JOURNAL OF CLINICAL MICROBIOLOGY, May 2007, p. 1669–1670 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.00396-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 5
Letter to the Editor Evaluation of the Wayson Stain for the Rapid Diagnosis of Melioidosis䌤 Melioidosis is a significant cause of morbidity and mortality in east Asia and northern Australia (8). Early diagnosis is vital if patients are to receive effective treatment. Definitive laboratory diagnosis is based on the isolation of Burkholderia pseudomallei, but culture is often slow or not available in these regions. Techniques such as direct immunofluorescence (DIF) (9) and urinary antigen enzymelinked immunosorbent assays (5) allow a rapid provisional diagnosis to be made with acceptable levels of accuracy but are expensive and not widely available. Rapid, cheap diagnostic methods are still needed. Direct microscopy using Gram staining may be helpful if typical slender, pale, bipolar-staining bacilli are seen, but this technique lacks specificity (7). The Wayson stain is a simple, cheap, modified methylene blue stain, originally used for diagnosing murine plague (6). Wayson staining gave better sensitivity than Gram staining did in suspected meningitis cases in one study (3). U.S. Army pathologists in Vietnam recommended the use of the Wayson stain (with the Gram stain) in cases of melioidosis (1) but did not report a formal assessment. We report a randomized, blind comparison of the Wayson and Gram stains for rapid diagnosis of melioidosis in an area of endemicity. Clinical specimens were collected from patients with suspected melioidosis who had been admitted to Sappasithiprasong Hospital, Ubon Ratchathani, Thailand (Table 1). Air-dried, heatfixed smears were prepared for staining or DIF. B. pseudomallei was isolated and identified as described previously (4, 7). Paired slides were stained either with a modified Gram stain (with a neutral red counterstain) (7) or with the Wayson stain (2) for 10 seconds, rinsed in water, and air dried. Randomized slides were examined blind by three separate observers. Interobserver measures of agreement were assessed by means of the kappa statistic. One hundred thirty-two consecutive specimens from 95 patients were included (Table 1). B. pseudomallei was isolated from 40 specimens (30%). The results for sensitivity, specificity, positive predictive value, negative predictive value, and Youden’s J index (10) based on determinations by three observers for each stain were generally poor compared to the results of culture and are shown in Table 2. Excluding sputum specimens or specimens with visible gram-positive diplococci because of a possible false-positive bipolarity did not enhance the diagnostic accuracy of either stain (data not shown). DIF results were available for 110 specimens. The results of comparisons between Gram and Wayson stain results and DIF results were similar to those of comparisons made between stain results and those of culture (data not shown). There was satisfactory agreement between the three observers regarding the results of the Gram stain (⌲ ⫽ 0.72) but not regarding those of the Wayson stain (⌲ ⫽ 0.36), suggesting considerable variability in interpretation of the latter. Our results suggest that the Gram and Wayson stains are both insufficiently sensitive and specific, compared to either
TABLE 1. Types of specimen examined Specimen type(s)
Total no. of specimens
No. (%) of culture-positive specimens
Sputum Urine (centrifuged deposit) Wound swab Pleural fluid Liver pus Splenic pus Pus (other sites) Other fluids
52 50 12 4 4 1 7 2
18 (35) 4 (2) 8 (67) 2 (50) 1 (25) 1 (100) 6 (86) 0
culture or to DIF, to be relied upon for rapid presumptive diagnoses in cases of suspected melioidosis. Even when corrected for the presence of gram-positive diplococci, the sensitivity approached approximately 50% at best. Combining Gram and Wayson stain results did not significantly improve the diagnostic sensitivity. We also found a wide variation in the reporting of the presence of bipolar rods based on the Wayson stain, which may represent a lack of familiarity with the stain but which further limits its potential utility.
TABLE 2. Comparison of Gram and Wayson stain results with culture resultsa Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
J index
Gramb 1 2 3
52.5 62.5 47.5
66.3 71.7 73.9
40.4 49.0 44.2
76.3 81.5 76.4
0.19 0.34 0.21
Mean
54.2
70.6
44.5
78.1
0.25
Wayson 1 2 3
47.5 37.5 32.5
68.5 77.2 85.9
39.6 41.7 50
75 74 74.5
0.16 0.15 0.18
Mean
39.2
77.2
43.8
74.5
0.16
Wayson and Gram combinedc 1 2 3
47.5 57.5 45
64.1 72.8 78.3
36.5 47.9 47.4
73.8 79.8 76.6
0.12 0.30 0.23
Mean
50.0
71.7
43.9
76.7
0.22
Stain(s) and observer
a b c
1669
PPV, positive predictive value; NPV, negative predictive value. Any gram-negative rods seen. Either or both stains positive for bipolar rods.
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J. CLIN. MICROBIOL.
LETTER TO THE EDITOR
We thank Professor Wipada Chaowagul and the staff of Sappasithiprasong Hospital, Ubon Ratchathani, for their assistance. We also gratefully acknowledge the contributions of Professor Nick White and Dr. Wirongrong Chierakul. The study was funded by the Wellcome Trust.
9. Wuthiekanun, V., V. Desakorn, G. Wongsuvan, P. Amornchai, A. C. Cheng, B. Maharjan, D. Limmathurotsakul, W. Chierakul, N. J. White, N. P. Day, and S. J. Peacock. 2005. Rapid immunofluorescence microscopy for diagnosis of melioidosis. Clin. Diagn. Lab. Immunol. 12:555–556. 10. Youden, W. J. 1950. Index for rating diagnostic tests. Cancer 3:32–35.
Elizabeth A. Sheridan* Division of Infection Royal London Hospital Pathology and Pharmacy Building 80 Newark Street, Whitechapel London E1 2ES, United Kingdom
REFERENCES 1. Brundage, W. G., C. J. Thuss, Jr., and D. C. Walden. 1968. Four fatal cases of melioidosis in U.S. soldiers in Vietnam. Bacteriologic and pathologic characteristics. Am. J. Trop. Med. Hyg. 17:183–191. 2. Chapin, K. C., and T.-L. Lauderdale. 2003. Reagents, stains, and media: bacteriology, p. 354–383. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, DC. 3. Daly, J. A., W. M. Gooch III, and J. M. Matsen. 1985. Evaluation of the Wayson variation of a methylene blue staining procedure for the detection of microorganisms in cerebrospinal fluid. J. Clin. Microbiol. 21:919– 921. 4. Dance, D. A. B., V. Wuthiekanun, P. Naigowit, and N. J. White. 1989. Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 2ONE. J. Clin. Pathol. 42:645–648. 5. Desakorn, V., M. D. Smith, V. Wuthiekanun, D. A. B. Dance, H. Aucken, P. Suntharasamai, A. Rajchanuwong, and N. J. White. 1994. Detection of Pseudomonas pseudomallei antigen in urine for the diagnosis of melioidosis. Am. J. Trop. Med. Hyg. 51:627–633. 6. Meyer, K. F., and A. P. Batchelder. 1926. A disease in wild rats caused by Pasteurella muricida, n. sp. J. Infect. Dis. 39:386–412. 7. Walsh, A. L., and V. Wuthiekanun. 1996. The laboratory diagnosis of melioidosis. Br. J. Biomed. Sci. 53:249–253. 8. White, N. J. 2003. Melioidosis. Lancet 361:1715–1722.
Andrew R. Ramsay Jennifer M. Short Kasia Stepniewska Vanaporn Wuthiekanun Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme Faculty of Tropical Medicine Mahidol University Bangkok, Thailand Andrew J. H. Simpson Centre for Clinical Vaccinology and Tropical Medicine Nuffield Department of Clinical Medicine University of Oxford Oxford, United Kingdom *Phone: 44 20 3246 0368 Fax: 44 20 3246 0303 E-mail:
[email protected] 䌤
Published ahead of print on 14 March 2007.
