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4. Costa AR, Porto E, da Lacaz S, de Melo NT, Calux M de JF, Valente NYS: Cutaneous and ungual phaeohyphomycosis caused by species of ChaetomiumKunze (1812) ex Fresenius, 1829. Journal of Medical and Veterinary Mycology 1988, 26: 261-268. 5. Hoppin EC, McCoy EL, Rinaldi MG: Opportunistic mycotic infection caused by Chaetomium in a patient with acute leukemia. Cancer 1983, 52: 555-556. 6. Arx JA, Figueras MJ, Guarro J: Sordariaceous ascomycetes without ascospore ejaculation. Beihefte zur Nova Hedwigia, 1988, 94: 1-104. 7. de Almeida F, Gomes JM, Salles C: Considera~6es s6bre una amostra de "Chaetomiurrf' isolada de una les&o epidarmica. Anais da Faculdade de Medicina da Universidade de S&o Paulo 1944, 20: 145-154. 8. Huppert M, Oliver DJ, Sun SH: Combined methenaminesilver nitrate and hematoxylin and eosin stain for fungi in tissues. Journal of Clinical Microbiology 1978, 8: 598603. 9. Van Cutsem J, Van Gerven F: Activit~ antifongique in vitro de I'itraconazole sur les champignous filamenteux opportunistes. Traitement de la keratomycose et de la p6nicilliose exparimentales. Journal de Mycologie M6dicale 1991, 118: 10-15. 10. Barale T, Fumey MH, Reboux G, Mallea M: Septicemie a Chaetomium sp. Iors d'une autogreffe de moelle pur leucose aigue lymphoblastique. Bulletin de la Soci~t~ Frangaise de Mycologie M~dicale 1990, 19: 43-46. 11. van Koch HA, Haneke H: Chaetomiumfunicolum Cooke als m6glicher Erreger einer tiefen Mykose. Mykosen 1965, 9: 23-28. 12. Barthez JP, Pierre D, de Bievre C, Arbeille M: Peritonite & Chaetomiumglobosum chez un insuffisant renal traite par D.P.C.A. Bulletin de la Sociat6 Frangaise de Mycologie M~dicale 1984, 13: 205-208. 13. Rippon JW: Medical mycology. The pathogenic fungi and pathogenic acetinomycetes. 3rd edition. Saunders, Philadelphia, 1988, p. 797. 14. Anandi V, Jacob JT, Walter A, Shastry JCM, Lalitha MK, Padhye AA, Ajello L, Chandler FW: Cerebral phaeohyphomycosis caused by Chaetomiumglobosumin a renal transplant recipient. Journal of Clinical Microbiology 1s 27: 2226-2229. 15. Naidu J, Singh SM, Pouranik M: Onychomycosis caused by Chaetomiumglobosum Kunze. Mycopathologia 1991, 113: 31-34. 16. Stiller MJ, Rosenthal S, Summerbell RC, Pollack J, Chan A: Onychomycosis of the toenails caused by Chaetomium globosum. Journal of the American Academy of Dermatology 1992, 26: 775-776.
Eur. J. Clin. Microbiol. Infect. Dis.
Detection of Pathogenic Fungi in Human Blood by the Polymerase Chain Reaction A . M . P o l a n c o , J.L. R o d r f g u e z - T u d e l a , J.V. M a r t f n e z - S u f i r e z *
The ability of the polymerase chain reaction (PCR) to detect pathogenic fungi in human blood was investigated. A DNA fragment of about 300 bp from the 18S rDNA, highly conserved in all fungi, was amplified with target DNA from 18 different species of fungi commonly isolated from clinical samples. The presence of PCR products was confirmed by hybridization with a fluorescein-labelled internal probe (21-mer). The PCR assay described is sensitive enough to detect 125 fg of purified Candida albicans DNAand 10 to 100 yeast cells per millilitre of blood.
During the last decade there has been an increase in reports of serious fungal infections, especially in immunocompromised and surgical patients. Candida spp. account for 10 to 15 % of hospitalacquired bloodstream infections, and the percentage has increased over the past decade by almost 500 % in large medical centres (1). Both morbidity and mortality are extremely high in these kind of infections. Mortality rates among fungaemia patients have been estimated at 50 % for Candida albicans (2) and can be even higher with other fungal groups (Candida glabrata, Fusarium spp.) (1, 2). Blood cultures remain the basic tool for diagnosing fungaemia. Nevertheless, they may fail to detect as many as 75 % of disseminated candidiasis cases (2). Even when blood cultures are positive, growth of fungal organisms requires several days before isolation and identification can be attempted. Serologic diagnosis of fungaemia is problematic since it is neither sensitive nor specific (2). New alternative methods are needed in order to allow prompt and appropriate treatment that could improve the prognosis of affected patients. Unidad de Micologfa, Centro Nacional de Microbiologfa, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
Vol. 14, 1995
In the present study, a method based on the polymerase chain reaction (PCR) amplification of funga118S rDNA sequences was tested for the detection of fungi in blood samples.
Materials and Methods. Strains of bacteria and Leishmania spp. from the American Type Culture Collection (ATCC) and fungal strains from our mycology unit collections were used (Table 1). The latter strains were isolated from clinical sources and identified by conventional methods (3). Purified lambda phage DNA was purchased from Pharmacia Biotech, Spain. All fungal strains were grown to early logarithmic phase in YEPD (1% yeast extract, 2 % peptone, 2 % dextrose) broth, harvested and washed in phosphatebuffered saline. For Candida spp. and Saccharomyces cerevisiae cells, spheroplasts were made by a method described previously (4) using 1,000 U/ml of [3glucuronidase (Sigma Quimica, Spain) as lytic enzyme for i h. Spheroplasts were lysed with 0.1% sodium dodecyl sulfate (SDS) and the DNA was purified by phenol-chloroform extraction and ethanol precipitation (5). For Cryptococcus neoformans, mutanase (Sigma) instead of [~glucuronidase was employed (6). DNA purification from mycelial fungi was achieved by a nonenzymatic method in which the mycelial tissue, previously frozen at -70~ was ground mechanically with glass beads (7). Genomic DNA was also obtained from human lymphocytes and bacterial and protozoan cells by conventional methods described previously (5). Blood was collected in tubes containing EDTA as an anticoagulant and was artificially inoculated with different numbers of Candida albicans cells. Candida albicans-spiked blood specimens were initially processed as described previously with non-ionic detergents for blood cell lysis and DNase I hydrolysis of human DNA (8). Subsequently, the yeasts were pelleted and DNA was prepared by the same protocol as for fungal cultures. The oligonucleotides used for amplification were the previously described primer system NS5 (5'A A C T T A A A G G A A T T G A C G G A A G - 3 ' ) and NS6 (5'-GCATCACAGACCTGTTATTGCCTC-3') (9). The oligonucleotide used as endlabelled probe was GL167 (5'-AGCGTTAATTCGTCTGTTTAG-3'), an internal sequence of the expected PCR product conserved in different fungi (10). All oligonucleotides were synthesized by standard phosphoramidite chemistry on a DNA synthesizer (Applied Biosystems, Spain).
Notes
619
Using 0.5 gM of each primer and i gl of the different dilutions of purified DNA or processed blood samples, PCR reactions were performed with 2.5 units of Taq DNA polymerase under standard conditions (Perkin-Elmer Hispania, Spain). Amplification was performed in a thermal cycler (Gene Ataq Controller, Pharmacia, Sweden) by using 40 cycles as follows: 1 min denaturation at 94~ 2 min annealing at 42~ and 3 min primer extension at 74~ After electrophoresis on 1.5 % agarose gels, the PCR products were transferred by Southern blotting onto a nylon membrane (Hybond-N+, Amersham Iberica, Spain) (5). Labelling of the oligonucleotide probe GL167 with fluorescein (ECL 3'-oligolabelling system, Amersham) hybridization, washing and detection by chemiluminiscence (ECL detection system) were performed according to the manufacturer's instructions. Results and Discussion. All 18 fungal species tested (Table 1) yielded a product of about 300 bp by PCR with primers NS5-NS6. Neither human (blood lymphocytes) nor microbial DNAs gave an amplified product with this pair of primers. In all cases amplimers were confirmed by hybridization with the fluorescein-labelled GL167 oligonucleotide probe corresponding to an internal and conserved sequence from the amplified region.
The samples were processed as described above, and purified Candida albicans DNA was added to these extracts in serial tenfold dilutions ranging from 125 ng to 1.25 fg. In all cases the limit of PCR detection was 125 fg.
Table 1: PCR results with DNA from different organisms. Positive (300 bp amplified product) Absidia corymbifera Aspergillus fumigatus Aspergillus versicolor Candida albicans Candida glabrata Candida guilliermondii Candida krusei Candida lusitaniae Candidaparapsilosis Candida tropicalis Candida zeylanoides Cryptococcus neoformans Curvularia lunata Fusarium solani Saccharomyces cerevisiae Scedosporium apiospermum Scedosporiumprolificans Trichosporon beigelii
Negative (no amplified product) Staphylococcus aureus Staphylococcus epidermidis Escherichia coil Klebsiella pneumoniae Pseudomonasaeruginosa Leishmaniaspp. Lambda phage Human lymphocytes
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Eur. J. Clin. Microbiol. Infect. Dis.
multiple copies per genome, which allow fungal DNA to be specifically detected with high sensitivity. We have chosen this last group with primers NS5NS6 (9), used previously by other authors (14), although to our knowledge this is the first time that blood samples have been tested with them. The specificity obtained was extremely high (100 %). We have also confirmed the specificity of the 300 bp amplimer by hybridization with an oligonucleotide probe within the amplified sequence. A good alternative method to conventional diagnosis of fungaemia must be rapid and sufficiently sensitive to allow the detection of less than 100 yeast cells per millilitre of blood (2). We obtained a high sensitivity when we processed blood samples in vitro, similar to that obtained by some authors (12-15) and higher than that obtained by others (8). Our findings suggest that PCR amplification methods for the different pathogens causing fungaemia are feasible, findings which could stimulate the development of a clinically useful test. Meanwhile, the role of PCR could be confirmatory and complementary to the traditional culture and identification of fungal organisms.
Acknowledgements Figure 1: PCR-amplified products from Candida albicans ATCC 64548-spiked blood samples in serial tenfold dilutions ranging from 108 cfu/ml (lane 3) to 10 cfu/ml (lane 10). Lane 1,100 bp ladder size-marker (Pharmacia); lane 2, fluorescein-labelled Hindlll digest of lambda phage DNA size marker (Amersham). (A) ethidium bromidestained agarose gel. (B) Southern blot probed with the fluorescein-labelled 21-mer GL167.
This work was supported in part by grants BIO94-0181 from the Comisi6n Interministerial de Ciencia y Tecnologfa and 93/0002/01 from the Fondo de Investigaciones Sanitarias, both of Spain. A.M.R is a fellow of the Fondo de |nvestigaciones Sanitarias of Spain. We thank Dr. Ingrid M. Outschoorn for the English version of the manuscript.
When blood samples were spiked with Candida albicans, the limit of PCR detection was between 10 and 100 cfu/ml (Figure 1).
References
Several previous studies have used PCR technology to detect fungi in clinical specimens. The primers used have amplified three kinds of target DNA: (i) Sequences coding for antifungal targets, such as an enzyme in the pathway for ergosterol biosynthesis (8), or fungal-specific regions of conserved proteins such as actin (11). Among the possible disadvantages of this approach are a lack of selectivity, as well as low sensitivity due to these sequences being present as single copy genes per haploid genome. (ii) Candida albicansspecific repeat sequences (2, 12). (iii) Highly conserved rDNA sequences (2, 13-15) present in
1. Pfaller M, Wenzel R: Impact of changing epidemiology of fungal infections in the 1990s. European Journal of Clinical Microbiology & Infectious Diseases 1992, 11: 287-291. 2. Reiss E, Morrison CJ: Non culture methods for diagnosis of disseminated candidiasis. Clinical Microbiology Reviews 1993, 6: 311-323. 3. McGinnis, MR: Laboratory handbook of medical mycology. Academic Press, New York, 1980, p. 103-410. 4. Forte MA, Fangman WL: Naturally occurring cross-links in yeast chromosomal DNA. Cell 1976, 8: 425-431. 5. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, p. 9.1-9.62. 6. Varma A, Kwon-Chung KJ: Rapid method to extract DNA from Cryptococcus neoformans. Journal of Clinical Microbiology 1991, 29: 810-812.
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7. Aufauvre-Brown A, Cohen J, Holden DW: Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. Journal of Clinical Microbiology 1992, 30: 2991-2993. 8. Buchman TG, Rossier M, Merz WG, Gharache P: Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery 1990, 108: 338-347. 9. White TJ, Bruns T, Lee S, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed): PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA, 1990, p. 315-322. 10. Neefs JM, Van der Peer Y, De Rijk P, Goris A, De Wachter R: Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Research 1991, 19, Supplement: 1987-1992. 11. Kan VL: Polymerase chain reaction for the diagnosis of candidemia.Journal of Infectious Diseases1993, 168: 338-347. 12. Miyakawa Y, Mabuchi T, Fukazawa Y: New method for detection of Candida albicans in human blood by polymerase chain reaction. Journal of Clinical Microbiology 1993, 31: 3344-3347. 13. Holmes AR, Cannon RD, Shepherd MG, Jenkinson HF: Detection of Candida albicans and other yeasts in blood by PCR. Journal of Clinical Microbiology1994, 32: 228231. 14. Hopfer RL, Walden P, Setterquist S, Highsmith WE: Detection and differentiation of fungi in clinical specimens using polymerasechain reaction (PCR) amplificationand restriction enzyme analysis. Journal of Medical & Veterinary Mycology 1993, 31: 65-75. 15. Makimura K, Murayama SY, Yamaguchi H: Detection of a wide range of medically important fungi by the polymerase chain reaction. Journal of Medical Microbiology 1994, 40: 358-364.
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Detection of HIV-1 Proviral Sequences in Lymphocytes Using a Qualitative Polymerase Chain Reaction Assay M . G . M a r i n 1., E Lillo 2, O . E . V a r n i e r 2, S. B r e s c i a n i 1, A . M o l i n e l l i 3, C. A b e c a s i s 2, R A . B o n i n i 2, A . A l b e r t i n i I
The performance and clinical relevance of a qualitative PCR-based assay for the detection of HIV-1 DNA sequences in peripheral blood mononuclear cells (PBMCs) was evaluated by two different laboratories. Four hundred and one samples were obtained from 397 individuals from different risk populations. All blood donors tested had negative results; positive signals were obtained from all infected patients. HIV-1 DNA was detected in 3 of 17 infants born to seropositive mothers; Western blot indeterminate blood donors and exposed healthcare workers had negative results. Our results demonstrate that this PCR assay provides both sensitive and specific results and is suitable for testing large numbers of samples and for rapid identification of HIV-1 infection.
The application of the polymerase chain reaction (PCR) as a laboratory technique represents a significant improvement in the diagnosis of diseases caused by infectious agents, including human immunodeficiency virus type I (HIV-1). Several studies have demonstrated that HIV-1 proviral sequences can be detected by PCR directly from cells obtained from infected patients (1, 2). Currently, HIV-1 infection is identified, using screening and confirmation tests, by demonstrating the presence of specific antibodies (3). Nevertheless, in some situations serological diagnosis is inadequate, as when determining the status of infectivity in individuals with an indeterminate Western blot (4) or in the diagnosis of infection in babies born to seropositive mothers, in l Institute of Chemistry,Schoolof Medicine,University of Brescia, III Laboratorio Analisi, Spedali Civili, R le Spedali Civili 1, 25123 Brescia, Italy. ZLaboratory of Virology, "Centro San Luigi;" IRCCS, Hospital S. Raffaele, Milan, Italy. 3Roche Diagnostic Systems,Milan, Italy.
