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A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular and 1
Improving Detection of Extracellular Mycobacterium Tuberculosis in 2
Cerebrospinal Fluid 3
4
Ping Chen1†, Ming Shi1†, Guo-Dong Feng1†, Jia-Yun Liu2†, Bing-Ju Wang1, 5
Xiao-Dan Shi1, Lei Ma1, Xue-Dong Liu1, Yi-Ning Yang1, Wen Dai1, Ting-Ting Liu1, 6
Ying He1, Jin-Ge Li3, Xiao-Ke Hao2, Gang Zhao1* 7
8
1 Department of Neurology, 2 Center for Clinical Laboratory Medicine, and 3 9
Department of Infectious Diseases, Xijing Hospital, the Fourth Military Medical 10
University, 15 West Changle Road, Xi'an, Shaanxi 710032, China 11
12
Running title: A modified Ziehl-Neelsen stain for intracellular Mtb 13
14
† These authors contributed equally to this work. 15
16
* Corresponding author: 17
Prof. Gang Zhao 18
Department of Neurology, Xijing Hospital, the Fourth Military Medical University, 15 19
West Changle Road, Xi’an, Shaanxi 710032, China; TEL: +86-29-8477 5361, Fax: 20
+86-29-8255 1806. Email: [email protected] 21
22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.05756-11 JCM Accepts, published online ahead of print on 11 January 2012
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Abstract 23
Tuberculous meningitis leads to a devastating outcome, and early diagnosis and rapid 24
chemotherapy are vital to reduce morbidity and mortality. Since Mycobacterium 25
tuberculosis (Mtb) is a kind of cytozoic pathogen and its number is very few in the 26
cerebrospinal fluid, detecting Mtb in the cerebrospinal fluid from tuberculous 27
meningitis patients is still a challenge for the clinicians. Ziehl-Neelsen stain, the 28
current feasible microbiological method for diagnosis of tuberculosis, often needs a 29
large amount of cerebrospinal fluid specimen but shows a low detection rate of Mtb. 30
Here, we developed a modified Ziehl-Neelsen stain, involving cytospin slides with 31
Triton processing, in which only 0.5 ml of cerebrospinal fluid specimens was required. 32
This method not only improved the detection rate of extracellular Mtb significantly, 33
but also identified intracellular Mtb in the neutrophils, monocytes and lymphocytes 34
clearly. Thus, our modified method is more effective and sensitive than the 35
conventional Ziehl-Neelsen stain, providing clinicians a convenient and yet powerful 36
tool in rapidly diagnosing tuberculous meningitis. 37
38
Keywords: Tuberculous meningitis; Mycobacterium tuberculosis; Cerebrospinal fluid; 39
Ziehl-Neelsen stain 40
41
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Introduction 43
Tuberculous meningitis (TBM) is the most severe form of tuberculosis and causes 44
substantial morbidity and mortality (18). Early diagnosis of and prompt initiation of 45
chemotherapy for TBM are crucial to a successful outcome. However, early and 46
accurate detection of Mycobacterium tuberculosis (Mtb) in the cerebrospinal fluid 47
(CSF) of TBM patients still remains a challenge for clinicians, which is mainly due to 48
lack of rapid, efficient and practical detection methods (30). 49
Currently, mycobacterial culture is the gold standard for detecting Mtb, but it is 50
time-consuming and requires specialized safety procedures in laboratories (19, 26). 51
Serological methods are convenient, but lack sensitivity and specificity (4, 7). Though 52
the polymerase chain reaction technique is rapid, it is costly for routine use in 53
developing countries where most tuberculosis cases occur (5, 17, 21, 24). 54
Conventional smear microscopy with the Ziehl-Neelsen stain is a rapid and practical 55
method for detecting acid-fast bacilli (AFB), especially in low-income countries, due 56
to its rapidity, low cost and high positive predictive value for tuberculosis (14). 57
However, Ziehl-Neelsen method is severely handicapped by its low detection rate, 58
ranging from 0-20 % for CSF specimens (31-33). One of the main reasons behind this 59
is that Mtb can hardly be stained by acid-fast dyes once it enters the cells. Another 60
important reason is that Ziehl-Neelsen method requires a large volume of CSF for 61
TBM diagnosis as it is incapable of detecting bacilli that are fewer than 10,000 per 62
slide or per mL of specimen (32, 33). Therefore, it is important to develop an 63
alternative cost-effective method for detecting intracellular Mtb. Additionally, 64
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knowing which cell type is infected by Mtb in the CSF of TBM patients could help us 65
to unravel new anti-tuberculotic candidates (10). 66
To reveal the presence of intracellular Mtb and improve the detection of 67
extracellular Mtb from a small volume of CSF specimens, we developed a highly 68
efficient Ziehl-Neelsen stain involving the use of only 0.5 mL CSF specimens from 69
TBM cases. The formed elements in the CSF including the bacilli and cells were 70
compactly collected onto the slides by cytospinning followed by staining with 71
acid-fast dyes containing the detergent Triton X-100. Using this modified staining 72
method, AFB can be clearly revealed within the immune cells and the detection rate 73
of extracellular AFB was significantly improved as well. 74
75
Materials and Methods 76
Study subjects 77
The study protocol was approved by the Institutional Review Board of Xijing 78
Hospital, the Fourth Military Medical University, Xian, China and written informed 79
consent was obtained from all patients or their legal surrogates. Twenty-nine patients, 80
whose CSF specimens were culture positive for Mycobacterium by a MGIT 960 81
Mycobacteria Culture System, were diagnosed as TBM and enrolled in this study. For 82
identification of Mtb and differentiation of non-Mtb from positive cultures, 83
Ziehl-Neelsen stain and a commercial TB real-time PCR kit (Qiagen GmBH, Hilden, 84
Germany) were used. 85
Conventional Ziehl-Neelsen stain 86
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Two mL of CSF specimens were centrifuged at 3,000 × g for 15 min and the sediment 87
was smeared on slides as previously described (27). All smears were stained by the 88
conventional Ziehl-Neelsen method for the presence of AFB as depicted earlier (14) 89
and observed under a light microscope. 90
Modified Ziehl-Neelsen stain 91
Cytospin was used to collect the formed elements of CSF specimens. In brief, 0.5 mL 92
CSF specimen was loaded into the chamber in which poly-L-lysine-coated slides were 93
inserted and centrifuged at 1,000 g for 5-10 min. After the media were aspirated, the 94
cells were fixed with 4 % paraformaldehyde (pH 7.4) for 10 min at room temperature. 95
The cells on cytospin slides were then permeabilized with 0.3 % TritonX-100 for 30 96
min followed by conventional Ziehl-Neelsen stain, in which fast acid dye contained 97
0.3 % TritonX-100. The cells were counterstained with methyl blue for 5 min. 98
Combination of auramine-O stain and immunofluorescence 99
The cytospin slides were prepared as described above and then permeabilized with 100
0.3 % TritonX-100 for 30 min. Auramine-O (AO) (Sigma, St. Louis, MO) stain was 101
performed as described by the manufacturer. After AO stain, the cytospin slides were 102
incubated with the following primary antibodies in PBS containing 2 % normal 103
donkey serum at 4°C overnight: mouse anti-CD11b (Millipore, Bilerica, MA), mouse 104
anti-ED1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CD3, and mouse 105
anti-CD20 (MaiXin Biotechnology, China). After wash with PBS, the cells were 106
treated with biotinylated anti-mouse or anti-rabbit IgG antibody (Vector Laboratories, 107
Burlingame, CA) for 3 h and then with Cy3-conjugated streptavidin (Jackson 108
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ImmunoResearch, West Grove, PA) for 1 h at room temperature. The cells were 109
counterstained with Hoechst 33342 (Sigma). Immunofluorescent signals were 110
observed under a laser confocal microscope (LSM 510, Carl Zeiss Microscopy). 111
Data analysis 112
All the smear and cytospin slides stained by the conventional or modified method 113
were observed under oil immersion at the magnification of 1,000 ×. A total of 300 114
visual fields on each slide were observed, among which AFB-positive fields were 115
counted by three experienced observers independently. All the data were displayed as 116
mean ± SEM and analyzed using one-way ANOVA. Differences were considered 117
statistically significant when P < 0.05. 118
119
Results 120
A total of 48 CSF samples were collected from 29 TBM patients (Table 1). When 121
analyzed by the samples, thirty-five samples (35/48, 72.92%) were culture-positive, 122
and 8 (8/48, 16.67%) were Ziehl-Neelsen smear positive while all the samples (48/48, 123
100%) were positive by our modified Ziehl-Neelsen stain. The sensitivity of the 124
Ziehl-Neelsen stain was 22.9 % [95 % C.I 8.9 – 36.7] (n = 8/35) and 16.7% [95 % C.I 125
11.29 – 22.05] (n = 8/48) against culture and the modified Ziehl-Neelsen stain, 126
respectively. The sensitivity of culture was 72.92% [95 % C.I 66.51 – 79.33] (n = 127
35/48) against the modified Ziehl-Neelsen stain. The specificity of all techniques was 128
100 % (n = 48/48). When analyzed by patients, the sensitivity of culture and the 129
modified Ziehl-Neelsen stain was identical (100 %, 29/29) while that of the 130
Ziehl-Neelsen stain was 27.6 % [95 % C.I 9.29 – 35.89] (n = 8/29) against culture and 131
the modified Ziehl-Neelsen stain. The specificity of all techniques was 100 % (n = 132
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29/29). These data suggest a higher sensitivity of our modified Ziehl-Neelsen stain 133
than that of mycobacteria culture and the conventional Ziehl-Neelsen stain. 134
In the conventional method, a relatively large CSF volume (2 mL) was used for 135
smear slides. Ziehl-Neelsen stain showed that the AFB were sparsely distributed and 136
the integrity of immune cells was violated (Fig. 1A). In some regions of smear slides, 137
aggregated cells were often observed in AFB-positive fields (Fig. 1B). We noted that 138
in the conventional method, all the AFB observed were distributed extracellularly and 139
no AFB were found within the cells. By comparison, in the modified method, 0.5 mL 140
CSF samples were used for cytospin slides. Ziehl-Neelsen stain with Triton 141
processing showed that the immune cells were distributed evenly on the slides and 142
maintained the integrity of cellular morphology (Fig. 1C-F). Moreover, using this 143
method, we clearly observed AFB within the immune cells, including neutrophils, 144
monocytes and lymphocytes (Fig. 1D-F). Importantly, we also found that the number 145
of extracellular AFB was significantly increased in the modified method. In addition, 146
we noted that cells containing AFB displayed an abnormal morphology relative to 147
those free of AFB. For example, a larger percentage of neutrophils with more lobes 148
showed an obvious right shift (Fig. 1D, arrow) and monocytes displayed a larger cell 149
body and foam-like cytoplasm (Fig. 1E, arrow) while lymphocytes showed a smaller 150
cell body and nuclear pyknosis (Fig. 1F, arrow). These data suggest that upon and 151
following phagocytosis by the immune cells, intracellular Mtb also exerts effect on 152
the immune cells. 153
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To confirm intracellular localization of AFB and exclude the possibility that the 154
bacilli inadvertently adhere to the cell surface during cytospinning, we performed 155
double labeling of AO, a fluorescent dye used for staining tubercle bacillus (28, 31), 156
and the activated neutrophil marker CD11b, monocyte marker ED1, and T 157
lymphocyte marker CD3 or B lymphocyte marker CD20. Our results showed that 158
AO+ bacilli were indeed located in the cytoplasm of neutrophils, monocytes or 159
lymphocytes, but not on the surface of these cells (Fig. 2 and 3). 160
For quantitative analysis of extracellular AFB, the conventional method identified 161
extracellular AFB in 8 of 48 CSF specimens (16.7 %) while the modified method 162
identified all the specimens (100 %, 48/48). For quantification of intracellular AFB, 163
the modified method detected intracellular AFB in 45 of 48 CSF specimens (93.8 %) 164
while none was detected by the conventional method (Table 2). Furthermore, through 165
observation of 300 fields on each slide from 48 CSF samples, we found that the 166
number of extracellular and intracellular AFB-positive fields with the conventional 167
method was 20.0 ± 63.1 and 0 fields, respectively, and 73.5 ± 58.0 (P < 0.001) and 168
24.3 ± 22.0 (P < 0.001) fields with the modified method, respectively (Fig. 4). 169
170
Discussion 171
In the present study, we developed a modified Ziehl-Neelsen method on cytospin 172
slides with Triton processing. Only using a small amount of CSF samples, our 173
modified method yielded a 93.8 % detection rate of intracellular AFB while the 174
conventional method failed to detect any intracellular AFB. Also, this method can 175
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improve the detection rate of extracellular AFB from 16.7 % of the conventional 176
Ziehl-Neelsen stain to 100%. Moreover, our method had a higher sensitivity than the 177
mycobacterium culture method, and is easier to implement than AO staining, in which 178
expensive fluorescence microscopy is required. 179
We attributed the higher detection rate of Mtb by the modified method to two 180
possible reasons. First, cytospinning was employed, by which AFB within cells were 181
concentrated compactly in a small circular area on the slide with low speed 182
centrifugation. In contrast to the conventional method, by which the cells are mostly 183
destroyed, our modified method preserves the cellular integrity and consequently 184
prevents the loss of intracellular AFB from inside the cells. In addition, coating the 185
slides with poly-L-lysine further prevents cells and bacilli in the CSF from falling off 186
during staining. Second, we employed Triton X100, which permeates the cellular 187
membrane and facilitates the entry of fast-acid dye. Thus, the modified method 188
combining cytospin and Triton permeation significantly improves the efficiency of 189
Ziehl-Neelsen stain. Furthermore, the cytospin method concentrates extracellular AFB 190
in the CSF. Consequently, the CSF specimens from all the 48 specimens were found 191
positive for extracellular AFB by the modified stain, as compared to the low detection 192
rate (16.7 %, 8/48) by the conventional method. Triton processing also improves the 193
permeability of the unique bacterial wall of AFB (15), which resists staining by 194
acid-fast dyes. Thus, the modified method also improved the detection rate of 195
extracellular AFB. 196
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Accumulating evidence shows that the presence of intracellular Mtb is a crucial 197
indicator of the body’s immune response to tuberculosis (26, 28). Monocytes, 198
neutrophils and lymphocytes are three major immune cell types in the CSF of TBM 199
and play different roles in the pathogenesis of TBM. As is known, monocytes are 200
regarded as the main immune cells for host defense against Mtb. However, a group of 201
studies have shown that the antigen-presenting function of macrophages is 202
significantly impaired following infection with Mtb (9, 12, 20, 22). Thus, it is 203
proposed that other immune cells may be recruited to enhance the immune response 204
against Mtb infection (16). In the present study, we revealed that Mtb was present in 205
both neutrophils and lymphocytes, indicating that these two types of cells are indeed 206
involved in the host’s immune response against Mtb. Neutrophils have both 207
bactericidal and immunomodulatory functions (3, 13, 25). When infected with Mtb, 208
neutrophils can directly kill invading Mtb via generation of reactive oxygen species 209
and release of preformed oxidants and proteolytic enzymes, and/or indirectly 210
eliminate Mtb by releasing an array of cytokines and chemokines to attract other 211
inflammatory cells. On the other hand, infected neutrophils also provide a permissive 212
site for active replication of Mtb (6, 8), which in turn induces apoptosis of neutrophils 213
(1, 2). Macrophages are capable of phagocytizing apoptotic neutrophils, resulting in 214
the eventual elimination of intracellular Mtb (29). Unexpectedly, our results also 215
showed an intracellular distribution of Mtb in CD3+ T lymphocytes. Previous studies 216
have shown that dendritic cells, a derivative from lymphocyte precursor, can 217
phagocytize Mtb and exert their antigen-presenting functions (11, 23). These findings 218
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together prompt us to propose that lymphocytes may play a similar role in TBM, 219
compensating decreased antigen-presenting function of macrophages. Thus, our 220
present study suggests that in addition to monocytes, neutrophils and lymphocytes 221
may also participate in the host defense against Mtb infection. 222
223
Conclusion 224
Given the acute need for a simple, efficient and practical method for detecting Mtb 225
within the cells and from a small amount of CSF samples, our modified Ziehl-Neelsen 226
staining method can efficiently reveal the presence of AFB in the immune cells of a 227
small amount of CSF samples from TBM patients, and improve the detection rate of 228
extracellular AFB as well. Therefore, this method will be of tremendous value in 229
improving both the diagnosis and treatment of tuberculosis. 230
231
Acknowledgement 232
The authors are grateful to Prof. Bo-Quan Jin (Department of Immunology, the Fourth 233
Military Medical University) and Prof. Zhi-Kai Xu (Department of Microbiology, the 234
Fourth Military Medical University) for valuable comments on the manuscript, and Dr. 235
Bo Cui (Associate editor for Journal of Biomedical Research, Nanjing University) for 236
his laborious work on English correction. This work was supported by a grant from 237
Discipline-Boosting Program of Xijing Hospital (No. XJZT10Z03). 238
239
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Figure legends 353
Fig. 1. Comparison of the conventional and modified Ziehl-Neelsen stain for CSF 354
samples from tuberculous meningitis patients. (A, B) The conventional stain shows 355
damaged cellular structure (A) and cell aggregations (B). No intracellular acid fast 356
bacilli (AFB) are observed with the conventional method. (C-F) The modified method 357
can concentrate AFB in the CSF (C). Intracellular AFB are frequently observed in 358
neutrophils (D), monocytes (E) and lymphocytes (F). Arrows show acid fast 359
dye-positive AFB. Insets indicate higher magnification views of arrow-indicated AFB 360
or cells. Scale bars: 20μm (A-F), 5μm (Insets). 361
362
Fig. 2. Intracellular distribution of acid fast bacilli (AFB) in neutrophils and 363
monocytes on the modified Ziehl-Neelsen stain. Double-labeling of auramine-O (AO) 364
(A, E, green) with CD11b (B, red) and ED1 (F, red) shows the intracellular location of 365
AFB in neutrophils (A-D) and monocytes (E-H). AO, CD11b and ED1 label AFB, 366
neutrophils and monocytes, respectively. The nuclei are stained by Hoechst 33342 367
(blue). Panels (D’, D’’) and (H’, H’’) show higher magnification views of panel (D) 368
and (H) in Z-axis projections, respectively. Scale bars: 20 μm (A-H), 5 μm (D’, D’’, 369
H’, H’’). 370
371
Fig. 3. Intracellular distribution of acid fast bacilli (AFB) in lymphocytes on the 372
modified Ziehl-Neelsen stain. Double-labelling of auramine-O (AO) (A, D, green) 373
with CD3 (B, red) and CD20 (E, red) shows the intracellular location of AFB in 374
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19
lymphocytes (C, F). The nuclei are stained by Hoechst 33342 (blue). Scale bar: 5 μm. 375
376
Fig. 4. Comparison of acid fast bacilli (AFB)-positive fields by the conventional and 377
modified Ziehl-Neelsen (ZN) stain. Totally, 300 fields on each slide from 48 CSF 378
specimens were observed. Compared with the ZN stain, which reveals no intracellular 379
AFB-positive fields, the modified ZN stain definitely identifies AFB within the 380
immune cells. Moreover, the modified stain reveals more extracellular AFB-positive 381
fields than the conventional ZN stain. 382
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Table 1. Sensitivity of all techniques analyzed by sample (n = 48)*, patient (n = 29).
Specificity of all techniques in all cases was 100%.
Technique [% sensitivity (No. positive samples)]
Group Culture ZN stain Modified ZN stain
Analysis by patient (n = 29) 100 % (29) 27.6 % (8) 100 % (29)
Analysis by sample (n = 48) 72.9 % (35) 16.7 % (8) 100 % (48)
* 48 CSF samples were collected from 29 TBM patients. ZN, Ziehl-Neelsen.
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Table 2. Positive rate of intracellular and extracellular ABF in the CSF samples (n =
48) detected by Ziehl-Neelsen stain and modified Ziehl-Neelsen (ZN) stain,
respectively.
Technique [% positive rate (No. positive samples)]
Group ZN stain Modified ZN stain
Extracellular ABF (n = 48) 16.7 % (8) 100 % (48)
Intracellular ABF (n = 48) 0 % (0) 93.8 % (45)
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