
==== Front
Acta Cytol
Acta Cytol
ACY
ACY
Acta Cytologica
0001-5547
1938-2650
S. Karger AG Basel, Switzerland

38838662
539567
10.1159/000539567
00000
Nongynecologic Cytopathology
Digital Image Comparison of Cellular Yield in Bronchial Brushing: Pre- and Post-Biopsy Lavage Cytology
Cellular Yield of Pre- and Post-Biopsy Lavage
2743425
Li Joshua Jing Xi a
2743426
Ng Joanna Ka Man b
2743427
Chan Christopher c
2743428
Lau Charlotte Ho Ying c
2743429
Ng Joyce Ka Ching c
2743430
Lo Rachel Lai Ping c
2743431
Yip Wing Ho c
2743432
Ngai Jenny Chun Li c
2743433
Chan Ka Pang c d
a Department of Pathology, Queen Mary Hospital, School of Clinical Medicine, The University of Hong Kong, Hong Kong, Hong Kong, China
b Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, Hong Kong, China
c Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, Hong Kong, China
d Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Hong Kong, China
Correspondence to: Ka Pang Chan, chankapang@cuhk.edu.hk
5 6 2024
9 2024
68 4 368373
3 4 2024
26 5 2024
2024
© 2024 The Author(s). Published by S. Karger AG, Basel
2024
https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission.
Abstract

Introduction

Bronchoscopy is a useful diagnostic tool capable of performing core biopsy, forceps biopsy, bronchoalveolar lavage, and bronchial brushing. This study compares the cellularity of bronchial cytology including pre- and post-biopsy lavage by digital image analysis, aiming to increase diagnostic and tumor yield by optimizing the sequence and combination of bronchial biopsy and cytology.

Methods

Alveolar macrophage, bronchial epithelium, and tumor cell cellularity from liquid-based cytology preparations of bronchial brushing and pre-biopsy and post-biopsy bronchoalveolar lavage were annotated on digitized whole-slide images and compared. Secondary analysis on the relationship of tumor cell and non-lesional cell yield was performed.

Results

Overall, 118 cytology specimens from 43 patients were retrieved in total. Bronchial epithelium count was higher in pre-biopsy than post-biopsy lavage (p < 0.01) but not for alveolar macrophages nor tumor cell (p > 0.05). Tumor cell count was higher for bronchial brushing cytology samples than lavage (p = 0.018). The alveolar macrophage count was higher in post-biopsy lavage than bronchial brushing (p = 0.033); otherwise, brushing showed consistently higher bronchial epithelium and tumor cell counts. There were 33 false negative (tumor cell absent) specimens, and the combination of bronchial brushing and pre-biopsy lavage yielded the lowest false negative cases. Correlation between bronchial epithelium and alveolar macrophage counts with tumor cell count was weak (correlation coefficient = −0.168–0.203) except for post-biopsy lavage (correlation coefficient = 0.412–0.479, p < 0.05).

Conclusion

Bronchial brushing yields a greater amount of tumor cell than lavage, and timing lavage before or after core biopsy does not affect tumor cell yield. Combining bronchial brushing and pre-biopsy lavage results in the lowest false negative rate.

Keywords

Digital pathology
Bronchial cytology
Bronchoalveolar lavage
Bronchial brushing
Lung cancer
No funding was received for the current study.
==== Body
pmcIntroduction

Bronchoscopy is a flexible diagnostic and therapeutic modality employed in the management of lung lesions. Tissue diagnosis can be obtained through core biopsy, forceps biopsy, bronchoalveolar lavage, and bronchial brushing. Studies have compared the diagnostic and detection rates varying the timing of bronchoalveolar lavage and brushing with respect to core biopsy but largely reported negative results [1–5]. In this study, instead of a dichotomous comparison of cytologic diagnosis [1–5], tumor cell counts, a more sensitive parameter, bronchoalveolar lavage before and after core biopsy, and bronchial brushing were assessed. Cell counting was performed on digitized scanned whole-slide images with annotations for improved reproducibility and accuracy. The yield of bronchial epithelium and alveolar macrophages and the correlation between these non-lesional cells and tumor cells were explored. The false negative rate of combinations of cytologic specimens was compared. Findings of this study aim to optimize the sequence of tissue procurement and adequacy assessment of bronchial cytology.

Methods

Bronchial brushing, bronchoalveolar lavage (pre- and post-core biopsy), and bronchial core biopsy were performed by board accredited respiratory physicians through bronchoscopic means in patients with suspicion of pulmonary malignancies and bronchoscopically visible tumor from October 2022 to October 2023. The sequence of procedures was (1) pre-biopsy bronchoalveolar lavage, (2) core biopsy, (3) bronchial brushing, and (4) post-biopsy bronchoalveolar lavage. Cases where the core biopsy and/or resection pathology reports did not confirm a malignant diagnosis were excluded. The bronchial cytology (bronchoalveolar lavage and brushing) specimens were retrieved and were prepared using Cytospin IV (Shandon, Life Science International [Europe] Limited) with double circle printing (two circular monolayer cell deposits on a single glass slide). The surfaces of the slides were physically cleaned to ensure the thorough removal of debris and then scanned and digitized using the Leica AT2 slide scanner. Slides that were physically damaged, not retrievable, showing significant artifacts (thick bloodied specimens, drying and crushing effects), and of poor staining quality were excluded.

The digitized slides were viewed and annotated using QuPath (version 0.3.2, for Windows) [6]. Two circular high-power fields hotspots of equivalent size to ×400 magnification with a 20 mm eyepiece generated in QuPath. One circular hotspot, with representative cellular yield and avoiding areas with thick cellular stacks, foreign material, and artifacts, was placed in each circle (circular monolayer cell deposit) (Fig. 1). The location of the hotspot was determined by a cytopathologist and reviewed by another independent cytopathologist. Bronchial epithelial cells (columnar cells with either cilia or a terminal bar identified), alveolar macrophages (macrophages with cytoplasmic dark carbon pigments), and malignant tumor cells were annotated and counted separately within each hotspot (Fig. 2). Annotation was performed by a cytopathologist and reviewed by another independent cytopathologist. All discrepancies were resolved by viewing the digitized slides together until a consensus was reached.

Fig. 1. One circular hotspot corresponding to one high-power field, upper left corner shows overview of the slide with double circle printing on a single slide.

Fig. 2. Annotation of tumor cells (maroon), bronchial epithelium (blue), and alveolar macrophages (pink).

Statistical analysis was performed using SPSS (version 23.0, for Windows). The paired t test was used to compare cell counts of each cell type in different cytology specimens for the average and maximum cell count of the two hotspots, whereas cross-tabulation was used for a binary comparison of false negative and true positive cytology specimens. Secondary analysis in the relationship between tumor cell yield and other cell types was performed by using the Pearson correlation coefficient between bronchial epithelium, alveolar macrophage count, and tumor cell count. A p value of <0.05 was considered significant.

Results

A total of 118 cytology specimens from 43 patients were retrieved, comprising 39 bronchial brushing, 37 pre-core biopsy bronchoalveolar lavage, and 42 post-core biopsy bronchoalveolar lavage. There were 36 cases of non-small cell carcinoma, with the majority being adenocarcinoma (n = 14/36, 38.9%), 4 cases of small cell carcinoma, and 1 case of lymphoepithelial-like carcinoma, adenoid cystic carcinoma, and metastatic colorectal carcinoma (Table 1).

Table 1. Composition of the cohort

Number of cytology specimen (patients)	118 (43)	
 Pre-biopsy lavage	37	
 Post-biopsy lavage	42	
 Bronchial brushing	39	
Histologic diagnosis	
 Non-small cell carcinoma	36	
   Adenocarcinoma	(14)	
   Squamous cell carcinoma	(8)	
   Not specified	(14)	
 Small cell carcinoma	4	
 Lymphoepithelial-like carcinoma	1	
 Adenoid cystic carcinoma	1	
 Metastatic colorectal carcinoma	1	

Paired comparisons of pre- and post-biopsy lavage show that the cell count of bronchial epithelium was higher in pre-biopsy than post-biopsy lavage (maximum: p = 0.001, average: p = 0.003), but there were no significant differences for the number of alveolar macrophages nor tumor cell count (p > 0.05). Comparing bronchial brushing to pre-biopsy and post-biopsy lavage, the tumor cell count yielded by bronchial brushing was consistently higher than pre-biopsy (maximum: p = 0.018, average: p = 0.018) and post-biopsy lavage (maximum: p = 0.006, average: p = 0.009). The bronchial epithelium cell count in post-biopsy lavage was higher than that of bronchial brushing (maximum: p = 0.015, average: p = 0.030), while the alveolar macrophage cell count was lower (maximum: p = 0.033, average: p = 0.052) (Table 2).

Table 2. Paired comparison of cell counts between bronchial brushing, pre-biopsy lavage, and post-biopsy lavage

	Bronchial epithelium (maximum)	Alveolar macrophage (maximum)	Tumor cell (maximum)	Bronchial epithelium (average)	Alveolar macrophage (average)	Tumor cell (average)	
Pre-biopsy lavage	21.47	20.58	28.25	16.79	15.57	19.64	
Post-biopsy lavage	5.53	16.89	22.31	4.58	13.22	16.75	
p value	0.001	0.597	0.293	0.003	0.678	0.457	
Pre-biopsy lavage	20.21	21.45	30.45	15.50	16.36	21.21	
Bronchial brushing	21.24	3.03	53.24	15.12	2.17	41.50	
p value	0.851	0.182	0.018	0.928	0.180	0.018	
Post-biopsy lavage	5.87	16.37	20.97	4.86	13.05	15.50	
Bronchial brushing	19.11	2.61	48.74	13.87	1.84	37.84	
p value	0.015	0.033	0.006	0.030	0.052	0.009	

Cross-tabulation of false negative cytology specimens (no tumor cells present) with other cytology preparations show that there were 12 false negative bronchial brushing and post-biopsy lavage specimens and nine pre-biopsy lavage specimens. Addition of one cytologic specimen to a false negative specimen decreases the false negative rate by at least 30% to above 60%, and the combination of bronchial brushing and pre-biopsy lavage yielded the lowest count of false negative cases (n = 3) compared to other combinations, with those 3 cases also negative on post-biopsy lavage (Table 3).

Table 3. Cross-tabulation of false negative (no tumor cells) cytology specimens

False negative	Bronchial brushing	Pre-biopsy lavage	Post-biopsy lavage	
Total false negative	12 (30.7%, 12/39)	9 (24.3%, 9/37)	12 (28.6%, 12/42)	
Bronchial brushing positive	–	4 (57.1%, 4/7)	4 (36.3%, 4/11)	
Pre-biopsy lavage positive	6 (66.7%, 6/9)	–	4 (40%, 4/10)	
Post-biopsy lavage positive	7 (58.3%, 7/12)	3 (37.5%, 3/8)	–	
Negative in all specimens	3 (25%, 3/12)	3 (33.3%, 3/9)	3 (25%, 3/12)	

The correlation between alveolar macrophage and tumor cell count was very weak for pre-biopsy lavage, post-biopsy lavage, and bronchial brushing with correlation coefficients of 0.033–0.203. Except for post-biopsy lavage (correlation coefficient 0.412–0.479, p < 0.05), correlation between bronchial epithelium and tumor cell count was also weak for pre-biopsy lavage and bronchial brushing (correlation coefficient −0.168 to −0.003) (Table 4).

Table 4. Correlation between (a) alveolar macrophage and (b) bronchial epithelium and tumor cell count

	Pre-biopsy lavage	Post-biopsy lavage	Bronchial brushing	
(a) Alveolar macrophage (maximum)	20.03 (SD 41.28)	16.10 (SD 27.80)	2.69 (SD 5.25)	
Tumor cell (maximum)	27.49 (SD 52.14)	20.38 (SD 42.38)	48.08 (SD 80.69)	
Correlation coefficient	0.125	0.019	0.203	
Alveolar macrophage (average)	15.15 (SD 5.22)	12.73 (SD 3.63)	1.91 (SD 0.62)	
Tumor cell (average)	19.11 (SD 6.73)	15.13 (SD 5.29)	37.23 (SD 11.28)	
Correlation coefficient	0.101	0.033	0.198	
(b) Bronchial epithelium (maximum)	21.03 (SD 26.62)	5.57 (SD 7.09)	19.51 (SD 30.46)	
Tumor cell (maximum)	27.49 (SD 52.14)	20.38 (SD 42.38)	48.08 (SD 80.69)	
	−0.028	0.412 (p = 0.007)	−0.150	
Bronchial epithelium (average)	16.46 (SD 22.89)	4.62 (SD 6.38)	14.13 (SD 22.94)	
Tumor cell (average)	19.11 (SD 40.94)	15.13 (SD 34.27)	37.23 (SD 70.47)	
	−0.003	0.479 (p = 0.001)	−0.168	

Discussion

Bronchoscopy and transthoracic biopsy are the mainstay of obtaining tissue diagnosis in lung neoplasms [7]. Bronchoscopy is preferred for central, tracheobronchial, and mucosal lesions [8]. Methods of tissue procurement during bronchoscopy include core biopsy (endobronchial, transbronchial, with or without ultrasound guidance), forceps biopsy, bronchoalveolar lavage, and bronchial brushing, of which each is different in terms of invasiveness, tissue yield, and complications [9–12].

There are studies comparing the diagnostic yield of different sequences of sampling techniques [1–5]. The majority of these studies failed to demonstrate improvement in optimizing the sequence of different types of tissue procurement [1, 3–5], except for Hou et al. reporting superior diagnostic rates for bronchial brushing specimens obtained before biopsy [5]. However, these studies only addressed the cytologic diagnosis of the specimens, mostly in a dichotomous fashion with either a positive/concordant or negative/discordant categorization. A binary classification is less sensitive in detecting subtle differences in the tissue yield between different cytologic preparations [13], which may have led to the lack of significant findings.

The current study aims to compare not only the cytologic diagnosis but also the diagnostic yield in terms of cell count of bronchial brushing, pre-biopsy, and post-biopsy bronchoalveolar lavage cytology. Cell counting is traditionally performed on glass slides based on a low-power impression or manual counting of limited high-power fields [14], which lacks reproducibility. Counted cells are not recorded and cannot be validated by another assessor, and manual counting under a microscope is prone to repetition and overlooking of cells. As demonstrated by studies on assessment of ki-67 and mitotic figures [15, 16], counts made on digital images are more dependable and reproducible, leading to recommendations preferring monitor and/or printout-image-based assessment over microscopy [15–17]. As such, the current study adopted digital cell counting with annotation and review by multiple assessors.

In terms of tumor cell count, there were no significant differences between pre-biopsy and post-biopsy lavage, but bronchial brushing demonstrated significantly higher cellular yield. Bronchial brushing is considered as an abrasive method that is associated with complications such as bleeding and airway trauma [18, 19], while increasing the detection rate for lung lesions [12]. The bronchial epithelium count was lower for post-biopsy lavage, which may be a result of the exhaustion of surface epithelial cells after repeated manipulation.

Of note, the alveolar macrophage count was higher for bronchoalveolar lavage compared to bronchial brushing. Alveolar macrophages are present deeper within the respiratory tract and mostly in the terminal alveoli [20]. The increased alveolar macrophage count supports the postulation that bronchoalveolar lavage fluid is able to reach the alveolar spaces effectively for diagnosing conditions affecting the distal bronchoalveolar tree [21]. Alterations in the composition of inflammatory cells within the alveoli can be indicative of immune and inflammatory disease, for example, asthma, interstitial lung disease, and drug-induced lung disease [22, 23].

Comparing the false negative rates (samples with no tumor cell yield) of different types of specimen, bronchial brushing was most commonly associated with a false negative result, followed by post-biopsy and pre-biopsy lavage, but differences did not reach statistical significance. The summated diagnostic rate of two specimens were compared to find the optimal combination of procedures. The lowest false negative rate was achieved by the combination of bronchial brushing and pre-biopsy lavage, at 9.1% (n = 3/33). Post-biopsy lavage was also falsely negative in those 3 cases, indicating that omission of post-biopsy lavage may not reduce the diagnosis rate for malignant lung lesions.

Secondary analysis was performed to investigate the correlation between bronchial epithelium and alveolar macrophage versus tumor cell count. Adequate non-lesional epithelial cell count has been regarded as a prerequisite for an adequate specimen in exfoliative cytology [24, 25], whereas presence of alveolar macrophage was considered as an indicator of lower respiratory tract sampling and specimen quality [26]. However, the correlation coefficients between these non-lesional cells and tumor cells were low to negative. Alveolar macrophage was not confirmed to be useful indicators in assessment of specimen adequacy in this cohort. The strongest correlation was seen between bronchial epithelial cell count and tumor cell count; in post-biopsy lavage specimens (Kappa >0.4), and at a bronchial epithelial cell count of at least 6 per high-power field, an adequacy rate of 81.8% was achieved.

Conclusion

Bronchial brushing yields higher tumor cell counts than bronchoalveolar lavage, and timing bronchoalveolar lavage before or after bronchial core biopsy does not affect tumor cell yield. Combining bronchial brushing and pre-biopsy lavage results in the lowest false negative rate, over the other combinations, and is not improved by further adding post-biopsy lavage, indicating an optimal sequence of bronchoalveolar lavage, and brushing before core biopsy. Correlation between the number of bronchial epithelial cells and alveolar macrophages versus tumor cell count was low to negative, indicating that the numbers of bronchial epithelial cells and alveolar macrophages were not useful in assessment of specimen adequacy.

Statement of Ethics

The study was approved by the Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee (Reference No. 2022.61). Exemption of the requirement of written informed consent was granted by the Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee (Reference No. 2022.61).

Conflict of Interest Statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding Sources

No funding was received for the current study.

Author Contributions

J.J.X.L.: conceptualization, formal analysis, investigation, methodology, visualization, and writing – original draft. J.K.M.N.: investigation and validation. C.C., C.H.Y.L., J.K.C.N., R.L.P.L., W.H.Y., and J.C.L.N.: investigation. K.P.C.: conceptualization, investigation, methodology, resources, supervision, validation, and writing – review and editing.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.
==== Refs
References

1. Fernández-Villar A , GonzálezA, LeiroV, RepresasC, Isabel BotanaM, BlancoP, . [Effect of different bronchial washing sequences on diagnostic yield in endoscopically visible lung cancer]. Arch Bronconeumol. 2006;42 (6 ):278–82.16827976
2. Hou G , MiaoY, HuXJ, WangW, WangQY, WuGP, . The optimal sequence for bronchial brushing and forceps biopsy in lung cancer diagnosis: a random control study. J Thorac Dis. 2016;8 (3 ):520–6.27076949
3. Yigla M , NagivD, SolomonovA, MalbergerE, Ben-IzhakO, RubinA-HE, . Timing of collecting bronchoscopic cytologic specimens in endobronchial malignant neoplasms. J Bronchology Interv Pulmonology. 2002;9 (4 ):272–5.
4. van der Drift MA , van der WiltGJ, ThunnissenFB, JanssenJP. A prospective study of the timing and cost-effectiveness of bronchial washing during bronchoscopy for pulmonary malignant tumors. Chest. 2005;128 (1 ):394–400.16002962
5. Lee HS , KwonSY, KimDK, YoonHI, LeeS-M, LeeJH, . Bronchial washing yield before and after forceps biopsy in patients with endoscopically visible lung cancers. Respirology. 2007;12 (2 ):277–82.17298463
6. Bankhead P , LoughreyMB, FernándezJA, DombrowskiY, McArtDG, DunnePD, . QuPath: open source software for digital pathology image analysis. Sci Rep. 2017;7 (1 ):16878.29203879
7. Yu Lee-Mateus A , ReisenauerJ, Garcia-SaucedoJC, Abia-TrujilloD, BuckarmaEH, EdellES, . Robotic-assisted bronchoscopy versus CT-guided transthoracic biopsy for diagnosis of pulmonary nodules. Respirology. 2023;28 (1 ):66–73.36104312
8. Faurschou P , MilmanN, DirksenA, GarsdalP, JarnvigIL, NissenF. Classification of pulmonary lesions into central and peripheral with a template applied on chest X-ray. Respir Med. 1996;90 (6 ):349–52.8759478
9. Tomar V , VijayN, NuwalP, DixitR. Comparative study of bronchoalveolar lavage, bronchial brushing, and FNAC in diagnosing malignant neoplasms of lungs. J Cytol. 2016;33 (4 ):210–3.28028336
10. Lee P , MehtaAC, MathurPN. Management of complications from diagnostic and interventional bronchoscopy. Respirology. 2009;14 (7 ):940–53.19740256
11. Ng JKM , ChanKP, TseGM, LiJJX. Bronchial cytology of pulmonary adenoid cystic carcinoma: a multi-institute series with emphasis on immunocytochemistry. Ann Diagn Pathol. 2023;64 :152132.36963153
12. Ng JKM , CheungW, LiJJX, ChanKP, YipWH, TseGM. Detection of early (T1) lung cancers and lepidic adenocarcinomas in sputum and bronchial cytology. Ann Diagn Pathol. 2023;67 :152191.37579536
13. Poon IK , ChanRCK, ChoiJSH, NgJKM, TangKT, WongYYH, . Reply to: a comparative study of diagnostic accuracy in 3026 pleural biopsies and matched pleural effusion cytology with clinical correlation: a methodological issue. Cancer Med. 2023;12 (4 ):4365–6.36478178
14. Kitchener HC , GittinsM, DesaiM, SmithJH, CookG, RobertsC, . A study of cellular counting to determine minimum thresholds for adequacy for liquid-based cervical cytology using a survey and counting protocol. Health Technol Assess. 2015;19 (22 ):1–64.
15. Chow ZL , ThikeAA, LiHH, NasirNDM, YeongJPS, TanPH. Counting mitoses with digital pathology in breast phyllodes tumors. Arch Pathol Lab Med. 2020;144 (11 ):1397–400.32150458
16. Dogukan FM , Yilmaz OzguvenB, DogukanR, KabukcuogluF. Comparison of monitor-image and printout-image methods in ki-67 scoring of gastroenteropancreatic neuroendocrine tumors. Endocr Pathol. 2019;30 (1 ):17–23.30367334
17. Cree IA , TanPH, TravisWD, WesselingP, YagiY, WhiteVA, . Counting mitoses: SI(ze) matters. Mod Pathol. 2021;34 (9 ):1651–7.34079071
18. Urabe N , SakamotoS, ItoA, SekiguchiR, ShimanukiY, KanokogiT, . Bronchial brushing and diagnosis of pulmonary nontuberculous mycobacteria infection. Respiration. 2021;100 (9 ):877–85.34044411
19. Stahl DL , RichardKM, PapadimosTJ. Complications of bronchoscopy: a concise synopsis. Int J Crit Illn Inj Sci. 2015;5 (3 ):189–95.26557489
20. Joshi N , WalterJM, MisharinAV. Alveolar macrophages. Cell Immunol. 2018;330 :86–90.29370889
21. Stanzel F . Bronchoalveolar Lavage. Principles and practice of interventional pulmonology. 2012; p. 165–76.
22. Allen JN , DavisWB, PachtER. Diagnostic significance of increased bronchoalveolar lavage fluid eosinophils. Am Rev Respir Dis. 1990;142 (3 ):642–7.2389917
23. Smith H . Asthma, inflammation, eosinophils and bronchial hyperresponsiveness. Clin Exp Allergy. 1992;22 (2 ):187–97.1571812
24. Birdsong GG , DaveyDD. Specimen adequacy. In: NayarR, WilburDC, editors. The Bethesda System for Reporting Cervical Cytology: Definitions, Criteria, and Explanatory Notes. Cham: Springer International Publishing; 2015. p. 1–28.
25. Renshaw AA , GouldEW. Adequacy criteria for voided urine cytology using cytospin preparations. Cancer Cytopathol. 2019;127 (2 ):116–9.30561909
26. Courcol RJ , DamienJM, RamonP, VoisinC, MartinGR. Presence of alveolar macrophages as a criterion for determining the suitability of sputum specimens for bacterial culture. Eur J Clin Microbiol. 1984;3 (2 ):122–5.6373252
