1 L Disseminated Cryptococcosis Diagnosed on Peripheral Blood Smear in a Patient with Acquired Immunodeficiency Syndrome JOSEPH D.C. YAO, M.D., CHARLES F. ARKIN, M.D., JOHN P. DOWEIKO, M.D., Scorr
ryptococcosis occurs in 2% to 9% of patients with the acquired immunodeficiency syndrome (AIDS) C [l-4] and most commonly takes the form of meningitis, pneumonia, or disseminated disease. The disseminated form of the disease may, at times, present as a fulminant, life-threatening illness suggestive of bacterial septic shock. In this setting, prompt diagnosis is crucial in permitting initiation of appropriate therapy to improve the chance of survival. Rapid diagnosis relies on the demonstration of organisms on smear of body fluids (e.g., cerebrospinal fluid [CSF], broncholavage specimens) and/or the presence of cryptococcal antigen in CSF or blood. Culture is often confirmatory to these more immediate diagnostic techniques. In this report, we wish to alert physicians to the utility of the peripheral blood smear in diagnosing fulminant cryptococcosis on presentation and to the fact that the presence of cryptococci in peripheral blood may cause a falsely elevated white blood cell count when it is determined by a quantitative, automated hematology analyzer. CASE REPORT A 32-year-old HIV-seropositive homosexual man, who had been doing well following an episode of Pneumocystis carinii pneumonia 18 months earlier, was admitted with 2 weeks of malaise, anorexia, progressive dyspnea, and productive cough, and 1 week of nonbloody diarrhea and fever. Examination revealed an emaciated man with hyperpigmented facies, fever of 39OC, pulse rate of 124/minute, supine blood pressure of 85/50 mm Hg, and respiratory rate of 32/minute. Inspiratory crackles at both lung bases, tender hepatosplenomegaly, and guaiac-positive stool were noted. Initial laboratory studies showed a hemoglobin level of 102 g/L, hematocrit of 37.5%, platelet count of 105 X log/L, and leukocyte count of 37.0 X log/L, with an automated “three-part” differential count of 0.22 granulocytes, 0.64 lymphocytes, and 0.14 monocytes, as determined by the hematology analyzer (Model SPLUS VI, Coulter Electronics, Inc., Hialeah, Florida). Other abnormal values included serum sodium, 127 mmol/L; serum potassium, 6.0 mmol/L; prothrombin time, greater than 45 seconds; activated partial thromboplastin time, 79 seconds; D-dimer level, greater than 1 x 10e3 g/L; aspartate aminotransferase, 14.60 ukat/ L (876 U/L); alanine aminotransferase, 8.10 ukat/L (486 U/L); and lactate dehydrogenase, 11.52 ukat/L (691 U/L). Admitting room-air arterial blood gas meaFrom the Infectious Disease Section, Departments of Medicine and Pathology, lololo westar,campNew England Deaconess Hospital, and Harvard Medical School, Boston. Massachusetts. Requests for reprints should be England Deaconess 02215. Manuscript
100
July
Hospital, submitted
185 Pilgrim Road, Boston, Massachusetts September 11. 1989, and accepted in re-
1990 The American
Journal
of Medicine
Volume
89
M. HAMMER, M.D., BOSTON, Massactwsetts
surements were as follows: oxygen partial pressure, 84 mm Hg; partial carbon dioxide pressure, 16 mm Hg; bicarbonate, 10 mEq/L; and pH, 7.38. His urine contained seven red cells, two white cells, and a few yeast cells per high-power field. Bilateral lower pulmonary interstitial infiltrates were present on chest roentgenogram. Lumbar puncture was not performed due to the presence of coagulopathy. Fluid hydration, stress replacement doses of corticosteroids, and intravenous antibiotics (oxacillin, ceftizoxime, and trimethoprim-sulfamethoxazole) were initiated. His hypoxemia and metabolic acidosis progressed rapidly soon after admission, and he required endotracheal intubation with mechanical ventilation 6 hours later. Repeat chest roentgenogram revealed marked, diffuse, bilateral alveolar infiltrates. Numerous encapsulated yeast cells suggestive of cryptococci, some budding and a few intra-leukocytic, were seen on peripheral blood smear (Figure 1). After manual counting, the initial leukocyte count was adjusted to 23.9 X log/L with 0.46 segmented neutrophils, 0.21 bands, 0.09 lymphocytes, 0.17 monocytes, 0.06 basophils, and 0.01 eosinophils. His unstable hemodynamic state prevented initiation of amphotericin B therapy. Despite maximum cardiorespiratory support, he died 10 hours after admission. Permission for postmortem examination was refused. Methenamine silver stain of his endotracheal aspirate was negative for Pneumocystis but showed encapsulated yeast cells. Routine cultures of his admission blood, urine, and sputum eventually yielded Cryptococcus neoformans.
METHODS To verify the observation that overwhelming cryptococcemia can result in a falsely elevated peripheral leukocyte count, we carried out the following experiment. C. neoformans isolated from the patient’s blood cultures was subcultured onto Sabouraud dextrose agar. After 48 hours of incubation, a dense suspension of the organism was prepared by inoculating several yeast colonies into 2 mL of balanced electrolyte solution (Isoton III, Coulter Diagnostics, Hialeah, Florida). A Neubauer hemocytometer (Bright-Line, American Optical Corp., Buffalo, New York) was used to determine the actual number of cryptococci per liter of suspension. An aliquot of the suspension was tested directly by the automated Coulter hematology analyzer. Another aliquot was admixed in a 1:l volume ratio with a peripheral blood sample (from a randomly selected patient) containing a known platelet count of 225 X log/ L, and leukocyte count of 12.0 X log/L, with automated differential counts of 0.84 granulocytes, 0.10 lymphocytes, and 0.06 monocytes. The “leukocyte count” of the mixture was then determined by the same machine.
PERIPHERAL
BLOOD
SMEAR
IN DISSEMINATED
CRYPTOCOCCOSIS
/ YAO ET AL
RESULTS The suspension of cryptococci contained 34.0 f 2.3 X log organisms/L as determined by manual counting. Results of the automated “leukocyte count” procedure for the same suspension and the cryptococci-peripheral blood admixture are shown in Table I. The automated analyzer recorded the cryptococcal suspension count as 22.5 X log/L for a 66% recovery (ratio of automated to manual count), with 90% of these reported as lymphocytes. The fact that only about two thirds of the cryptococci were counted as leukocytes is probably due to their variability in size. A relatively small number (2 X log/L) were counted as platelets, but specific quantitation of the number recorded as red blood cells, if any, could not be derived as this was below the level of precision of the automated analyzer. Interestingly, the addition of cryptococci to peripheral blood in a 1:l mixture falsely elevated the leukocyte count to a number predicted by the counts obtained separately [i.e., predicted count = (12.0 X log/L f 2) + (22.5 X log/L + 2) = 17.3 X log/L; observed count = 17.7 X log/L, Table I]. COMMENTS To our knowledge, this case is the first report of disseminated cryptococcosis diagnosed antemortem by peripheral blood smear in a patient with AIDS. Prior reports of fungi seen on peripheral blood smears in patients with AIDS have been those of disseminated histoplasmosis [5,6]. The rapid recovery of C. neoformans (within 36 hours of incubation) from routine cultures of the peripheral blood, urine, and sputum reflected the presence of an overwhelming disseminated infection [7]. Of note was the patient’s hyperpigmented facies, hyponatremia, and hyperkalemia, suggestive of adrenal insufficiency most likely due to cryptococcosis [8]. His rapid cardiorespiratory decompensation and eventual demise were consistent with a previous report of disseminated cryptococcosis manifested as adult respiratory distress syndrome [9]. This patient’s initial peripheral leukocyte count was falsely elevated as a result of the marked cryptococcemia. The leukocyte count was corrected after carefully examining the Wright-Giemsa-stained peripheral blood smears, which revealed the presence of cryptococci. Occasionally, small cryptococcal cells in these smears may be mistaken for platelets by inexperienced observers. To improve accurate detection, the mucicarmine stain could be used to directly highlight the cryptococcal capsule as bright red, often with a spiny or scalloped appearance. The cryptococci, usually varying from 4 to 15 pm in diameter, were “miscounted” by the Coulter automat-
Figure 1. Peripheral blood smear demonstrating the presence of yeast (4- to 8-pm diameter), some budding (top), and one with a well-demarcated capsule engulfed by a monocyte (bottom). Cultures of peripheral blood yielded C. neoformans (Wright-Giemsa stain; original magnification, X 1,000; top, reduced by 35%; bottom, reduced by 40%.)
ed hematology analyzer as lymphocytes that measure 7 to 10 pm in diameter. The cryptococcal suspension and peripheral blood admixture experiments suggest that about two thirds of the cryptococci can be falsely counted as white blood cells, while a much smaller proportion (
ryptococcosis occurs in 2% to 9% of patients with the acquired immunodeficiency syndrome (AIDS) C [l-4] and most commonly takes the form of meningitis, pneumonia, or disseminated disease. The disseminated form of the disease may, at times, present as a fulminant, life-threatening illness suggestive of bacterial septic shock. In this setting, prompt diagnosis is crucial in permitting initiation of appropriate therapy to improve the chance of survival. Rapid diagnosis relies on the demonstration of organisms on smear of body fluids (e.g., cerebrospinal fluid [CSF], broncholavage specimens) and/or the presence of cryptococcal antigen in CSF or blood. Culture is often confirmatory to these more immediate diagnostic techniques. In this report, we wish to alert physicians to the utility of the peripheral blood smear in diagnosing fulminant cryptococcosis on presentation and to the fact that the presence of cryptococci in peripheral blood may cause a falsely elevated white blood cell count when it is determined by a quantitative, automated hematology analyzer. CASE REPORT A 32-year-old HIV-seropositive homosexual man, who had been doing well following an episode of Pneumocystis carinii pneumonia 18 months earlier, was admitted with 2 weeks of malaise, anorexia, progressive dyspnea, and productive cough, and 1 week of nonbloody diarrhea and fever. Examination revealed an emaciated man with hyperpigmented facies, fever of 39OC, pulse rate of 124/minute, supine blood pressure of 85/50 mm Hg, and respiratory rate of 32/minute. Inspiratory crackles at both lung bases, tender hepatosplenomegaly, and guaiac-positive stool were noted. Initial laboratory studies showed a hemoglobin level of 102 g/L, hematocrit of 37.5%, platelet count of 105 X log/L, and leukocyte count of 37.0 X log/L, with an automated “three-part” differential count of 0.22 granulocytes, 0.64 lymphocytes, and 0.14 monocytes, as determined by the hematology analyzer (Model SPLUS VI, Coulter Electronics, Inc., Hialeah, Florida). Other abnormal values included serum sodium, 127 mmol/L; serum potassium, 6.0 mmol/L; prothrombin time, greater than 45 seconds; activated partial thromboplastin time, 79 seconds; D-dimer level, greater than 1 x 10e3 g/L; aspartate aminotransferase, 14.60 ukat/ L (876 U/L); alanine aminotransferase, 8.10 ukat/L (486 U/L); and lactate dehydrogenase, 11.52 ukat/L (691 U/L). Admitting room-air arterial blood gas meaFrom the Infectious Disease Section, Departments of Medicine and Pathology, lololo westar,campNew England Deaconess Hospital, and Harvard Medical School, Boston. Massachusetts. Requests for reprints should be England Deaconess 02215. Manuscript
100
July
Hospital, submitted
185 Pilgrim Road, Boston, Massachusetts September 11. 1989, and accepted in re-
1990 The American
Journal
of Medicine
Volume
89
M. HAMMER, M.D., BOSTON, Massactwsetts
surements were as follows: oxygen partial pressure, 84 mm Hg; partial carbon dioxide pressure, 16 mm Hg; bicarbonate, 10 mEq/L; and pH, 7.38. His urine contained seven red cells, two white cells, and a few yeast cells per high-power field. Bilateral lower pulmonary interstitial infiltrates were present on chest roentgenogram. Lumbar puncture was not performed due to the presence of coagulopathy. Fluid hydration, stress replacement doses of corticosteroids, and intravenous antibiotics (oxacillin, ceftizoxime, and trimethoprim-sulfamethoxazole) were initiated. His hypoxemia and metabolic acidosis progressed rapidly soon after admission, and he required endotracheal intubation with mechanical ventilation 6 hours later. Repeat chest roentgenogram revealed marked, diffuse, bilateral alveolar infiltrates. Numerous encapsulated yeast cells suggestive of cryptococci, some budding and a few intra-leukocytic, were seen on peripheral blood smear (Figure 1). After manual counting, the initial leukocyte count was adjusted to 23.9 X log/L with 0.46 segmented neutrophils, 0.21 bands, 0.09 lymphocytes, 0.17 monocytes, 0.06 basophils, and 0.01 eosinophils. His unstable hemodynamic state prevented initiation of amphotericin B therapy. Despite maximum cardiorespiratory support, he died 10 hours after admission. Permission for postmortem examination was refused. Methenamine silver stain of his endotracheal aspirate was negative for Pneumocystis but showed encapsulated yeast cells. Routine cultures of his admission blood, urine, and sputum eventually yielded Cryptococcus neoformans.
METHODS To verify the observation that overwhelming cryptococcemia can result in a falsely elevated peripheral leukocyte count, we carried out the following experiment. C. neoformans isolated from the patient’s blood cultures was subcultured onto Sabouraud dextrose agar. After 48 hours of incubation, a dense suspension of the organism was prepared by inoculating several yeast colonies into 2 mL of balanced electrolyte solution (Isoton III, Coulter Diagnostics, Hialeah, Florida). A Neubauer hemocytometer (Bright-Line, American Optical Corp., Buffalo, New York) was used to determine the actual number of cryptococci per liter of suspension. An aliquot of the suspension was tested directly by the automated Coulter hematology analyzer. Another aliquot was admixed in a 1:l volume ratio with a peripheral blood sample (from a randomly selected patient) containing a known platelet count of 225 X log/ L, and leukocyte count of 12.0 X log/L, with automated differential counts of 0.84 granulocytes, 0.10 lymphocytes, and 0.06 monocytes. The “leukocyte count” of the mixture was then determined by the same machine.
PERIPHERAL
BLOOD
SMEAR
IN DISSEMINATED
CRYPTOCOCCOSIS
/ YAO ET AL
RESULTS The suspension of cryptococci contained 34.0 f 2.3 X log organisms/L as determined by manual counting. Results of the automated “leukocyte count” procedure for the same suspension and the cryptococci-peripheral blood admixture are shown in Table I. The automated analyzer recorded the cryptococcal suspension count as 22.5 X log/L for a 66% recovery (ratio of automated to manual count), with 90% of these reported as lymphocytes. The fact that only about two thirds of the cryptococci were counted as leukocytes is probably due to their variability in size. A relatively small number (2 X log/L) were counted as platelets, but specific quantitation of the number recorded as red blood cells, if any, could not be derived as this was below the level of precision of the automated analyzer. Interestingly, the addition of cryptococci to peripheral blood in a 1:l mixture falsely elevated the leukocyte count to a number predicted by the counts obtained separately [i.e., predicted count = (12.0 X log/L f 2) + (22.5 X log/L + 2) = 17.3 X log/L; observed count = 17.7 X log/L, Table I]. COMMENTS To our knowledge, this case is the first report of disseminated cryptococcosis diagnosed antemortem by peripheral blood smear in a patient with AIDS. Prior reports of fungi seen on peripheral blood smears in patients with AIDS have been those of disseminated histoplasmosis [5,6]. The rapid recovery of C. neoformans (within 36 hours of incubation) from routine cultures of the peripheral blood, urine, and sputum reflected the presence of an overwhelming disseminated infection [7]. Of note was the patient’s hyperpigmented facies, hyponatremia, and hyperkalemia, suggestive of adrenal insufficiency most likely due to cryptococcosis [8]. His rapid cardiorespiratory decompensation and eventual demise were consistent with a previous report of disseminated cryptococcosis manifested as adult respiratory distress syndrome [9]. This patient’s initial peripheral leukocyte count was falsely elevated as a result of the marked cryptococcemia. The leukocyte count was corrected after carefully examining the Wright-Giemsa-stained peripheral blood smears, which revealed the presence of cryptococci. Occasionally, small cryptococcal cells in these smears may be mistaken for platelets by inexperienced observers. To improve accurate detection, the mucicarmine stain could be used to directly highlight the cryptococcal capsule as bright red, often with a spiny or scalloped appearance. The cryptococci, usually varying from 4 to 15 pm in diameter, were “miscounted” by the Coulter automat-
Figure 1. Peripheral blood smear demonstrating the presence of yeast (4- to 8-pm diameter), some budding (top), and one with a well-demarcated capsule engulfed by a monocyte (bottom). Cultures of peripheral blood yielded C. neoformans (Wright-Giemsa stain; original magnification, X 1,000; top, reduced by 35%; bottom, reduced by 40%.)
ed hematology analyzer as lymphocytes that measure 7 to 10 pm in diameter. The cryptococcal suspension and peripheral blood admixture experiments suggest that about two thirds of the cryptococci can be falsely counted as white blood cells, while a much smaller proportion (
