Detection of pneumococcemia by quantitative buffy coat analysis.

Abstract

Quantitative buffy coat (QBC) analysis is a well accepted tool for the sensitive and rapid diagnosis of malaria [1]. It can also detect several other microorganisms in human specimens, including microfilaria, trypanosomes, Leishmania amastigotes, Trichomonas trophozoites, Babesia piroplasms, Borrelia spirochetes and leptospires [2, 3, 4, 5, 6, 7, 8]. Here, we present a case in which QBC analysis facilitated the rapid detection of pneumococcemia. A 35-year-old man presented at the emergency department with severe headache and cold extremities. He had arrived in the Netherlands from Burkina Faso 3 weeks earlier and had no permanent living address. He was conscious but confused and disorientated. Initial examination revealed a heart rate of 100 bpm, unstable blood pressure (ranging from 80/40 to 200/110 mmHg), and a temperature of 36.4 C. Further physical examination revealed nuchal rigidity and paresis of the left leg. Petechiae were not observed. Lung sounds were normal. Laboratory investigations revealed a leukocyte count of 8.1 109/l (78% polymorphs, 8% bands, 10% metamyelocytes). Numerous Howell-Jolly bodies were observed. Erythrocyte sedimentation rate was 2 mm/first hour, platelet count 33 109/l, prothrombin time 54.6 s, activated partial thromboplastin time 140 s, fibrinogen level 0.3 g/l. C-reactive protein level was 211 mg/l and creatinine level 362 mol/l. Lumbar puncture was not performed because of presumed disseminated intravascular coagulation. A presumptive diagnosis of bacterial sepsis with meningitis was made. Empirical antibacterial therapy with amoxicillin, ceftriaxone and gentamicin was commenced after blood cultures were drawn. As part of the patient’s laboratory work-up, the parasitology lab was asked to perform diagnostic tests for malaria. Thick and thin blood smears were prepared from finger-prick blood. Microscopic investigation of smears according to World Health Organization standard methods did not reveal malaria parasites. Malaria pigment was, however, observed in some peripheral blood phagocytes, indicating that the patient had been infected with malaria in the recent past. EDTA-anticoagulated blood obtained by venepuncture was used to perform QBC analysis. A QBC tube (Becton Dickinson, USA) was filled with blood (approximately 55–65 l) and mixed with acridineorange dye, which coats the interior of the tube [7]. The tube was stoppered and a plastic float was inserted. The tube was centrifuged at 12,000 rpm for 5 min in a capillary tubes centrifuge (Becton Dickinson) and examined by fluorescence microscopy (Olympus BH-2; Olympus America, USA) with a 50 oil immersion lens. The entire erythrocyte layer was examined by revolving the tube and examining all sections. No malaria parasites were observed. The leucocyte, platelet, and plasma layers were additionally examined for signs of other infections e.g. borreliosis, trypanosomiasis and leishmaniasis. Just above the plasma-platelet interface, a dense concentration of brightly luminescent structures was observed, which morphologically resembled diplococci (Fig. 1A). No other abnormalities were observed. Subsequent to the QBC analysis, direct Gram stain was performed on a thin blood smear prepared from fingerprick blood. After a thorough search, a few encapsulated gram-positive diplococci resembling Streptococcus pneumoniae were observed both extracellularly and intracellularly in granulocytes (Fig. 1B). The remainder of the EDTA-anticoagulated blood was inoculated onto blood agar plates with antibiotic-impregnated disks. Overnight culture yielded penicillin-sensitive Streptococcus pneumoniae. P. C. Wever ()) · E. R. Heddema · M. D. de Jong · T. van Gool Section of Parasitology, Department of Medical Microbiology, Academic Medical Centre, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands e-mail: p.c.wever@amc.uva.nl Tel.: +31-20-5669111 Fax: +31-20-6979271