Rapid diagnosis of bacteremia in adults using acridine orange stained buffy coat smears

The use of anidine orange s tained buffy coat smears was assessed as a rapid screening test for bacteremia in adults. A total of 356 consecutive blood cultures were submitted with simulta neous anticoagulated blood samples. from which a buffy coal s mear was prepared and stained with acridine orange (1 00 mg/ L: pH 3.0). Forty-one of 356 blood samples ( 12%) yie lded organisms in the blood cult ure system. Compa red to blood culture. the overall sensitivity of acridine orange IN A PATIENT WITII SUSPECTED BACTEREMIA. RAPID demons tration of circu la ting organisms in the blood. identification of lhe pathogen. and prompt treatment with appropriate antimicrobia l drugs are essential for optimal care. The time between patient blood sampling and initial detection of infectious agents has been shown to have important prognostic s ignificance (1 ). Department of Microbiology. McGill Unil)ersity: and Dil)iSion of lrifeclious Diseases (Depanment of Medicine). Jewish General Hospital. Montreal. Quebec Correspondence and reprims: Or Mark Miller. Department of Microbiology. Montreal General I-Iospital. .1650 Cednr Avenue. Montreal. Quebec H3G 11\4. Telephone (514) 934·8074 This paper was presemed in part at the annual meeting qf the Association of Medical Microbiologists of Quebec. held in June 1988 in Quebec City. Quebec Receil)edfor publication December 20. / 989. Accepted March 2. 1990 CAN J INFECT D IS V OL 1 No 1 SPRING 1990 s tained bu !Ty coal smears was 16%. s pecifici ty 88%. and positive predictive value 13%. Th ere was no statistically si~nificant difference in perfonnance of the lest a mong patients who had fever greater than 39°C and/or shocl<. The low sens itivity a nd specificity of the lest makes it unsuitable as a means of rapid screening for adults with suspected bacteremia. Can J Infect Dis 1990;1(1):7-10

Standard detection of bacteremia requires blood sampling from the patien t. inoculation of lhe sample into any of the cun-enlly ava ilable blood cu lture system s.an incubation lime of hours lo days during w hich t he organisms mu ltiply.and detection of growth by various mechanisms.Innumerable studies h ave evaluated methods of decreasing lhe lime to detection of positive b lood cultures.All of these methods rely on active replication of the organisms present in the culture system with subseq u ent detection of either organism density.nutrient consumption, or metabolite production (2) .Therefore.detection of bactet-ial growth may lake from several hours lo severa l days .
Faster confirmation of bacteremia is desirable because of serious sequelae in patients with c irculating microorganisms.particularly in the immunocompromised.the elderly.and patients with multiple organ dysfunction.Rapid detection methods devised thus far have depended on the identification of circulating microbia l compon ents in serum (3)(4)(5).or the direct visualization of microorganisms in buJTy coat smears from periphe ra l blood samples us ing a va1iety of n onnuorcscent staining techniques (6-8) .
In one prospective study.evaluation of acridine orange stained buJTy coat smears in 89 neonates was able to identify quickly eight of nine episodes of clinical septicemia (9).The test h ad an overall sensitivity of 88%, a specificity of 96%. and a positive predictive valu e of 67%.The a uthors decided to evaluate this meth od in an adult population.

MATERIALS AND METHODS
Over a five week period at the S ir Mortimer B Davis/Jewish Gen era l Hospita l. the role of acridine orange stained buffy coat smears in diagnosing bacteremia in adults was evalua ted.Physicians and nurses responsible for perfom1ing blood cultures were made aware of the study through posters and ward meetings .They were instructed that. in a ny patient with suspected bacteremia on whom blood cultures were being performed.blood was to be routinely inoculated as usual into one aerobic and one anaerobic blood culture boltlc (Frappier Diagnostic Inc. and Fisher Scientific.respectively).However.they were asked to place si multaneous ly an extra 5 to 7 mL of blood in a s terile tube containing EDTA.available in a ll a reas of the hospital.Al l he laboratory.the specimen in EDTA was ke pt at 4°C until the buffy coat s mear was made (a minimum of 30 mins and a maximum of 10 h a fter collection): the blood culture bottles were processed by the standard h ospital technique .This consisted of incubation of both aerobic a nd anaerobic bottles at 35°C for one week prior to discard ing as n egative.wiU1 blind Gram s tains and s ubcultures onto both chocolate and 5% sheep blood agar (in 5% carbon dioxide and anaerobically.respectively) on days 1 a nd 5 of incubation: a nd daily visual ins pection of bottles for turbidity.Visibly turbid bollles were immedia tely Gram-stained and subcultured as above.
The specimen in EDTA was processed to obtain a buffy coal smear by a modification of the method of Brooks et a l (7).In brief.the blood was centrifuged a t 700 g for 10 mins and the serum removed with a sterile Pasteur pipette.T he buffy coat was then removed wilh a sterile pipette and two drops were spread on a sterile glass s lide.The s lides were marked with numbers only. in order to b lind the microscopist as to the origin of the smear.
The smears were air dried.heat ftxed.and subsequently s ta ined with acridine orange a t a con-ccnlration of 100 mg/L buffered a t pH 3.0 according to the method of Kronvall and My hre (10).The solution was overlaid on the slides for a duration of 2 mins.The s lides were then rinsed with distilled water for 15 s. a nd a ir dried .
The smears were read within l h of sta ining on a Ouorescent microscope using 630x magnification.Suspicious nuorescence was confirmed us ing 1000x magnification.The bufiy coat smears were graded 'positive' if one or more bacteria or fungi were visualized per s lide.Each smear was a lso graded on the basis of the number of white b lood cells present: 'leukocyte-poor' if less than lO while blood cells were seen per 630x fie ld, and 'leukocyte-rich' oth erwise.In order to standa rdize the microscopy.each slide was read for 5 mins .On e investigator (MM) read a ll the smears.
Sensitivity.s pecificity and positive a nd n egative predictive values of the test were calculated using the standard formulae (ll) .Con fiden ce intervals for proportions were calcula ted assuming a binomial distribution (12) .

RESULTS
Three hundred and fifty-six consecu tive blood samples were received in the laboratory for inclusion in the s tudy.The distribution of patient locations was as follows: medical or surgical wards 195 (55%).e mergen cy room 148 (41%).and intensive car e units 13 (4%).
Forty-one of th e 356 blood samples (12%) yielded positive b lood cultures.The distribution of organisms was as follows: aerobic Gram-negative rods 28 (68%).aerobic Gram-positive cocci 10 (25%), anaerobes two (5%).yeast one (2%).and mixed zer o.Three of the 10 samples which yielded Gram-positive cocci grew Staphylococcus epidermidis.The three patients were not treated.and the organism was considered by the L realing physicians to be a contaminant.Their blood cultures were considered nega tive for the purpose of this study.
F'orly-five buffy coat smears .wereread as •positive'.Table 1 s hows the distribution of positive blood cultures and buffy coat smears.The opera ting c haracteristics of the fluorescent s mear a re as follows: sensitivity 16% (95% confidence interval 6 to 31%).specificity 88% (84 to 92%).positive predictive value 13% (5 to 27%).and n egative predictive value 90% (86 lo 93%).The performance of the Lest was secondarily analyzed in a group of high risk patients \vilh fever greater than or equal to 39°C and/or a diagnosis of shock (systolic blood pressure less lhan l 00 mmi lg and presence of lactic acidosis).The sensitivity oflhe test in lhis population was 29% (95% confidence interval 8 to 58%) which is not significantly different from the overall sensitivity of 16% (P=0.34;exact binom ia l test).
There was greater pred ictive value in finding fluorescent rods than cocci on the smear.Visualization of fluorescent rods con•eclly predicted Gram-negative rod bacteremia in four of six patients (67%).whereas fluorescent cocci were correctly predictive of bacteremia in only two of 39 individuals (5%).

DISCUSSION
It is estimated that a large concentration of organisms (10 5 to 10 6 /mLofwhole blood) must be present in order to be detected with light microscopy (13).Because of this.Gram staining ofbuffy coats in adults has d emonstrated a sensitivity of only 12% (14).Fluorescent evaluation of such smears has been estimated to be approximately 10 limes more sensitive.based on studies comparing fluorescent and Gram stained smears of blood culture bottles (15) or clinical specimens such as cerebrospinal fluid (16.I 7).
Acridine orange (3.6-b is[dimeU1ylamino] acridine).a basic fluorescent dye.has been shown to bind to both RNA and DNA (1 8).It docs this by intercalating into double stranded chains as well as binding to the outside of the double helix (19).In addition to binding to bacterial nucleic acids.il b inds to the nucleic acids of fungi.Mycoplasma species (20).trichomonads (21).s pirochetes (22) .mycobacte1ia.and mala rial parasites (23).There is a striking difference between lhe fluorescence of the organisms noted above (bright orange) and that of somatic cells (green or yellow) when the slain is buffered at pH 3 to 4 (10) .This differential staining was the basis for assessment of its usefulness in delecling pathogens in buffy coat smears.
The results of lhis study show lhat an ac1idine orange stained buffy coat smear has limited cl in ical usefulness as a screening lest in an adult hospital selling because of ils low sensiUvity.The overall sensilivily of 16% is s imilar to lhal of the Gram-staining of buffy coals previously rcpoi-led ( 14), and is only slightly higher U1an th e l l % prior probability of bacteremia in the authors' adu lt patients.The test's sensitivity probably resu lts from lhe low number of bacteria that normally circulate in most bactere mic adults (24).
The low specificity of the test can be altributed to the 39 'positive• buffy coal smears from nonbactcrcmic patien ts which d isplayed fluorescence that resembled bacteria.The probable reason for the test's low specificity is Ouorescence of rou nd intracytoplasmic granules which resemble cocci in the leukocytes.These granules fluoresce brightly.proba bly due to hig h RNA content.Although on most preparations it was easy to differentiate between coccal bacteria and these granules (the latter being less round in shape , less fluorescent.and a darker orange).many granules sufncicntly resembled bacteria to be so mistaken.This accounts for lhe almost complete predominance of Ouorescent •cocci' seen on false-positive buffy coat smears.
The low sensitivity and specificity of acridine orange stained buffy coat smears in adults wilh suspected bacteremia makes il unsu itable as a rapid screening test.munodiagnosis of candidiasis and aspergillosis.Rev Infect Dis 1984:6:301 -12. 5. Brooks.JB.Use of frequency-pulsed.electron-capture gas-liquid chromatography to selectively detect certain types of baC'terial products produced both in vivo and in vitro.In: