis
Research Institute, People's Republic of China. Samples were from
patients, who lived in at least 17 different localities around Beijing
city and counties or the surrounding counties of Hebei province, had a
clinical diagnosis of pulmonary tuberculosis, and were undergoing
pneumonectomy from 1956 to 1990. Ten of the samples with positive
spologotyping patterns were from Hebei province, and 17 from Beijing
had no district or counties recorded. In addition, specimens were
selected based on the pathological confirmation of pulmonary
tuberculosis. Samples were grouped in 10-year intervals according to
the time when preparation was performed: 1956-1960 (group 1,
n = 25), 1969-1970 (group 2, n = 18), 1979-1980
(group 3, n = 23), and 1989-1990 (group 4,
n = 19). The age and sex of the patients were chosen randomly.
Each tissue sample was prepared with the following procedures. Once the
microtome had been well cleaned using 10% freshly diluted bleach, the
first section was taken to expose a "PCR-clean" surface by
sectioning a companion negative control tissue (14 µm), a healthy
lung tissue. Two 3-µm specimen tissues were then sectioned. One of
them was subjected to acid-fast staining and one to conventional
hematoxylin and eosin (H&E) staining. This procedure allowed
confirmation of the original histological and clinical diagnosis. Six
14-µm sections were then sectioned successively; four of them were
placed separately in 1.5-ml screw-cap microcentrifuge tubes, and the
remaining two were placed in one tube for the purpose of doubling the
concentration of the M. tuberculosis DNA for better detection. The blade and gloves were discarded after each sample was
handled, and a new blade was replaced for sectioning the next sample.
To avoid several steps of centrifugation and washes and multiple tube
transfers, a one-step DNA extraction method was developed and applied
based on the Chelex 100 method for extraction of DNA (9).
Briefly, one tissue section (14 µm thick) was placed into a 1.5-ml
microcentrifuge tube. A total of 300 µl of extraction solution,
including 5% Chelex 100 (Bio-Rad), 0.1% (wt/vol) lauryl sulfate
(Sigma), 1% (vol/vol) Nonidet P-40 (Sigma), and 1% (vol/vol) Tween 20 (Sigma), was added. The mixture was then mixed and heated at 100°C
for 30 min. The paraffin then appeared floating on the surface of the
solution. After centrifugation at 13,000 rpm for 10 minutes (Eppendorf
centrifuge model 5415C), the supernatant under the solidified paraffin
layer was transferred to a new tube carefully to avoid taking Chelex
particles. The supernatant then was centrifuged for 10 s at 13,000 rpm. Two volumes, 10 and 2 µl of the suspension, were used as DNA
templates for PCR.
Spoligotyping (spacer oligotyping) exploits the DNA polymorphism at a
unique chromosomal locus, the direct repeat (DR) region, of M. tuberculosis complex bacteria (5). Details of the
method were described by Kamerbeek et al. (6). As described
previously (11), a typical Beijing family strain contained
only 9 out of the 43 spacers, located near the 3' end of the DR region
of strain H37Rv. The samples with all nine spacers were counted as
having the complete Beijing pattern (Fig.
1, samples: 89-21532, 79-11571 Aa and Ab,
and 69-6532), and those with one to eight spacers within the
nine-spacer region were counted as having the incomplete pattern (Fig.
1, samples 79-11571 Ba, 59-1216, and 59-1214). Among the 49 samples
giving positive spoligotyping patterns, 45 (92%) showed the Beijing
patterns (Table 1). However, there were
four positive samples demonstrated the non-Beijing patterns (Fig. 2).
Although one sample (89-20934) exhibited the complete spoligotyping
pattern, the other three (89-22129, 69-6401, and 59-1213) showed
truncated patterns. Spoligotyping patterns of M. tuberculosis strains were managed and analyzed using Microsoft
Excel for Windows 95, version 7.0a. Statistical analysis of data was
performed using EPIINFO 6.0.
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FIG. 1.
Hybridization patterns (spoligotypes) of five
M. tuberculosis strains from preserved lung biopsy
samples (89-21532, 79-11571, 69-6532, 59-1216, and 59-1214). Numbers on
the top correspond to the 43 applied spacers (6). The
numbers: 89-, 79-, 69-, and 59- represent the year when the tissues
were preserved. For each sample, duplicated sections (A and B) were run
simultaneously for spoligotyping PCR. For each section, two volumes (10 µl [a] and 2 µl [b]) of DNA templates were used as templates
for PCR. A black dot represents the presence of the particular spacer
within amplified DNA of the corresponding sample. Between each sample,
there is a negative tissue control. Typical complete Beijing family
patterns are shown as 89-21532 A and B. The truncated Beijing patterns
are demonstrated as 59-1216 and 59-1214. M. tuberculosis H37Rv and Mycobacterium bovis BCG P3 DNAs
were used as the hybridization pattern controls. One normal lung tissue
specimen without acid-fast bacilli was used as the negative tissue
control. Another lung tissue specimen with confirmed acid-fast bacilli
was used as the positive lung tissue control (10 µl [upper lane]
and 2 µl [lower lane]).
|
|
As shown in Table 1, we found that the Beijing family M. tuberculosis strains have been dominant since the mid-1950s (90%) and have remained so to the 1990s, with an average of 92% of the positive biopsies being positive for the Beijing family pattern over
the 34-year time period. Statistically, there are no difference between
the percentages of the Beijing family strains in four time groups
(
2 = 2.1, P = 0.55). However, older
samples had a lower positive rate of spoligotyping reactions (Table 1).
In the first time period (1956-60) only 40% (10 of 25) of the samples
were spoligotyped positive. Samples from the second time period
(1969-70) were 50% (9 of 18) positive, and samples from 1979-80 were
78% (18 of 23) positive. Samples from the most recent time period
(1989-90) were positive 63% (12 of 19) of the time. These findings
demonstrated an increased detection rate for DR spacer DNA with the
more recent samples. Comparison of the positive reactivity or detection
rate of groups 1 and 2, with a mean of 44.2%, and that of groups 3 and
4, with a mean of 71.4%, revealed that specimens in the latter groups
gave a significantly higher positive rate than the former groups
(2 = 6.46, P = 0.011). The
truncated/incomplete spoligotyping patterns (Fig. 1 and Fig.
2) frequently found in older samples
indicated that the old DNA templates might have become damaged and
fragmented during the course of storage. Humidity, pH, temperature, and
nuclease activity, etc. could be the possible factors associated with
DNA damage. We considered restoration of intact DNA templates, such as
using DNA ligation method(s), which might have been helpful for
restoration of original spoligotyping patterns. However, artifact(s) created after the DNA ligation might have resulted in false-positive reactions. Therefore, we did not use this technique. For the negative samples, we thought that using a smaller volume of DNA template in the
PCR might give better detection of M. tuberculosis
DNA, based on the postulate that the dilution of the DNA template will also cause the dilution of inhibitor(s) of DNA polymerase(s) and therefore increase the PCR sensitivity. The DNA templates were tested
at two volumes (10 and 2 ml) to limit the possible presence of an
inhibitor. We found no significant difference between the two volumes
in the development of the spoligotyping patterns (labeled a and b in
Fig. 1 and Fig. 2).
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FIG. 2.
Spoligotypes of four M. tuberculosis strains from paraffin-embedded lung samples
(89-22129, 89-20934, 69-6401, and 59-1213). Compared with the Fig. 1,
these four samples demonstrate the Non-Beijing patterns. For each
sample, either section A (89-20934, 69-6401, and 59-1213) or B
(89-22129) is shown. For each section, two volumes (10 µl [a] and 2 µl [b]) of DNA templates were used respectively as templates for
PCR. M. tuberculosis H37Rv and Mycobacterium
bovis BCG P3 DNAs were used as the hybridization pattern controls.
One normal lung tissue specimen without acid-fast bacilli was used as
the negative tissue control.
|
|
The Beijing family was highly prevalent in at least 17 different areas
around Beijing, as far back as 1956 to 1960. Although randomly chosen,
most of the subjects from this period were under the age of 30 (65%)
and males (81%). These data suggested a relatively recent tuberculosis
infection among this group. The more recent groupings (1970, 1980, and
1990) were gradually skewed toward older individuals and an increasing
proportion of females. This might suggest a shift in the mixture of
recent infections and reactivated cases during the past 20 years toward
more reactivated cases, indicating an improved living standard and
tuberculosis control activities. The dominance of the Beijing family
genotype was well established based on the fact that 92% of the total
and a similar percentage of positive samples for each 10-year time period were of this type. If we factor in the possibility of
reactivation in older patients found in the more recent groupings, we
still come up with a limit in our study to the 1950s.
In summary, we demonstrated that the Beijing family has been dominant
in the Beijing area since the middle of 1950s. Our efforts to improve
sensitivity by increasing the dilution factor of the DNA templates did
not enhance detection. Finally, we demonstrated that the spoligotyping
method is a valuable and sensitive tool for providing typing
information on stored specimens. Further studies of older biopsies or
older patients from Beijing or other areas where the Beijing family
strains are currently dominant, such as Taipei, Taiwan (our unpublished
data), may allow us to determine the time when the Beijing family
became dominant in corresponding areas. Also, studies using these
methods would give us some insights into the dynamics of tuberculosis
strains within and between populations in the past.
| |
ACKNOWLEDGMENTS |
We are indebted to the personnel of the Laboratory of Medical
Microbiology, Deventer, The Netherlands, and the Department of
Pathology, Beijing Tuberculosis Research Institute and National Tuberculosis Control Center, Beijing, People's Republic of China, whose assistance have made this work possible. We thank Annelies Bunschoten and Ria Hoentjen for technical assistance in spoligotyping. Norbert Tiemessen is acknowledged for help in sectioning the lung biopsy. Haiqing Zhang is acknowledged for assistance with the selection
and organization of the lung biopsies.
Lishi Qian was financially supported by the Leahi Trust, Hawaii
Community Foundation, Pacific Health Institute, Honolulu, Hawaii, and
the Art and Science Advisory Council Award, University of Hawaii.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Hawaii at Manoa, Department of Microbiology, 2538 The Mall (Snyder Hall 101), Honolulu, HI 96822. Phone: (808) 956-4639. Fax: (808) 956-5339. E-mail: jdouglas{at}hawaii.edu.
| |
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Journal of Clinical Microbiology, February 1999, p. 471-474, Vol. 37, No. 2
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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