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J Med Microbiol 53 (2004), 1201-1206; DOI: 10.1099/jmm.0.45742-0
© 2004 Society for General Microbiology
ISSN 0022-2615

Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA

Muhammad Amjad1, Najla Kfoury1, Raymond Cha2,4 and Reem Mobarak3

1,2Clinical Laboratory Science Program1 and Department of Pharmacy Practice2, Eugene Applebaum College of Pharmacy and Health Services, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA 3,4Medical Research Program3 and School of Medicine4, Wayne State University, 540 E. Canfield Street, Detroit, MI 48201, USA

Correspondence Muhammad Amjad m.amjad{at}wayne.edu

Received May 11, 2004
Accepted August 12, 2004

Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1–10 c.f.u. ml–1 of the sample. No amplification was observed from up to105 heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Cryptococcus neoformans is an opportunistic human pathogen with worldwide distribution. It infects the central nervous system causing meningitis, which is fatal if untreated (Warren & Hazen, 1999). C. neoformans infections, as well as many other fungal diseases, start with the inhalation of airborne fungi from an environmental source. The infectious strain may then remain in a dormant phase in the host (Garcia-Hermoso et al., 1999; Goldman et al., 2001). As soon as some form of immune defect occurs, which for most patients is AIDS, the fungus is able to multiply, disseminate and cause infection (Abadi et al., 1999). Cryptococcosis is a leading, life-threatening, opportunistic infection in AIDS patients, with widespread dissemination involving the kidneys, lungs, skin and eyes (Saag et al., 2000). In some cases, individuals with no apparent immune defect can also develop cryptococcosis.

C. neoformans produces a prominent polysaccharide capsule, which, with the production of melanin, is an important virulence factor (Buchanan & Murphy, 1998). There are four C. neoformans serotypes based on the capsular antigen reaction (A–D), and three varieties have been defined: serotype A corresponds to the variety grubii, serotype D to the variety neoformans and variety gatti encompasses serotypes B and C (Franzot et al., 1999; Kwon-Chung & Bennett, 1984). The teleomorphic (sexual) reproductive phase of C. neoformans, which occurs in nature, constitutes the genus Filobasidiella. If the strains of serotype A or D mate with each other, Filobasidiella neoformans var. neoformans is the teleomorph (Warren & Hazen, 1999).

Cryptococcal meningitis is diagnosed by staining cerebrospinal fluid with India ink, by antigen detection or by culture method. But these methods can be difficult for other clinical specimens such as bronchoalveolar lavage fluids, lung biopsies or blood (Kralovic & Rhodes, 1998). While antigen detection and EIA (Gade et al., 1991) are sensitive and specific diagnostic tools, false-positive as well as false-negative results have been described, and they are unreliable for monitoring the efficacy of antifungal treatment (Blevins et al., 1995; Kralovic & Rhodes, 1998; Tintelnot et al., 2000), and recurrence of infection in AIDS and other immunocompromised patients.

Several PCR-based assays have been developed to detect and diagnose cryptococcal infection (Mitchell et al., 1994; Prariyachatigul et al., 1996; Rappelli et al., 1998; Tanaka et al., 1996). The amplification of a targeted DNA sequence by PCR does not, however, indicate the viability of the source organisms. Determination of the viability of the causative agent is important in determining the active infection and in the evaluation of the efficacy of a particular therapy. The amplification and detection of mRNA by RT-PCR has been used to assess the viability of several pathogenic micro-organisms (Kaucner & Stinear, 1998; Maher et al., 2001; Okeke et al., 2000).

In this study, we established a method of quantification and assessment of viability of C. neoformans by a one-step LightCycler amplification of capsule gene mRNA. The cryptococcal capsule plays an important role in the pathogenesis and the expression of mRNA indicates the presence and viability of the organism.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
C. neoformans isolates.

C. neoformans reference strain (ATCC 32045T), six clinical isolates and Candida spp. cultures were maintained on Sabouraud's dextrose agar (SDA) (Difco). Identification of all isolates was confirmed by conventional morphological and physiological methods (Warren & Hazen, 1999). Single colonies of C. neoformans cultures from SDA plates were inoculated into 5 ml Sabouraud dextrose broth (SDB), and were incubated at 30 °C for 48 h. The yeast cells were harvested by centrifugation at 3000 g at 4 °C for 5 min and were washed three times in PBS.

Microscopic organism enumeration and viability assessment.

Before RNA extraction the organism's density and viability were determined by fluorescence microscopy using live/dead yeast viability stains (Molecular Probes). An aliquot from the C. neoformans preparation was placed in 200 µl PBS with fluorescent dyes FUN1 and Calcofluor White M2R at final concentrations of 10 µM and 25 µM, respectively. This suspension was incubated at 30 °C for 30 min, after which the cells were examined by fluorescence microscopy. Dead cells exhibited diffuse yellow-green fluorescence while metabolically active viable yeast cells contain cylindrical, red-fluorescent structure in their vacuoles.

RNA extraction.

Total RNA from C. neoformans and Candida cultures were extracted by using a commercially available RNA extraction kit (Roche Applied Science). The harvested 5 x 107 yeast cells were resuspended in 200 µl PBS and 10 µl lyticase (0.5 mg ml–1) was added and incubated for 30 min at 30 °C to generate spheroplasts. Spheroplasts were lysed by lysis/binding buffer containing 4.5 M guanidine hydrochloride, 40 mM Tris/HCl and 30 % Triton X-100 (w/v) and RNA was extracted using High Pure collection tubes containing glass-fibre fleece according to the manufacturer's instruction. The genomic DNA (gDNA) was hydrolysed by adding 90 µl DNase (18.2 KU ml–1) in DNase buffer containing 1 M NaCl, 20 mM Tris/HCl and 10 mM MnCl2. The collection tube was washed three times with wash buffer containing 20 mM Tris/HCl and ethanol, and RNA was eluted in 50 µl RNase-free water. The concentration of RNA was calculated by measuring the absorbance at A260. The isolated RNA was used directly for RT-PCR analysis or was stored at –20 °C until used.

PCR primers and fluorescent hybridization probes.

Specific primer pairs and fluorescent probes were designed based on the sequence of C. neoformans CAP10 capsule gene mRNA (GenBank accession no. AF144574) (Chang & Kwon-Chung, 1999). The forward primer spanned the first exon–exon boundary of the gene, thus excluding the possibility of gDNA amplification. The forward primer was CAP10 F 5'-TCTTCTCTTGGTATTGAACACGTC-3', and the reverse primer was CAP10 R 5'-GGAAGAAAAAGTCTTCAGAAGGAG-3'. The first probe CAP10 FL 5'-ACGCCTCCCAGACGTTACCATC-3' was labelled with fluorescein. The second probe CAP10 LC 5'-CCTCGCCAATCGG GAATGAATT-p-3' was labelled with LightCycler Red 640 fluorophore (TIB Molbiol LLC). To ensure the specificity of the primers, an alignment of similar genes from related organisms was performed using GenBank BLAST.

Cloning and in vitro transcription of capsule gene mRNA.

A 223 bp portion of C. neoformans capsule gene mRNA was amplified by CAP10 primers. The PCR product was purified by using the Wizard PCR clean-up system (Promega). The purified DNA fragment was cloned into the pCR2.1 plasmid vector and transformed into One Shot cells using TOPO TA cloning kit (Invitrogen). The plasmid was isolated from transformed cells using High Pure plasmid isolation kit (Roche Applied Science). The purified linearized pCR2.1 containing T7 RNA polymerase promoter sites was subjected to in vitro transcription by adding 2 µl plasmid DNA to RNA polymerase mix along with nucleotides in the 20 µl final reaction volume and by incubation at 37 °C for 4 h (Ambion). The concentration of RNA was calculated from the A260 reading.

LightCycler RT-PCR assay and quantification of target RNA.

The one step LightCycler hot-start RT-PCR in glass capillaries was performed by a Tth DNA polymerase combined with Aptamers (Roche Applied Science). The DNA polymerase was a thermostable enzyme with RNA-dependent reverse transcriptase activity and DNA-dependent polymerase activity, allowing the combination of RT and PCR in a single tube. The RT-PCR mixture contained Tth DNA polymerase, reaction buffer, dNTP mixture, 0.5 µg total RNA, 0.5 µM concentration each of CAP10 primers, 0.2 µM each of fluorescein- and LC-Red 640-labelled probes and 50 mM Mn(OAc)2 in a volume of 20 µl. The reaction was carried out using the following thermal cycling program: one cycle of RT at 61 °C for 20 min, one cycle of cDNA/RNA hybrid denaturation at 95 °C for 30 s, 45 cycles of amplification of cDNA with melting at 95 °C for 1 s, annealing at 55 °C for 15 s and elongation at 72 °C for 13 s, and one final cycle of cooling and holding at 40 °C for 30 seconds. As a positive control a cytokine RNA (Roche Applied Science) containing a 322 bp fragment of in vitro-transcribed cytokine mRNA was amplified with specific primers (Roche Applied Science). The negative control contained PCR-grade water only instead of template RNA. The LightCycler products were resolved in a 1.0 % agarose gel in TBE buffer (Promega) at 100 V and were visualized and photographed after ethidium bromide staining.

Sensitivity and specificity of the LightCycler assay.

In order to determine the sensitivity of the assay and detect viable and dead cells, RNA extracted from serially diluted live and heat/UV radiation-killed C. neoformans cultures was used. As a control, a serial dilution of 2.2 pg µl–1 (106 copies) of a 223 bp fragment of an in vitro-transcribed capsular CAP10 mRNA was amplified using a specific pair of primers and hybridization probes labelled with LightCycler Red-640 and fluorescein. RNA isolated from several bacterial or fungal pathogens and human DNA/RNA was also subjected to LightCycler RT-PCR assay for the determination of specificity.


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
In recent years several rapid and sensitive diagnostic assays based on PCR, LightCycler and real-time PCR have been used to identify fungal pathogens (Luo & Mitchell, 2002) including C. neoformans (Bialek et al., 2002; Mitchell et al., 1994; Prariyachatigul et al., 1996; Rappelli et al., 1998; Tanaka et al., 1996). These methods are based on the detection of DNA of specific genes or unique ribosomal DNA (rDNA) sequences (Luo & Mitchell, 2002) encoding highly conserved 28S rRNA, 18S rRNA and 5.8S rRNA genes. All these methods were based on the detection of DNA sequences, which does not indicate the presence of active live organisms. The determination of the viability of the source organism is particularly important in several situations, for example in evaluating the efficacy of a particular therapy or the determination of recurrent or asymptomatic infection.

In order to overcome this problem several RT-PCR methods have been developed based on the detection of mRNA of a particular gene indicating viability of pathogenic micro-organisms (Kaucner & Stinear, 1998; Maher et al., 2001; Okeke et al., 2000, 2001). The rationale for the viability assay is that mRNA molecules, as opposed to DNA, are usually unstable following the death of an organism and indicate the viability of the organism. Regular quantitative RT-PCR has been used to detect and assess the viability of Pneumocystis carinii targeting heat-shock protein 70 mRNA (Maher et al., 2001), Giardia cysts and Cryptosporidium oocysts (Kaucner & Stinear, 1998). A LightCycler-based two step RT-PCR method has been used to quantify and assess the viability of Candida albicans by amplification of the actin mRNA gene (Okeke et al., 2001).

In this study C. neoformans capsule mRNA-specific primers and probes were designed to detect, quantify and assess the viability of the yeast cells by a real-time LightCycler RT-PCR assay. Among several genes involved in the capsule formation we tested primers designed from CAP10 gene mRNA (Chang & Kwon-Chung, 1999). Primers CAP10 F and CAP10 R and LightCycler fluorogenic probes detected C. neoformans ATCC 32045T and six clinical isolates (Fig. 1a), whereas no amplification was observed with RNA isolated from different bacterial and fungal pathogens and human genomic DNA or RNA (Figs 1b and 2). Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp (Fig. 2).



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  		Fig. 1. (a) Evaluation of LightCycler assay for detection and quantification of C. neoformans ATCC 32045T and six clinical isolates, and (b) control organisms using capsular CAP10 gene mRNA primers.

 


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  		Fig. 2. Agarose gel electrophoresis of LightCycler-generated products of C. neoformans and control organisms. Lanes: 1, C. neoformans ATCC 32045T; 2–7, C. neoformans clinical isolates; 8–16, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Candida glabrata, Rhodotorula rubra, Klebsiella pneumoniae, Escherichia coli and human genomic DNA; M, molecular mass markers.

 

In order to determine the sensitivity of the assay, RNA extracted from serially diluted cultures of C. neoformans with known c.f.u. ml–1 was subjected to LightCycler amplification using specific primers and probes (Fig. 3). As a control, a serial dilution of 2.2 pg µl–1 (106 copies) of a 223 bp fragment of an in vitro-transcribed capsular CAP10 mRNA was amplified using specific primers and hybridization probes labelled with LightCycler Red-640 and fluorescein. The number of RNA copies was determined by using the calculated molecular mass of in vitro-transcribed capsular CAP10 mRNA, converting to moles and multiplying with Avagadro's number (6.02 x 1023). The amplification of in vitro-transcribed capsular CAP10 mRNA was monitored by the log of concentration versus cycle number and a standard curve was plotted (Fig. 4). Copy number and quantity of C. neoformans mRNA was calculated and quantified from the standard curve. It was found that LightCycler RT-PCR detected as little as 0.472 fg capsule mRNA corresponding to 1–10 C. neoformans c.f.u. ml–1 in the samples (Fig. 3a). The assessment of viability of C. neoformans was determined by assaying tenfold serial dilutions containing 105, 104, 103, 102 and 101 live or heat/UV-killed yeast cells ml–1. The loss of viability and viable cell counts were performed by fluorescent microscopy, and RNA isolates were amplified. Amplification of the gene was observed in samples containing as little as 101 c.f.u. ml–1 of the live yeast cells (Fig. 3a), whereas no amplification was observed in any of the heat/UV-killed samples (Fig. 3b).



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  		Fig. 3. LightCycler amplification: (a) live C. neoformans; (b) dead C. neoformans. Agarose gel electrophoresis: (c) live C. neoformans; (d) dead C. neoformans; M, molecular mass markers.

 


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  		Fig. 4. (a) Detection and quantification of serially diluted standard in vitro-transcribed RNA by the LightCycler RT-PCR System. (b) Standard curve. (c) Agarose gel electrophoresis of the amplified product of the standard RNA. Lanes: 1, 2.2 pg µl–1 (106 copies); 2, 0.22 pg µl–1 (105 copies); 3, 0.022 pg µl–1 (104 copies); 4, 0.0022 pg µl–1 (103 copies); 5, 0 pg µl–1; M, molecular mass marker.

 

The potential of fluorescence-based real-time RT-PCR quantification of mRNA is increasing with the emergence of better RNA isolation procedures, enzymes, chemistries and instrumentation (Bustin, 2002; Peters et al., 2004). Besides detection and quantification of infectious agents (Okeke et al., 2001) and drug-resistance gene expression of infectious agents (Frade et al., 2004; Okeke et al., 2001) real-time RT-PCR has also been used in the molecular assessment of tumour stage (Bustin, 2000; Gelmini et al., 2003), monitoring the response to cancer treatment (Miyoshi et al., 2002), analysis of tissue-specific gene expression (Bustin, 2000) and cytokine mRNA profiling (Stordeur et al., 2002).

In our study the detection of capsule mRNA by a single step LightCycler RT-PCR represents a sensitive, specific and quantitative means of assessing the viability of C. neoformans as compared to PCR methods targeting DNA and rRNA genes. This LightCycler-based real-time RT-PCR assay can be useful in the detection and quantification of viable yeast cells in clinical specimens and in the evaluation of therapeutic efficiencies of antifungal drugs in the treatment of C. neoformans meningitis, especially in immunocompromised and AIDS patients. Further work is in progress to determine the efficacy of antifungal agents and quantify the viable C. neoformans in an in vitro pharmacodynamic model.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
We would like to thank Dr Michael A. Pfaller of the University of Iowa for providing the clinical isolates of C. neoformans, and VA Hospital, Detroit, MI for the use of the LightCycler equipment.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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