ROLE OF BUFFY COAT EXAMINATION IN THE DIAGNOSIS OF NEONATAL CANDIDEMIA

Abstract

In recent years fungal infections have become an important cause of morbidity and mortality in neonates, especially preterm infants.1 They are more prone to develop fungal infection because of their immunocompromised state and modern intensive care practices. An early diagnosis can help in early institution of therapy. Modalities that may possibly be useful in early diagnosis include buffy coat smear microscopy and buffy coat culture.2-5 However, a prospective evaluation of these modalities in a high risk population of neonates is not available. We have therefore evaluated the role of buffy coat smear microscopy and buffy coat culture in the diagnosis of fungemia in a high risk population of neonates. Materials and methods. Twenty-nine neonates who were considered at high risk of fungemia were included in the study. These were selected from neonates admitted to pediatric wards of the Nehru Hospital Post Graduate Institute of Medical Education and Research, Chandigarh, from January, 1994, to January, 1995. Informed consent was obtained from the parents of all patients. The study was approved by the Institution's ethics committee. Inclusion criteria. Neonates were included in the study if they fulfilled the following criteria: (1) presence of at least one of the following predisposing factors: very low birth weight babies (<1500 g), prolonged antibiotic therapy (≥2 weeks), prolonged use of intravenous catheters (≥1 week), prolonged endotracheal intubation (≥3 days) or granulocytopenia (≤500/mm3); along with (2) at least one of the following clinical features: poor response to antibacterial therapy (defined as inadequate or no clinical improvement within 48 h of initiation of therapy in a neonate suspected of having sepsis), oral thrush, cutaneous candidiasis, unresolving infiltrates on chest roentgenogram and secondary unexplained deterioration while receiving antibiotic therapy. The later was defined as the appearance of signs suggestive of sepsis such as lethargy, tachypnea, respiratory distress, apnea, etc., in spite of continued antibacterial therapy in a neonate who had initially shown definite clinical improvement with the same antibacterial therapy. Not all infants meeting the inclusion criteria during the study period could be enrolled because of problem of logistics, especially on weekends and nights. Sample. Blood (5 ml) was drawn aseptically from a peripheral vein; 2.5 ml of blood were immediately inoculated into biphasic brain-heart infusion broth with agar and the remaining 2.5 ml of blood were placed in a sterile EDTA vial and centrifuged at 3500 rpm for 15 to 30 min. The plasma was pipetted off with a Pasteur pipet and a buffy coat smear was prepared. The remaining portion of the buffy coat was inoculated into another biphasic brain-heart infusion broth with agar. Direct microscopy. The buffy coat smear slide was stained with a drop of Calcofluor white (a fluorescent dye that stains chitin), covered with a coverslip and observed by fluorescent microscopy using a 340 to 400 nm filter for the presence of yeast cells, hyphae and pseudohyphae. The dye can also enter the phagocytes and highlight the intracellular yeast. Blood cultures. Biphasic blood culture bottles were incubated at 37°C for 48 h. The first subculture was made by shaking the bottle and tilting it in such a way that the liquid phase covered the solid phase and was kept for 30 min at room temperature. The bottle were further incubated for 1 week; then the second subculture was made and a week later third subculture was made in the same manner.6 Bottles were checked daily during the first week and twice a week later for 4 weeks for any suspected growth. Any suspected growth was subcultured on Sabouraud dextrose agar and identified by conventional yeast identification methods such as the germ tube method, tetrazolium reduction medium, urease production, sporulation on cornmeal agar/rice starch agar, sugar fermentation and sugar assimilation.7 Statistical methods. The sensitivity, specifcity and positive and negative predictive values of buffy coat smear microscopy and buffy coat culture were compared with the whole blood culture results. Results. Of the 29 neonates included in the study 13 neonates had evidence of candidemia by conventional blood culture. Candida albicans8 was the most common organism isolated followed by Candida guillermondii,2Candida tropicalis2 and Candida parapsilosis.1 Buffy coat smear microscopy revealed Candida in 8 neonates. Figure 1shows brightly fluorescent Candida pseudohyphae and yeast cells, some inside phagocytes. The outline of phagocytes, however, are not seen clearly. Blood culture was positive in all of these infants. Buffy coat cultures grew Candida species in 12 neonates, 11 of which had grown Candida species on whole blood culture. Sensitivity, specificity and positive and negative predictive values of buffy coat smear microscopy and buffy coat culture are shown in Table 1. Buffy coat cultures grew yeast species after the same interval as that of conventional whole blood culture (≈48 h). One infant had a positive buffy coat culture of C. tropicalis but a negative blood culture. This was a preterm, 1.2-kg birth weight infant, who had respiratory distress syndrome, neonatal jaundice requiring exchange transfusion and Staphylococcus aureus sepsis (on Day 7). The infant was sampled because of oral thrush on Day 28 of life; simultaneously obtained suprapubic urine, rectal swab and pharyngeal swab all grew C. tropicalis. Discussion. Early diagnosis of fungal infection remains a challenge. Although our population may not be representative of at risk population in other parts of world, the finding that buffy coat smear microscopy had a sensitivity of 62% for detecting candidemia detected by whole blood culture remains relevant to early diagnosis of candidemia elsewhere. This sensitivity is better than 44% reported with use of peripheral blood smear examination for the diagnosis.3 Importantly, we found buffy coat smear microscopy to have a 100% specificity, no false positivity and 100% positive predictive value. Because the result may be available within 1 or 2 h it could be used as a simple bedside test for early diagnosis of candidemia for initiating antifungal therapy. The method can be safely used even for very low birth weight infants because 0.5 to 1.0 ml of blood is sufficient to obtain a buffy coat smear if a Wintrobe's tube is used. In making therapeutic decisions, however, it should be appreciated that a negative smear does not exclude the possibility of candidemia. Buffy coat culture had a 85% sensitivity and 94% specificity for candidemia detected by whole blood culture. Buffy coat culture also helped in the isolation of Candida in one infant from whom conventional blood culture failed to grow the organism. All positive buffy coat culture results were available by 48 h of incubation. Whole blood culture reports were also available at the same time. It may therefore appear that buffy coat cultures failed to yield a faster diagnosis than the routine blood culture. This is explained, however, by the early isolation of Candida from blood at our center as compared with other cultures.4 Ascuitto et al.4 reported that buffy coat culture is faster than blood culture on the basis of two cases of fungemia (one case each of Cryptococcus neoformans and C. albicans). Cryptococcus neoformans was isolated at 48 h by buffy coat culture in contrast to 10 days by blood culture and C. albicans was isolated at 36 h and 3 days, respectively. We found that clinical features of illness, namely poor response to antibacterial therapy and unexplained clinical deterioration while receiving antibacterial therapy, were predictive of candidemia in a majority of our cases. This is in agreement with studies in adults with candidemia.8 It is concluded that buffy coat smear microscopy is a reliable, rapid diagnostic tool for the diagnosis of candidemia. In view of 100% specificity and a 100% positive predictive value, empirical antifungal therapy can be started in patients showing Candida on buffy coat smear microscopy. Thimmapuram Chandra Sekhara Reddy, M.D.; Arunaloke Chakrabarti, M.D.; Meenu Singh, M.D.; Sunit Singhi, M.D. Department s of Pediatrics (TCSR, MS, SS) and Medical Microbiology (AC) Postgraduate Institute of Medical Education and Research Chandigarh, IndiaFIG. 1: Calcofluor white stain showing yeast cells and pseudohyphae of Candida.