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 ~  Abstract
 ~  Materials and Me...
 ~  Extraction of DNA:
 ~  Amplification:
 ~  Electrophoresis:
 ~  Controls:
 ~  Results
 ~  Discussion
 ~  References

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SPECIAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 3  |  Page : 132-136
 

Polymerase chain reaction based diagnosis of systemic fungal infections and sensitivity testing of the fungal isolates


Department of Pathology and Microbiology, Sir Hurkisondas Nurrotamdas Hospital and Research Centre, Raja Rammohan Roy Road, Mumbai- 400 004, India

Correspondence Address:
Department of Pathology and Microbiology, Sir Hurkisondas Nurrotamdas Hospital and Research Centre, Raja Rammohan Roy Road, Mumbai- 400 004, India

 ~ Abstract 

PURPOSE: To highlight the usefulness of polymerase chain reaction (PCR) for the rapid diagnosis of systemic fungal infections. METHODS: Clinical samples were collected from 50 clinically suspected cases of systemic mycosis and subjected to smear, culture, antifungal sensitivity and PCR (based on 18S rRNA gene). RESULTS: Of the 50 clinical specimens tested by PCR, 39 were found to be positive. PCR gave more positive results than smear and culture examination. Out of the 50 clinical specimens 35 were found to be fungal culture positive. The sensitivity testing results of these fungal isolates showed that there was a good correlation between the in vitro results and the clinical response of the patient to antifungal therapy. Itraconazole exhibited maximum antifungal activity followed by fluconazole, ketoconazole and amphotericin B. CONCLUSIONS: PCR technology provides rapid and accurate diagnosis of fungal infection, however, it must be used with caution to avoid false positives.

How to cite this article:
Iyer R S, Pal R B, Patel R Y, Banker D D. Polymerase chain reaction based diagnosis of systemic fungal infections and sensitivity testing of the fungal isolates. Indian J Med Microbiol 2002;20:132-6


How to cite this URL:
Iyer R S, Pal R B, Patel R Y, Banker D D. Polymerase chain reaction based diagnosis of systemic fungal infections and sensitivity testing of the fungal isolates. Indian J Med Microbiol [serial online] 2002 [cited 2019 Sep 17];20:132-6. Available from: http://www.ijmm.org/text.asp?2002/20/3/132/6936


Fungi are opportunistic organisms, which are ubiquitous in nature. The last few decades have seen a steady increase in the incidence of systemic fungal infections especially those due to opportunistic fungi. Prolonged antibiotic therapy, invasive therapeutic procedures, immunosuppressive therapy and the Acquired Immunodeficiency Syndrome (AIDS) pandemic have all contributed to the rise in systemic fungal infections.[1],[2],[3] The conventional methods of diagnosis are time consuming and laborious. Molecular biology based methods on the other hand are not only rapid but more specific and sensitive. The Polymerase Chain Reaction (PCR) technology is being increasingly applied in the laboratory diagnosis of infections. Detection of fungal DNA by PCR enables early initiation of antifungal therapy and better therapeutic results. Here, we report the use of PCR in the rapid diagnosis of systemic fungal infections. We carried out the study in order to highlight the usefulness of the technique for the rapid diagnosis of systemic fungal infections. The conventional method of culture based identification of the fungus and antifungal sensitivity testing of the fungal culture isolates was also carried out. The PCR results were compared with the conventional culture based method of diagnosis.

 ~ Materials and Methods Top

A total of 50 clinical specimens from patients with clinically suspected systemic fungal infections were studied. The specimens were subjected to smear and culture studies. The specimens were microscopically screened for the presence of fungi using Gram stain, and lactophenol cotton blue and potassium hydroxide (KOH) mounts. Sabouraud dextrose agar with and without chloramphenicol and brain heart infusion agar supplemented with blood were used for culture studies. Cultures were identified using standard methods.[4],[5] Yeast cultures were identified on the basis of germ tube and chlamydospore formation and carbohydrate assimilation and fermentation reactions. The mould cultures were identified on the basis of the colony characteristics and microscopically using the slide culture technique.
Antifungal Susceptibility Testing:
Antifungal drug sensitivity testing of the isolated cultures was carried out by determining the minimum inhibitory concentration of four antifungal drugs namely amphotericin B, ketoconazole, fluconazole and itraconazole using the broth macrodilution method as described by the National Committee for Clinical and Laboratory Standards [NCCLS].[6],[7] Amphotericin B and itraconazole were obtained in pure form from HiMedia Laboratories and Janssen Research Foundation respectively. The pharmaceutical preparations of fluconazole and ketoconazole were used for the sensitivity testing. Stock solutions containing 1600 g/mL of the drugs in dimethyl sulphoxide were stored at - 20oC and used within one month. Buffered RPMI 1640 medium was used as the growth medium. Serial two-fold dilutions of the drugs were prepared so as to obtain concentrations ranging from 16 g/mL to 0.016 g/mL.
Polymerase Chain Reaction (PCR)
The methodology used for detecting the fungal DNA by the PCR is described below.

 ~ Extraction of DNA: Top

DNA was extracted from isolated fungal cultures as well as from clinical specimens using a lysis buffer containing Tris-HCL, EDTA, sodium dodecyl sulphate (SDS), proteinase K, lysozyme and Tween 20. All the chemicals required for DNA extraction were obtained from Sigma Chemicals. Equal quantities (100mL) of the culture suspension or the clinical specimen and the lysis buffer were mixed and incubated at 550C for 2 hours. Proteinase K was then inactivated, by heating the mixture to 950C for 10 minutes. DNA was then extracted by the phenol-chloroform method and precipitated with isopropanol. Briefly, 500 l of saturated phenol was added along with 250 l of a mixture of chloroform and isoamyl alcohol in the ratio 24:1 to the above mixture and centrifuged at 14000 rpm for 15 minutes at 4oC. The DNA was then precipitated from the aqueous phase using isopropanol. The DNA was dissolved in 15 mL of Tris-EDTA buffer for use as a template.

 ~ Amplification: Top

A 197 bp fragment of the 18S rRNA gene, which is common in all medically important fungi was amplified. Primers RIBIF - AGC TCT TTC TTG ATT TTG TGG and NS6 - GCA TCA CAG ACC TGT TAT TGC CTC, were used to achieve amplification.[8] The reaction was performed in a total volume of 50 mL with 10 mM TAPS, 1.5 mM MgCl2, 50mM KCl, 1mM of each dNTP, 0.02 mg/mL of each primer, 2.5U of Taq polymerase (Bangalore Genei Pvt. Ltd.) and 5 mL of the DNA template which was preheated to 95oC for 2 minutes. The amplification reaction involved an initial denaturation cycle of 940C for 5 mins followed by 30 cycles of 940C for 1 min, 550C for 2 mins, 720C for 2 mins and a final extension step of 720C for 7 mins. The primers were obtained from the National Chemical Laboratory, Pune.

 ~ Electrophoresis: Top

The amplified product was electrophoresed on a 2% agarose gel containing 0.5 mg/mL of ethidium bromide and the product size was verified [Figure] by comparison with Hinf I digested pBR-322 molecular size marker (Bangalore Genei Pvt. Ltd.).

 ~ Controls: Top

A set of four standard strains obtained from the Institute of Microbial Technology, Chandigarh, namely Candida albicans MTCC 227, C. tropicalis MTCC 184, Aspergillus fumigatus MTCC 2550 and Rhizopus arrhizus MTCC 2233 served as standards for determining the specificity of PCR. Besides, a set of 18 fungal cultures isolated from clinical specimens namely four strains each of Candida albicans and C. tropicalis, three strains each of Aspergillus spp. and Rhizopus spp., three stains of Fusarium spp. and one strain of Paecilomyces spp. were also used as positive controls.
A set of 10 clinical specimens from age and sex matched individuals not known to have fungal infections and a set of three bacterial cultures namely one strain each of  Escherichia More Details coli, Staphylococcus aureus and  Mycobacterium tuberculosis  i>served as negative controls. The positive and negative culture controls served to determine the specificity of the test. The sensitivity of the test was determined in comparison with the conventional culture method. A negative control, without target DNA which had gone through all the sample preparation steps was included with each lot of samples tested.

 ~ Results Top

The PCR analysis of the 50 clinical specimens showed the presence of the 197 bp product of fungal origin in 39 (78%) cases [Table - 1].
The standard fungal cultures as well as the other fungal isolates, which were tested for PCR, were found to be positive. PCR carried out with fungal primers on the DNA extracted from the 3 bacterial cultures did not yield any amplified product, indicating that the primers were specific for fungi. All the negative controls were found to be negative for PCR.
Factors predisposing to systemic fungal infections were recorded. Major factors that predisposed to systemic fungal infections in the study group included immunosuppressive therapy (16%), the Acquired Immunodeficiency Syndrome (26%), the use of broad-spectrum antibiotic treatment during post-operative care (44%) and diabetes (14%).
From the 50 clinical specimens 35 fungal isolates were obtained in culture [Table - 1]. Yeasts were the predominant isolates constituting 52% of the total isolates. Most of the isolates of Candida spp. were obtained from post-operative patients. Candida albicans constituted 14% followed by C.tropicalis (8%) and C. pseudotropicalis (2%) of all the clinical specimens cultured. All the isolates of Cryptococcus neoformans were from cases of cryptococcal meningitis in AIDS patients constituting 26% of the specimens cultured. All five isolates of Aspergillus spp. were obtained from the five renal transplant recipients on immunosuppressive drugs comprising 10% of the cultures isolated. Of these three isolates were obtained from cases with aspergilloma of the lung and two from fungal balls in the lung. All five Rhizopus isolates were obtained from zygomycosis patients with diabetes constituting 10% of the isolates.
The results of antifungal drug sensitivity testing are detailed in [Table - 2].

 ~ Discussion Top

The last few decades have recorded a remarkable rise in fungal infections, especially due to opportunistic fungi. Unfortunately, the big technological and therapeutic advances in the field of medicine have themselves become factors predisposing to these infections. Yeasts have been reported to be the common causative agents of opportunistic fungal infections[9] as was observed in this study. The fact that candidiasis is often encountered in critically ill surgical patients[10] was also noted by us. AIDS is an important predisposing factor for cryptococcosis[11],[12] as is diabetes to zygomycosis.[13]
The conventional culture based methods of diagnosis involve recovery of the fungus from the clinical specimen and identification of the fungal species, which is time consuming. As a result of greater stress being laid on rapid diagnosis of infections, molecular biology based methods are gaining importance. Molecular methods like PCR and the use of DNA probes specific for the fungi being tested are being developed for the rapid diagnosis of systemic fungal infections.[14] PCR was successfully used in the present study for diagnosis and confirmation of results. Early diagnosis is the key to proper treatment and favourable clinical outcome. The conventional methods of culture and identification of fungi would require a minimum of a week, whereas PCR results could be obtained in a few hours. PCR can detect extremely small quantities of DNA, i.e. 1-15 fungal cells with great accuracy[15] enabling diagnosis during the early stages of infection. Thus early diagnosis and appropriate treatment would help to reduce morbidity and mortality.
In the present study, we found 39 (78%) out of 50 clinical specimens to be positive by PCR as against 70% by culture, showing that the PCR assay was more sensitive. The assay was 100% specific as it amplified only fungal DNA, while DNA of non-fungal origin remained unamplified. There have been various reports in literature where PCR technology has been found to be highly specific and sensitive in the detection of fungal DNA.[16],[17] The fungal DNA could be detected in a variety of clinical specimens in our study and the method used for extraction of the fungal DNA was relatively simple. Most of the work on PCR for the diagnosis of fungal infections have been aimed at determining the DNA of Candida and Aspergillus.[18],[19],[20] Since the primers used in our study were common to the medically important fungi, DNA from a number of different species of fungi could be amplified. Further analysis of the amplified product by techniques like restriction enzyme analysis and enzyme immunoassay[21],[22] can facilitate speciation of the fungus without the use of expensive probes for the same.
The current applicability of in vitro antifungal sensitivity testing is limited to some extent due to inadequate standardization and insufficient correlation of in vitro results with clinical outcome.[23] However, the emergence of antifungal drug resistance has made susceptibility testing important.[24],[25],[26] Our results showed that itraconazole exhibited good activity against the isolates tested followed by fluconazole, ketoconazole and amphotericin B. In our study, we found a good correlation between the in vitro results and the clinical response of the patient to antifungal therapy. Infection with strains exhibiting in vitro resistance to the antifungal drugs resulted in mortality of the patients in spite of antifungal drug therapy. Patients infected with the sensitive fungal strains responded well to the treatment with the corresponding antifungal drug.
In conclusion, the PCR technology provides rapid and accurate diagnosis of fungal infections, facilitating early initiation of therapy. However, since fungal spores are common contaminants it is important to take necessary precautions to avoid false positive results. 

 ~ References Top

1.Advani SH. Incidence of fungal disease increasing in cancer patients. Fungal Challenge 1992;1:1-2.  Back to cited text no. 1    
2.Mohapatra LN. Study of Medical Mycology in India- an overview. Indian J Med Res 1989;89:351-61.  Back to cited text no. 2    
3.Diamond RD. The growing problem of mycoses in patients infected with the Human Immunodeficiency Virus. Rev Infect Dis 1991;13:480- 86.  Back to cited text no. 3    
4.Rippon JW. Medical Mycology. The pathogenic fungi and the actinomycetes. 2nd ed. (Philadelphia, WB Saunders) 1982:795.  Back to cited text no. 4    
5.Myers RM, Koshi G. Diagnostic procedures in medical microbiology and immunology/serology. (Microbiology Laboratories, Christian Medical College and Hospital) 1982:229-232.  Back to cited text no. 5    
6.National Committee for Clinical and Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard M27-A. NCCLS 1997;17:1-29.  Back to cited text no. 6    
7.National Committee for Clinical and Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium- forming filamentous fungi; Proposed standard M38-P. NCCLS 1998;18:1-21.  Back to cited text no. 7    
8.Walsh TJ, Francesconi A, Kasai M, Chanock SJ. PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi. J Clin Microbiol 1995;33:3216-20.   Back to cited text no. 8    
9.Doebeling BN, Hollis RT, Isenberg HD, Wenzel RP, Pfaller MA. Restriction fragment analysis of a Candida tropicalis outbreak of sternal wound infection. J Clin Microbiol 1991;29:1268-1270.  Back to cited text no. 9    
10.Cornwell EE, Belzberg H, Offne TV, Dougherty WR, Morates IR, Asensio J, Demetriades D. The pattern of fungal infections in critically ill patients. Am Surg 1995; 61:847-850.  Back to cited text no. 10    
11.Dismukes WE. Management of cryptococcosis. Clin Infect Dis 1993;17 (Suppl 2):507-512.  Back to cited text no. 11    
12.Deodhar L. Immunological diagnosis of cryptococcal meningitis in Human Immunodeficiency Virus infected patients. Indian J Med Microbiol 2000;18(2):85.  Back to cited text no. 12    
13.Harris LF, Hon JK, Schreeder MT. Mucormycosis: A community hospital perspective. Ala Med 1990;6:38, 40.  Back to cited text no. 13    
14.Chakrabarti A, Ravjot Kaur, Das S. Molecular methods in diagnosis and epidemiology of fungal infections. Indian J Med Microbiol 2000;18(4):146-152.  Back to cited text no. 14    
15.Makimura K, Mayurama SY, Yamaguchi H. Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 1994;40:358- 364.  Back to cited text no. 15    
16.Melchers WJG, Verweij PE, van den Hurk P, van Belkum A, De Pauw BE, Hoogkamp-Korstange JAA, Meis JFGM. General primer mediated PCR for detection of Aspergillus species. J Clin Microbiol 1994;32:1710-1717.  Back to cited text no. 16    
17.Sandhu GS, Kline BC, Stockman L, Robert GD. Molecular probes for diagnosis of fungal infections. J Clin Microbiol 1995;33:2913-2919.   Back to cited text no. 17    
18.James MJ, Lasker BA, Mc Neil MM, Shelton M, Warnock DW, Reiss E. Use of a repetitive probe to type clinical and environmental isolates of Aspergillus flavus from a cluster of cutaneous infections in a neonatal intensive care unit. J Clin Microbiol 2000;38:3612-3618.  Back to cited text no. 18    
19.Elie CM, Lott TJ, Reiss E, Morrison CJ. Rapid identification of Candida species with species specific probes. J Clin Microbiol 1998;36:3260-3265.  Back to cited text no. 19    
20.Williams DW, Wilson MJ, Lewis MA, Potts AJ. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J Clin Microbiol 1995;33:2476-2479.  Back to cited text no. 20    
21.Morace G, Sanguinetti M, Posteraro B, LoCasio G, Fadd G. Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis. J Clin Microbiol 1997;35:667-672.  Back to cited text no. 21    
22.Fujita S, Lasker BA, Lott TJ, Reiss E, Morrison CJ. Microtitration plate enzyme immunoassay to detect PCR amplified DNA from Candida species in blood. J Clin Microbiol 1995;32:962-967.  Back to cited text no. 22    
23.Galgiani JN, Rinaldi MG, Polak AM, Pfaller MA. Standardization of antifungal susceptibility testing. J Med Vet Mycol 1992;30(Suppl 1):213-224.  Back to cited text no. 23    
24.Maenza JR, Keruly JC, Moore RD, Chaisson RE, Merz WG, Gallant JE. Risk factors for fluconazole-resistant candidiasis in Human Immunodeficiency Virus-infected patients. J Infect Dis 1996;173:219-225.  Back to cited text no. 24    
25.Merz WG, Sandford GR. Isolation and characterization of a polyene-resistant variant of Candida tropicalis. J Clin Microbiol 1979;9:677-680.  Back to cited text no. 25    
26.Fotedar R, Banerjee U. Changing pattern of Candida species in a bone marrow transplant recipient. J Infect 1996;32:343-345.  Back to cited text no. 26    
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