JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1990, p. 631-632 0095-1137/90/030631-02$02.00/0

Vol. 28, No. 3

Rapid Diagnosis by Buffy Coat Smear of Disseminated Mycobacterium avium Complex Infection in Patients with Acquired Immunodeficiency Syndrome JOSEPH M. NUSSBAUM,1* CLIFFORD DEALIST,2 WILLIAM LEWIS,2 AND P. N. R. HESELTINE' of Internal Medicine, Section of Infectious Disease,' and Department of Microbiology,2 Los Angeles Countyl University of Southern California Medical Center, Los Angeles, California 90033

Department

Received 5 June 1989/Accepted 1 December 1989

A smear of the buffy coat of peripheral blood for acid-fast bacilli was assessed for sensitivity and specificity in the diagnosis of disseminated Mycobacterium avium complex (MAC) infection in acquired immunodeficiency syndrome (AIDS) patients. Seventeen AIDS patients with blood cultures positive for MAC had simultaneous quantitative blood cultures and buffy coat smears performed, as did 4 patients later proven not to have disseminated MAC. The sensitivity of the buffy coat smear for the detection of MAC was 35%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 22%. We conclude that the buffy coat smear is a rapid, simple, and specific method of diagnosis of disseminated MAC infection in AIDS patients, although it is not very sensitive.

Disseminated Mycobacterium avium complex (MAC) is the most common mycobacterial infection occurring in patients with acquired immunodeficiency syndrome (AIDS) in the United States (1, 3, 4, 6). Estimates on the incidence of this infection range as high as 50% (4). The symptoms of infection are usually nonspecific. Fever, night sweats, diarrhea, and weight loss are found in many AIDS patients with this infection but are not uniformly present. Although current treatment results are disappointing, several new agents are under investigation, and it is possible that effective therapy may be developed in the near future (1, 7). In view of this possibility, a rapid method of diagnosis would be desirable. Symptomatic infection is usually characterized by sustained mycobacteremia, and the diagnosis is most frequently established by culture of the organism from the blood (7). Even with a radiometric methodology (BACTEC system; Johnston Laboratories, Inc., Towson, Md.), establishing the diagnosis requires awaiting a culture result that may be delayed from 1 to 7 weeks (1). We were intrigued by a report of visible acid-fast bacilli (AFB) on a smear of a buffy coat preparation from a patient with AIDS and disseminated MAC (2). We decided to evaluate the sensitivity of this rapid and simple technique in patients with the established diagnosis of disseminated MAC. All patients evaluated had a previous diagnosis of AIDS based on positive human immunodeficiency virus serology and an opportunistic infection other than mycobacterial disease and had either a clinical presentation suspicious of disseminated MAC or more commonly a report of the growth of AFB from a blood culture done within the preceding 2 to 4 weeks at the time of evaluation. Patients subsequently had the diagnosis of disseminated MAC confirmed by blood cultures positive for mycobacteria identified as belonging to the M. avium-M. intracellulare complex by standard culture and biochemical techniques (5). Evaluation included a quantitative blood culture for MAC and a smear of peripheral blood buffy coat, done simultaneously, by the following technique. The buffy coat smear was prepared from peripheral blood drawn into a 10-ml *

heparinized-blood collection tube (VACUTAINER; Becton Dickinson Vacutainer Systems, Rutherford, N.J.). The heparinized blood was centrifuged at 1,800 x g for 20 to 30 min, and the buffy coat was aspirated with a pipette, usually 0.1 to 0.2 ml in volume. Two drops were placed on each of three slides, which were air dried and heat fixed. Slides were stained with auramine-rhodamine (5), rinsed, decolorized with 0.5% acid alcohol for 2 min, rinsed, rinsed again with potassium permanganate, and examined by an experienced technician under a fluorescence microscope for 2 to 5 min per slide. The finding of any AFB on any of the three slides was recorded as a positive smear. The quantitative blood culture for mycobacteria was performed in the following manner. Ten milliliters of blood was obtained by a sterile technique in a lysis-centrifugation blood culture tube (Isolator; Du Pont Co., Wilmington, Del.). The tube was centrifuged at 3,000 x g for 30 min and aseptically capped, and 8 ml of supernatant was removed and discarded. The remaining 2 ml of sediment was vortexed, and 0.3 ml was transferred to each of a set of three screw-cap bottles with slants of Lowenstein-Jensen medium. A 0.5-ml portion of the remaining blood sediment was diluted in 4.5 ml of sterile isotonic saline to make a 1:10 dilution, and a similar portion of this dilution was diluted once again to make a 1:100 dilution. Aliquots (0.3 ml) of each dilution were inoculated as described above into sets of three slant bottles. The organisms that grew were confirmed as AFB by Ziehl-Neelsen staining (5) and identified as MAC by routine biochemical tests (5). The total number of visible colonies in a given set multiplied by the appropriate dilution factor was reported as the number of colonies per milliliter of blood. Confluent growth was not counted; confluent growth in all sets, including the 1:100 dilution, was reported as >10,000 colonies per ml. A comparison of buffy coat smears with simultaneous quantitative blood cultures is presented in Table 1. In summary, 6 (31%) of 19 buffy coat smears were positive in 17 patients with disseminated MAC. Two patients each had the procedure repeated once; patient 5 had a negative smear with 10 colonies per ml on the simultaneous quantitative culture. When the procedure was repeated 1 week later

Corresponding author. 631

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NOTES TABLE 1. Buffy coat smear and quantitative blood culture results

Patient

smear result

Buffy coat

Blood result (no. ofculture colonies/ml)

1 2 3 4 5

Negative Positive Negative Negative Negative Positive Negative Positive Negative Negative Negative Positive Negative Negative Positive Negative Positive Negative Negative

200 >10,000 il 10 10 180 25 >10,000 >10,000 2,400 25 20 50 260 33 5,000 800 3 900

6 7 8 9 10 il 12 13 14 15 16 17

without therapy, this patient had a positive smear with 180 colonies per ml. Patient 6 had a negative smear with 50 colonies of MAC per ml of blood on the quantitative culture. When the procedure was repeated 2 weeks later without intervening antibiotic therapy, this patient had a positive smear with >10,000 colonies of MAC per ml of blood on the quantitative culture. Of 8 patients with colony counts greater than 100 colonies per ml, 4 (50%) had positive smears. Four patients with AIDS and initially suspected of having MAC bacteremia were subsequently found to have other diseases. One patient was found to have miliary tuberculosis (positive bone marrow culture and 6 colonies per ml of blood obtained 3 weeks earlier), one patient had persistently negative blood cultures with MAC cultured only from stools, one patient had M. tuberculosis cultured from sputum and stools, and one patient had no mycobacteria cultured from any source during the evaluation. Buffy coat smears in all four were negative. The calculated sensitivity of the buffy coat smear for the detection of MAC in the blood of the patients tested was 35%, and the calculated specificity was 100%. The positive predictive value was 100%, and the negative predictive value was only 22%. Graham et al. (2) found AFB on Kinyoun-stained buffy

coat smèars from a patient with AIDS and disseminated MAC. They estimated that their patient had 150,000 organisms per ml by extrapolation from the number of organisms counted and the leukocyte count. The simplicity and rapidity of this technique are immediately appealing, and if effective therapy is developed, such a technique could become useful in patient management. As demonstrated here, a positive buffy coat smear is both a rapid and a specific test for disseminated MAC, albeit not very sensitive. Intuitively, the sensitivity of the buffy coat smear would depend on the degree of mycobacteremia, and patients early in the course of disseminated MAC may have low levels of mycobacteremia and negative buffy coat smears. However, it has been our experience that AIDS patients with this infection usually have progressively increasing mycobacteremia, and as seen in two of our patients, repeated smears may yield the diagnosis. The finding of AFB on a buffy coat smear is highly suggestive, but not definitive, of disseminated MAC; however, in our experience M. tuberculosis does not produce such a high degree of bacteremia, even in patients with AIDS. LITERATURE CITED 1. Gill, V. J., C. H. Park, F. Stock, L. L. Gosey, F. G. Witebsky, and H. Masur. 1985. Use of lysis-centrifugation (Isolator) and radiometric (BACTEC) blood culture systems for the detection of mycobacteremia. J. Clin. Microbiol. 22:543-546. 2. Graham, B. S., M. V. Hinson, S. R. Bennett, D. W. Gregory, and W. Schaffner. 1984. Acid-fast bacilli on buffy coat smears in the acquired immunodeficiency syndrome: a lesson from Hansen's bacillus. South. Med. J. 77:246-248. 3. Hawkins, C. C., J. W. M. Gold, E. Whimbey, T. E. Kiehn, P. Brannon, R. Cammarata, A. E. Brown, and D. Armstrong. 1986. Mycobacterium avium complex infections in patients with the acquired immune deficiency syndrome. Ann. Intern. Med. 105: 184-188. 4. Klatt, E. C., D. F. Jensen, and P. R. Meyer. 1987. Pathology of Mycobacterium avium-intracellulare infection in acquired immunodeficiency syndrome. Hum. Pathol. 18:709-714. 5. Sommers, H. M., and R. C. Good. 1985. Mycobacterium, p. 216-248. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 6. Wong, B., F. F. Edwards, T. E. Kiehn, E. Whimbey, D. Harrison, E. M. Bernard, J. W. M. Gold, and D. Armstrong. 1985.

Continuous high grade Mycobacterium avium-intracellulare bacteremia in patients with the acquired immune deficiency syndrome. Am. J. Med. 78:35-40. 7. Young, L. S. 1988. Mycobacterium avium complex infection. J. Infect. Dis. 157:863-867.

Rapid diagnosis by buffy coat smear of disseminated Mycobacterium avium complex infection in patients with acquired immunodeficiency syndrome.

A smear of the buffy coat of peripheral blood for acid-fast bacilli was assessed for sensitivity and specificity in the diagnosis of disseminated Myco...
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