Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Thu, 25 Feb 2016 19:55:00 Detection of Cryptococcus by conventional, serological and molecular methods Dolan Champa Saha, 1 Immaculata Xess, 1 Ashutosh Biswas, 2 Dipankar M. Bhowmik 3 and M. V. Padma 4 Correspondence Immaculata Xess immaxess@gmail.com 1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India 2 Department of Medicine, All India Institute of Medical Sciences, New Delhi, India 3 Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India 4 Department of Neurology, All India Institute of Medical Sciences, New Delhi, India Received 14 October 2008 Accepted 3 April 2009 The rising incidence of cryptococcosis in India is posing a serious threat. Due to lack of sensitive methods for diagnosis, high morbidity and mortality are associated with the disease. Early diagnosis is essential to prevent serious complications. Therefore, we attempted to find highly sensitive and specific detection methods. A comparative evaluation of the detection of cryptococcosis was done by conventional (direct microscopy and culture) and rapid diagnostic [latex agglutination test (LAT), enzyme immunoassay (EIA) and PCR] methods. The study was done on 359 samples from 52 positive patients and 30 negative controls in an Indian set-up. Evaluation was done for cerebrospinal fluid (CSF), serum and urine separately. The diagnostic value of the tests was assessed in pre-treatment samples, and follow-up tests were also done on samples obtained after initiation of treatment. PCR had the highest sensitivity, followed by EIA and LAT, both before and after treatment. The positive detection by LAT, EIA and PCR was the longest in CSF (.90 days), followed by serum (~65 days) then urine (~45 days) after initiation of treatment. Our results indicated that the sensitivity and specificity of PCR and EIA were comparable in urine, CSF and serum for diagnosis of cryptococcosis. INTRODUCTION Cryptococcus neoformans causes pulmonary infection and meningoencephalitis. The rising incidence of cryptococcosis in India is posing a serious threat (Banerjee et al., 1994, 2001, 2004; Banerjee, 2005; Chakrabarti & Gupta, 1997; Chakrabarti et al., 2000). With the onset of AIDS, its occurrence has escalated manyfold (Currie & Casadevall, 1994). Early diagnosis and institution of specific antifungal therapy are imperative to minimize the severity of infection. The laboratory diagnosis of cryptococcosis is based on direct demonstration, culture, and antigen detection by latex agglutination test (LAT). Microscopic methods and culture, though specific, show a sensitivity of 50–80% (Snow & Dismukes, 1975). Also, culture takes time and requires more labour and large volumes of samples. LAT is more sensitive but suffers from the limitation of false positivity (Boom et al., 1985; Heelan et al., 1991; Kornfeld & Worthington, 1980; MacKinnon et al., 1978; Millon et al., 1995; Sachs et al., 1991; Stoeckli & Burman, 2001; Whittier et al., 1994) as well as high rates of false negativity (Currie et al., 1993; Stamm & Polt, 1980; Coovadia & Solwa, 1987). The apparent subjectivity of reading and grading the LAT reaction poses additional problems, especially in borderline cases. Enzyme immunoassay (EIA) is another method for detection of C. neoformans capsular polysaccharide antigens in clinical samples. Studies have demonstrated the low cross-reactivity of this EIA (Casadevall et al., 1992; Frank et al., 1993; Scott et al., 1981; Sekhon et al., 1993). PCR offers an excellent alternative for the early diagnosis of cryptococcosis com- pared to conventional methods, as it is rapid, can detect low fungal load, and can be used for a small sample size (Paschoal et al., 2004; Imwidthaya & Poungvarin, 2000). Here, we have carried out a comparative study of conventional methods, LAT, EIA and PCR on a total of 359 samples from 82 patients. The comparative evaluation was done on cerebrospinal fluid (CSF), serum and urine samples separately. METHODS Standardization of diagnostic test systems. Normal saline and negative CSF, serum and urine were spiked with 10 3 –1 cell ml 21 of Cryptococcus neoformans (ATCC 24067 and clinical isolates), to establish the sensitivity of different test systems. For serum, only LAT, EIA and PCR were performed as per the standard protocol, while CSF and urine were tested by culture, direct microscopy, as well as by LAT, Abbreviations: CSF, cerebrospinal fluid; EIA, enzyme immunoassay; LAT, latex agglutination test. Journal of Medical Microbiology (2009), 58, 1098–1105 DOI 10.1099/jmm.0.007328-0 1098 007328 G 2009 SGM Printed in Great Britain