Comparative evaluation of (1, 3)-β-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia

BMC Infectious Diseases Comparative evaluation of (1, 3)--D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia Fasahat F Alam 0 Abu S Mustafa 0 Zia U Khan 0 0 Address: Department of Microbiology, Faculty of Medicine, Kuwait University , Safat 13110, P. O. Box 24923 , Kuwait Background: Candidemia is a major infectious complication of seriously immunocompromised patients. In the absence of specific signs and symptoms, there is a need to evolve an appropriate diagnostic approach. A number of methods based on the detection of Candida mannan, nucleic acid and (1,3)-betaD- glucan (BDG) have been used with varying specificities and sensitivities. In this retrospective study, attention has been focused to evaluate the usefulness of two or more disease markers in the diagnosis of candidemia. Methods: Diagnostic usefulness of Platelia Candida Ag for the detection of mannan, Platelia Candida Ab for the detection of anti-mannan antibodies, Fungitell for the detection of BDG, and of a semi-nested PCR (snPCR) for the detection Candida species-specific DNA have been retrospectively evaluated using 32 sera from 27 patients with culture-proven candidemia, 51 sera from 39 patients with clinically suspected candidemia, sera of 10 women with C. albicans vaginitis, and sera of 16 healthy controls. Results: Using cut-off values recommended by the manufacturers, the sensitivity of the assays for candidemia patients were as follows: Candida snPCR 88%, BDG 47%, mannan 41%, anti-mannan antibodies 47%, respectively. snPCR detected 5 patients who had candidemia due to more than one Candida species. The sensitivities of the combined tests were as follows: Candida mannan and anti-mannan antibodies 75%, and Candida mannan and BDG 56%. Addition of snPCR data improved the sensitivity further to 88%, thus adding 10 sera that were negative by BDG and/or mannan. In clinically suspected, blood culture negative patients; the positivities of the tests were as follows: Candida DNA was positive in 53%, BDG in 29%, mannan in 16%, and anti-mannan antibodies in 29%. The combined detection of mannan and BDG, and mannan, BDG and Candida DNA enhanced the positivity to 36% and 54%, respectively. None of the sera from Candida vaginitis patients and healthy subjects were positive for Candida DNA and mannan. Conclusion: The observations made in this study reinforce the diagnostic value of snPCR in the sensitive and specific diagnosis of candidemia and detection of more than one Candida species in a given patient. Additionally, in the absence of a positive blood culture, snPCR detected Candida DNA in sera of more than half of the clinically suspected patients. While detection of BDG, mannan and anti-mannan antibodies singly or in combination could help enhancing sensitivity and eliminating false positive tests, a more extensive evaluation of these assays in sequentially collected serum samples is required to assess their value in the early diagnosis of candidemia. - Background Candidemia is a major infectious and steadily increasing complication of seriously immunocompromised patients that is associated with high mortality rates [1-5]. Non-specific clinical presentation, low positivity of blood cultures even in autopsy-proven cases and rapid course of the disease have necessitated the need for developing sensitive methods for the early diagnosis of invasive candidiasis [1,6]. Among the different approaches that have been developed and evaluated in the recent years include detection of Candida mannan [7,8], arabinitol [9-11], and nucleic acids [12-15]. However, all these methods have limitations of sensitivity and/or specificity. The recent introduction of BDG detection assay, a fungus-specific marker, has provided a new diagnostic tool with encouraging results [16-18]. This has encouraged the investigators to evaluate the usefulness of BDG in tandem of mannan and/or DNA for the early and specific diagnosis of invasive mycoses [19]. In this communication, we have evaluated the diagnostic value of Candida species-specific DNA, BDG, Candida mannan, and Candida anti-mannan antibodies in sera samples obtained from culture-proven candidemia patients and clinically suspected cases of candidiasis. Methods Patients and sera samples Thirty-two sera from 27 culture-proven candidemia patients, 51 from 39 clinically suspected blood culture negative systemic candidiasis patients, 10 from Candida vaginitis patients and 16 from healthy subjects with no complaints of oral or vaginal Candida infection were included in the study. All the candidemia patients were admitted to the intensive-care unit and other wards of Mubarak Al-Kabeer Hospital, Kuwait for various conditions (Table 1). According to the European Organization for Research and Therapy of Cancer and Mycoses Study Group (EORTC/MSG) consensus revised definitions draft presented at ICAAC, 2005, all the patients yielding Candida species in blood cultures were considered as cases of candidemia. Besides yielding Candida species in blood culture, the patients had suggestive signs and symptoms of septicemia along with one or more risk factors, such as prolonged use of broad-spectrum antibiotics, presence of central venous catheter or extended period (> 2 weeks) of hospitalization. The other risk factors included gastrointestinal surgical procedures (n = 6), chronic renal failure (n = 3), multiple organ injury (n = 3), invasive urinary tract manipulation (n = 3), diabetes mellitus (n = 2) and pancytopenia (n = 2). In absence of microbiologic evidence of candidemia, 39 patients were included in the possible/suspected category of systemic candidiasis on the basis of suggestive host and clinical criteria. The patients included in this category had fever that did not respond to 4 days of broad-spectrum antibiotic therapy. In addition, they had at least three of the following risk factors: extended period of hospitalization (> 2 weeks), isolation of Candida species from one or more anatomic sites, presence of intravenous catheter/line, recent history of a surgical procedure, and administration of immunosuppressive therapy. Sera of healthy subjects were included as controls to derive baseline values. All the sera were stored at -20C until used. The study was approved by the Committee for Protection of Human Subjects in Research, Faculty of Medicine. Kuwait University and informed written consent was obtained from the patients involved in the study. Candida species isolation and identification Blood samples were processed for isolation of Candida species by BACTEC 9240 system (Becton Dickinson, Paramus, N.J. USA) using aerobic culture bottles. Aliquots from blood culture bottles yielding yeast growth were subcultured on Sabouraud dextrose agar plates with chloramphenicol (40 mg/L). A single representative colony was processed for identification by germ-tube test and Vitek 2 yeast identification system (bio Merieux Marcy l'Etoile (...truncated)