Medical Mycology, 2014, 52, 528–534 doi: 10.1093/mmy/myu017 Advance Access Publication Date: 9 June 2014 Original Article
Original Article
Evaluation of noninvasive methods for the diagnosis of fungal endocarditis Parisa Badiee1,∗ , Ahmad Ali Amirghofran2 and Mohammad Ghazi Nour2 1
*To whom correspondence should be addressed. E-mail: [email protected] Received 25 November 2013; Revised 19 January 2014; Accepted 25 February 2014
Abstract Fungal endocarditis (FE) is an uncommon disease with a high risk of morbidity and mortality. Here, we evaluated the different methods for diagnosing this infection. Cardiac valve, vegetation, and embolic materials obtained during surgery were examined for fungal infections by direct smear and culture. At least two blood samples were inoculated at the bedside into BACTEC medium. Galactomannan, mannan Ag enzyme-linked immunosorbent assay, and real-time polymerase chain reaction (PCR) assay were performed with serum samples. Of 25 patients with suspected infective endocarditis (IE), 8 were found to have proven FE according to the direct culture results. The etiologic agents were Aspergillus niger (three cases), A. flavus (two cases), A. fumigatus (one case), and Candida albicans (two cases). Blood culture was positive in only 1 case. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios of the results from the galactomannan test were 83.3%, 84.2%, 62.5%, 94.1%, 5.3, and 0.2; these same values, obtained from real-time PCR, were 87.5%, 94.4%, 87.5%, 94.4%, 15.6, and 0.14, respectively. Because mannan antigen was positive in samples from only one patient, we opted not to calculate the sensitivity. However, the specificity value in 23 cases without IE caused by Candida spp. was 100%. Based on our results, both the galactomannan test and real-time PCR can serve as reliable, noninvasive tests for the diagnosis of FE, compared with culture, which is considered to be the gold standard. Key words: fungal endocarditis, real-time PCR, fungal culture, galactomannan Ag test, mannan Ag test.
Introduction Fungal endocarditis (FE) is a life-threatening infection with a poor prognosis that requires early diagnosis. Treatment includes surgical valve replacement and aggressive antifungal therapy [1]. Many factors, including prolonged use of indwelling catheters, anatomical cardiac conditions, exposure to multiple broad-spectrum antibiotics, intravenous drug abuse, and previous history of surgery, have been 528
implicated as possible causes of fungemia and FE. In addition, prosthetic heart valves are the most frequent risk factor for these infections, and a few cases have been reported after heart transplantation [2]. Clinical signs are nonspecific and, as with other invasive fungal infections, the diagnosis of FE remains a challenge. Diagnosis is based on the isolation of etiologic agents from cardiac and blood samples or specimens from other
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
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Professor Alborzi Clinical Microbiology Research Center, Shiraz, Iran and 2 Cardiothoracic Surgery Unit, Shiraz University of Medical Sciences, Shiraz, Iran
Badiee et al.
Materials and methods Sample collection Patients with suspected clinical IE from two major university hospitals in Shiraz, Iran (Nemazee and Shahid Faghihi), were enrolled in the study between November 2009 and May 2011. Fungal infections were classified according to the Duke criteria [14], based on clinical, microbiological, and echocardiographic findings. In the present study, all patients with a vegetation on echocardiography who did not respond to antibacterial or antifungal agents and needed surgery were selected. Samples were examined at the Professor Alborzi Clinical Microbiology Research Center, Shiraz.
Culture The cardiac valve, vegetation, and embolic materials obtained during surgery were examined for fungal infection
by direct smear with potassium hydroxide and culture on Sabouraud glucose agar (Merck, Darmstadt, Germany). The plates were incubated at 30◦ C for 7–14 days. Identification was based on macroscopic and microscopic features of the isolated fungi. At least two blood samples were inoculated at bedside into BACTEC medium (Becton-Dickinson, Sparks, MD, USA).
Serologic examination GM and mannan Ag EIA tests were performed with the patients’ serum samples. Platelia Aspergillus EIA and Platelia Candida Ag (Bio-Rad Laboratories, Marnes-la-Coquette, France) were used to detect Aspergillus GM antigen and Candida mannan antigen based on an immune enzymatic sandwich microplate assay in serum, according to the manufacturer’s instructions. The optical absorbance of specimens and controls was determined with a microplate spectrophotometer equipped with 450-nm and 620-nm filters (Multiskan Ascent ELISA; Thermo Labsystems, Finland). Sera with a mannan concentration ≥0.5 ng/ml were considered positive. GM was reported as an index of optical density when two aliquots from the same sample had an optical density ≥0.5.
Real-time PCR For real-time PCR assay, 3–5 ml of serum was collected and stored at −20◦ C until PCR testing was done. Realtime PCR was conducted as previously described [15,16]. To avoid potential contamination, all samples were handled under sterile conditions in a laminar flow cabinet. For DNA extraction from the serum, a QIAmp DNA Mini kit (Qiagen, Hilden, Germany) was used in accordance with the manufacturer’s recommendations. Fungal amplification primers and probes and thermal cycling conditions for all Candida spp. and C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei and fluorogenic probes were designed by Shin and coworker [17]. In the first step, a blood sample was tested with all Candida spp. probes (universal). If it was positive, testing was continued in order to identify the Candida species. The probe and primers for Aspergillus used in this study and the setup, which could identify all Aspergillus spp., were described previously [18]. All primers and TaqMan probes were from Metabion (Martinsried, Germany). DNA from 200 µl of the patients’ sera was extracted using the QIAmp DNA Minikit (Qiagen); the elution volume was 50 µl. The Gene Amp 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) with TaqMan universal PCR master mix (Roche, Branchburg, NJ, USA) and 0.2 μmol/l of each primer and 0.2 μmol/l of
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sterile sites, as well as positive histopathological findings. Aspergillus spp. and Candida spp. are the most frequent etiologic agents of FE. Unfortunately, recovery of Aspergillus spp. from blood cultures is rare [3], and tests for Candida spp. detect only 45%–75% of all cases of systemic candidiasis [4,5]. Efforts have been made to develop a simple, objective, more sensitive and specific method that can serve as a better diagnostic assay than routine microbiological methods. This is of particular importance in situations when culture fails or when the sensitivity of histopathological identification is low [6]. Serological diagnostic methods such as the detection of mannan and galactomannan (GM) can be useful for the detection of fungal infections [7–10]. Mannan is the major component secreted by Candida spp., especially C. albicans. GM is a specific Aspergillus cell wall polysaccharide that is released during aspergillosis. The GM enzyme immunoassay (GM EIA) kit, which uses the rat monoclonal antibody EBA-2 against the Aspergillus GM antigen [8], can serve as a noninvasive method for the diagnosis of Aspergillus infections, but false positive results have been reported [11]. Molecular diagnostic tools for detecting fungal nucleic acid have resulted in high detection rates and can be used to identify specific species [12,13], but to date few studies of molecular tools for the diagnosis of FE have been conducted. The aim of this study was to evaluate GM EIA, mannan EIA, real-time polymerase chain reaction (PCR), and culture of blood and tissue samples for the diagnosis of FE in patients with suspected infective endocarditis (IE).
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Ethical considerations The Clinical Microbiology Research Center Ethics Committee, Shiraz University of Medical Sciences, approved this study, which was carried out in accordance with the 1975 Declaration of Helsinki, as revised in 1983. Written informed consents were obtained from all patients prior to the study.
Results During this 19-month study, 25 patients with large vegetations in echocardiography and clinical signs such as fever (38◦ C–38.5◦ C), leg and arm pain, finger gangrene, tachycardia, left pleural effusion, history of previous surgery (one to four), previous mitral valve regurgitation, or aortic valve regurgitation were entered into the study. Mean age of the patients was 35.5 years, the male to female ratio was 15:10, and the mean number of previous operations was two. Of the 25 patients with suspected IE, 8 had proven FE based on the tissue culture results. The etiologic agents were Aspergillus spp.in six cases (A. niger, three cases; A. flavus, two cases; A. fumigatus, one case) and C. albicans in two cases. Blood culture was positive in only one case with C. albicans (case 5; Table 1), and the Candida species isolated from the blood and tissue samples was the same. The GM antigen test was positive in 5 of 6 patients with documented aspergillosis and in 3 of 19 patients without signs or symptoms of Aspergillus endocarditis. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios for the GM test were 83.3%, 84.2%, 62.5%, 94.1%, 5.3, and 0.2, respectively.
The PCR assay for the diagnosis of Aspergillus and Candida was positive for seven of eight patients with FE; one patient with a negative culture was positive with molecular methods. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios for the PCR test were 87.5%, 94.4%, 87.5%, 94.4%, 15.6, and 0.14, respectively. Because mannan antigen was positive in only one patient, we opted not to calculate the sensitivity, but the specificity value in 23 cases without IE caused by Candida spp. was 100%. The characteristics of the patients and their corresponding clinical and laboratory data are presented in Tables 1 and 2.
Discussion The number of fungal infections with high rates of morbidity and mortality has been increasing in recent years and are difficult to diagnose in some patients. In this study, the rate of FE was found to be 32% because the sample collection criteria included only those patients with suspected IE who were not responsive to antibacterial or antifungal agents and had undergone surgical procedures. The purpose of this study was to compare various diagnostic methods with culture. Consequently, we needed to surgically obtain tissue specimens from the infected sites of patients. Since medical therapy and surgical intervention are needed in cases of Aspergillus FE, its rate was high in this study because we were able to obtain the appropriate clinical samples. FE accounts for 1.3%–6% of all IE cases [19], and 1%–10% of these patients have a prosthetic valve [20]. Aspergillus FE has increased during the last two decades [21], and the incidence rates of infection, as assessed by Rubinstein et al. using culture [22] and by Challa et al. using autopsy results [23], were reported to be 12%–20% and 37.5%, respectively. While early diagnosis and therapy can lead to good clinical outcomes, the use of blood culture to diagnose FE is not reliable, and use of culture of material from the infected site remains the “gold standard.” However, this method is invasive, requires major surgery for sample collection, and is not possible in patients in poor condition. The time between sampling and obtaining the results of culture may be as long as 7–10 days, which may be too long for the introduction of effective treatment. Blood culture is a major Duke criteria for the diagnosis of IE. Unfortunately, the sensitivity of this method for the isolation of fungi is low [3,4]. Therefore, these methods cannot identify the etiologic agent in the early stages of illness. Serological and molecular methods may be more accurate and reliable for the diagnosis of these infections. Candida spp. are most frequently responsible for FE. The detection of Candida mannan antigen is a reliable, sensitive, and noninvasive serological method for the diagnosis
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each species probe were used for samples analysis (5 µl of template volume in a final PCR volume of 20 µl). Thermal cycling conditions consisted of heating at 94◦ C for 10 min prior to a two-stage temperature profile of 30 s at 95◦ C and 90 s at 60◦ C for 40 cycles. The DNA extracted from A. fumigatus and C. albicans isolates was used as the positive control, and distilled water was used as the negative control in the real-time PCR assay. To determine the sensitivity of the real-time PCR assay, 100 copies/well of each A. fumigatus and C. albicans DNA was serially diluted and measured. The limitation of PCR assay was 10 copies/well. Statistical analyses were performed using SPSS software for Windows (version 15.0; SSPS Inc., Chicago, IL, USA). Negative control participants were selected from patients with the same conditions. Hence, patients with negative results for fungal elements in the microscopic examination or negative results in fungal cultures were considered to be the negative control group for the present study.
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17/F
4/M
50/M
27/F
31/M
67/F
19/M
21/M
1
2
3
4
5
6
7
8
IV drug abuser
Previous operation (3)
Previous operation (1)
IV drug abuser
Previous operation (2)
Previous operation (2)
Previous pacemaker insertion
Previous operation (3)
Risk factor
F, female; IV, intravenous; M, male.
Age (y)/sex
Large vegetation
Large mobile vegetation, Para, new valvular regurgitation
Large vegetation
Large mobile vegetation, para valvular abscess
Large vegetation
Large vegetation, new valvular regurgitation
Large vegetation, new valvular regurgitation
Large vegetation, para valvular abscess
Major Duke criteria
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Fever >38 C, septic pulmonary infarct Fever >38 C
Fever >38 C
Fever >38 C, conjuctival hemorrhage, glomerunephritis Fever >38 C, IV drug abuser Fever >38 C, prostatic heart valve, conjuctival hemorrhage Fever >38 C, conjuctival hemorrhage, septic pulmonary infarct Fever >38 C, IV drug abuser, septic pulmonary infarct
◦
◦
◦
◦
◦
◦
◦
◦
Potassium hydroxide solution
Minor Duke criteria
0.1
0.85
0.35
0.2
2.0
0.65
0.8
5.1
Galacto mannan
0.65
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Mannan test
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Case number
Table 1. Characteristics of patients with corresponding data for fungal endocarditis diagnosis. Tissue culture
Aspergillus flavus
A. flavus
A. fumigatus
A. niger
Candida albicans
A. niger
A. niger
C. albicans
Polymerase chain reaction result A+ C–
A+ C–
A+ C–
A+ C–
A– C–
A+ C–
A+ C–
A– C+
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Blood culture
Badiee et al. 531
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Medical Mycology, 2014, Vol. 52, No. 5
Table 2. Characteristics of patients with negative results for fungal endocarditis. Case number
Age (y)/sex
Major Duke criteria
Minor Duke criteria ◦
Fever >38 C, septic pulmonary infarct ◦ Fever >38 C, IV drug abuser
30/M
Large mobile vegetation
Fever >38 C, IV drug abuser, septic pulmonary infarct
5
29/M
Large vegetation
Fever >38 C, prostatic heart valve, conjuctival hemorrhage
6
67/M
Fever >38 C, glomerunephritis
7
54/M
Large vegetation, new valvular regurgitation Large vegetation, para valvular abscess
8
23/M
Fever >38 C, IV drug abuser
9
31/M
10
54/M
11
21/M
12
31/M
13
48/M
14
23/M
Small mobile vegetation, para valvular abscess Large vegetation, para valvular abscess Large vegetation, new valvular regurgitation Para valvular abscess, new valvular regurgitation Large mobile vegetation, para valvular abscess Small vegetation, para valvular abscess Large vegetation
15
32/M
Large mobile vegetation
16
18/F
17
52/M
Large mobile vegetation, new valvular regurgitation Large mobile vegetation
40/F
2
45/F
3
58/M
4
◦
Fever >38 C
◦
◦
◦
◦
Fever >38 C
◦
◦
Fever >38 C, IV drug abuser ◦
Fever >38 C, septic pulmonary infarct ◦ Fever >38 C ◦
Fever >38 C, IV drug abuser ◦
Fever >38 C ◦
Fever >38 C, prostatic heart valve, septic pulmonary infarct ◦ Fever >38 C, IV drug abuser, conjuctival hemorrhage ◦ Fever >38 C, prostatic heart valve ◦
Fever >38 C, prostatic heart valve, conjuctival hemorrhage
Chest pain, malaise, dry cough Malaise, anorexia, changing murmur, positive CRP test Malaise, chill, pacemaker endocarditis, repair of tricuspid valve Distraction of tricuspid valve, chest pain, left-side pleural effusion, multiple embolic lesions in lung Distraction of tricuspid wall, perforation of septum below the valve, cardiomegaly Elevated ESR, splenomegaly, dyspnea Diabetic mellitus, general body pain, positive CRP test, elevated ESR Edema, malaise, weight loss Chill, cough, malaise, cardiomegaly Diabetic mellitus, cardiomegaly, bilateral severe pleural effusion Malaise, weight loss Cardiomegaly, elevated ESR, positive CRP test Diabetic mellitus, chill, cardiomegaly Chest pain, dyspnea, cough, night sweats Night sweats, malaise, weight loss Edema, malaise, weight loss Malaise, weight loss
CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; F, female; IV, intravenous; M, male.
of systemic candidiasis [7,9,24], with an overall sensitivity of 90.9% and specificity of 46.2% [10]. In one study it was noted that Candida mannan antigen is rapidly cleared from the patient’s serum [25]. In the present study, the mannan antigen test was positive only with samples from one patient with Candida endocarditis and negative in all other patients (the specificity of this test was 100%). In this investigation, only two patients had Candida infection, and the result was positive in one of them. These low numbers prevent a discussion of the implications of this single positive result. However, based on specificity, in the patients
with suspected systemic candidiasis and negative results for the infection, systemic candidiasis could be ruled out. The detection of Aspergillus GM in serum has been reported to be a valuable tool for the diagnosis of invasive aspergillosis. Several factors affect the accuracy of this test, as high rates of false-positive results were reported in patients treated with amoxicillin–clavulanate [26], piperacillin–tazobactam, amoxicillin, and all fermentation products of Penicillium spp. [27], as well as in patients who received the intravenous electrolyte solution Plasmalyte [28,29]. In some instances, fungi other than
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Large mobile vegetation, para valvular abscess Large mobile vegetation, new valvular regurgitation Large vegetation, new valvular regurgitation
1
Other clinical signs and symptoms
Badiee et al.
Conclusion The rate of FE is low in clinical practice. Consequently, additional studies that involve different samples and different populations are needed in order to improve the accuracy of diagnostic methods and make the prompt diagnosis of this potentially life-threatening problem possible. Until these studies are completed, we suggest that for patients with a history of surgery, heart valve vegetation be sampled
and noninvasive methods that incorporate the GM antigen and PCR tests be used to improve the chances of detecting and identifying the etiologic agent in a timely manner. Despite some limitations, the combination of these two noninvasive methods can increase sensitivity and specificity and, in turn, facilitate accurate diagnosis and earlier initiation of specific therapy.
Acknowledgments Our thanks are due to Hassan Khajehei for his help with linguistic copyediting and to K. Shashok (AuthorAID in the Eastern Mediterranean) for improving the use of English in the manuscript.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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Aspergillus caused the infections [30]. In the current study, three patients with negative findings in both culture and molecular assays were GM antigen positive, probably due to use of antibacterial agents. The usefulness of the GM antigen test is reportedly related to population characteristics. For instance, there are reports of high sensitivity and specificity in patients with neutropenia and in stem cell transplant recipients [31,32]. There have been few investigations of this method for the diagnosis of FE. In one study of the use of the GM test to establish a diagnosis of aspergillosis FE, the result was positive (≥1 ng/ml) in only two of seven patients with endocarditis and mediastinitis [33]. In another study, only four of nine patients with Aspergillus endocarditis had positive GM test results [34]. Racil et al. found the sensitivity, specificity, and positive and negative predictive values of GM for invasive aspergillosis to be 88.2%, 95.8%, 75%, and 98.3%, respectively [28]. These values are close to the ones we obtained in our sample of patients Several researchers have reported the results obtained with molecular assays to detect invasive fungal infection [12,35] and Aspergillus endocarditis [36]. The sensitivity and specificity of molecular assays vary depending on the type of test (real-time PCR, nested PCR, PCR–enzymelinked immunosorbent assay). As with other diagnostic methods, molecular methods have limitations such as cost, lack of standardization at all laboratories, and potential contamination from environmental sources. The signs and symptoms of fungal infections are nonspecific, and the interpretation of radiological findings to distinguish between fungal and bacterial endocarditis can be problematic. These potential obstacles to a prompt, accurate diagnosis make new methods for diagnosing FE advisable. Based on the results our study, both the GM antigen test and real-time PCR can serve as reliable noninvasive tests for this purpose, compared with culture, which is considered to be the gold standard. However, because the positive likelihood ratio for real-time PCR is higher and the negative likelihood ratio is lower than with the GM antigen test, PCR-based methods are more valuable for the diagnosis of FE.
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11. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin Infect Dis 2006; 15: 1417–1427. 12. Badiee P, Alborzi A, Joukar M. Molecular assay to detect nosocomial fungal infections in intensive care units. Eur J Internal Med 2011; 22: 611–615. 13. Yeo SF, Wong B. Current status of nonculture methods for diagnosis of invasive fungal infections. Clin Microbiol Rev 2002; 15: 465–484. 14. Li JS, Sexton DJ, Mick N et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000; 30: 633–638. 15. Badiee P, Alborzi P, Vojdani R et al. Early diagnosis of systemic candidiasis in bone marrow transplant recipients. Exp Clin Transplant 2010; 8: 98–103. 16. Badiee P, Alborzi A . Detection of Aspergillus species in bone marrow transplant patients. J Infect Dev Ctries 2010; 4: 511– 516. 17. Shin JH, Nolte FS, Holloway BP, Morrison CJ. Rapid identification of up to three Candida species in a single reaction tube by a 5 exonuclease assay using fluorescent DNA probes. J Clin Microbiol 1999; 37: 165–170. 18. Kami M, Fukui T, Ogawa S et al. Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis. Clin Infec Dis 2001; 33: 1504–1512. 19. Karchmer AW. Infections on prosthetic valves and intra vascular devices. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases. Philadelphia, PA: Churchill Livingstone, 2000; 903– 917. 20. Giamarellou H. Nosocomial cardiac infections. J Hosp Infect 2002; 50: 91–105. 21. Rubinstein E, Lang R. Fungal endocarditis. Eur Heart J 1995; 16: 84–89. 22. Rubinstein E, Noriega ER, Simberkoff MS, Holzman R, Rahal JJ, Jr. Fungal endocarditis: analysis of 24 cases and review of the literature. Medicine 1975; 54: 331–334. 23. Challa S, Prayaga AK, Vemu L et al. Fungal endocarditis: An autopsy study. Asian Cardiovasc Thorac Ann 2004; 12: 95–98. 24. Persat F, Topenot R, Piens MA et al. Evaluation of different commercial ELISA methods for the serodiagnosis of systemic candidosis. Mycoses 2002; 45: 455–460.
Medical Mycology, 2014, Vol. 52, No. 5
Original Article
Evaluation of noninvasive methods for the diagnosis of fungal endocarditis Parisa Badiee1,∗ , Ahmad Ali Amirghofran2 and Mohammad Ghazi Nour2 1
*To whom correspondence should be addressed. E-mail: [email protected] Received 25 November 2013; Revised 19 January 2014; Accepted 25 February 2014
Abstract Fungal endocarditis (FE) is an uncommon disease with a high risk of morbidity and mortality. Here, we evaluated the different methods for diagnosing this infection. Cardiac valve, vegetation, and embolic materials obtained during surgery were examined for fungal infections by direct smear and culture. At least two blood samples were inoculated at the bedside into BACTEC medium. Galactomannan, mannan Ag enzyme-linked immunosorbent assay, and real-time polymerase chain reaction (PCR) assay were performed with serum samples. Of 25 patients with suspected infective endocarditis (IE), 8 were found to have proven FE according to the direct culture results. The etiologic agents were Aspergillus niger (three cases), A. flavus (two cases), A. fumigatus (one case), and Candida albicans (two cases). Blood culture was positive in only 1 case. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios of the results from the galactomannan test were 83.3%, 84.2%, 62.5%, 94.1%, 5.3, and 0.2; these same values, obtained from real-time PCR, were 87.5%, 94.4%, 87.5%, 94.4%, 15.6, and 0.14, respectively. Because mannan antigen was positive in samples from only one patient, we opted not to calculate the sensitivity. However, the specificity value in 23 cases without IE caused by Candida spp. was 100%. Based on our results, both the galactomannan test and real-time PCR can serve as reliable, noninvasive tests for the diagnosis of FE, compared with culture, which is considered to be the gold standard. Key words: fungal endocarditis, real-time PCR, fungal culture, galactomannan Ag test, mannan Ag test.
Introduction Fungal endocarditis (FE) is a life-threatening infection with a poor prognosis that requires early diagnosis. Treatment includes surgical valve replacement and aggressive antifungal therapy [1]. Many factors, including prolonged use of indwelling catheters, anatomical cardiac conditions, exposure to multiple broad-spectrum antibiotics, intravenous drug abuse, and previous history of surgery, have been 528
implicated as possible causes of fungemia and FE. In addition, prosthetic heart valves are the most frequent risk factor for these infections, and a few cases have been reported after heart transplantation [2]. Clinical signs are nonspecific and, as with other invasive fungal infections, the diagnosis of FE remains a challenge. Diagnosis is based on the isolation of etiologic agents from cardiac and blood samples or specimens from other
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
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Professor Alborzi Clinical Microbiology Research Center, Shiraz, Iran and 2 Cardiothoracic Surgery Unit, Shiraz University of Medical Sciences, Shiraz, Iran
Badiee et al.
Materials and methods Sample collection Patients with suspected clinical IE from two major university hospitals in Shiraz, Iran (Nemazee and Shahid Faghihi), were enrolled in the study between November 2009 and May 2011. Fungal infections were classified according to the Duke criteria [14], based on clinical, microbiological, and echocardiographic findings. In the present study, all patients with a vegetation on echocardiography who did not respond to antibacterial or antifungal agents and needed surgery were selected. Samples were examined at the Professor Alborzi Clinical Microbiology Research Center, Shiraz.
Culture The cardiac valve, vegetation, and embolic materials obtained during surgery were examined for fungal infection
by direct smear with potassium hydroxide and culture on Sabouraud glucose agar (Merck, Darmstadt, Germany). The plates were incubated at 30◦ C for 7–14 days. Identification was based on macroscopic and microscopic features of the isolated fungi. At least two blood samples were inoculated at bedside into BACTEC medium (Becton-Dickinson, Sparks, MD, USA).
Serologic examination GM and mannan Ag EIA tests were performed with the patients’ serum samples. Platelia Aspergillus EIA and Platelia Candida Ag (Bio-Rad Laboratories, Marnes-la-Coquette, France) were used to detect Aspergillus GM antigen and Candida mannan antigen based on an immune enzymatic sandwich microplate assay in serum, according to the manufacturer’s instructions. The optical absorbance of specimens and controls was determined with a microplate spectrophotometer equipped with 450-nm and 620-nm filters (Multiskan Ascent ELISA; Thermo Labsystems, Finland). Sera with a mannan concentration ≥0.5 ng/ml were considered positive. GM was reported as an index of optical density when two aliquots from the same sample had an optical density ≥0.5.
Real-time PCR For real-time PCR assay, 3–5 ml of serum was collected and stored at −20◦ C until PCR testing was done. Realtime PCR was conducted as previously described [15,16]. To avoid potential contamination, all samples were handled under sterile conditions in a laminar flow cabinet. For DNA extraction from the serum, a QIAmp DNA Mini kit (Qiagen, Hilden, Germany) was used in accordance with the manufacturer’s recommendations. Fungal amplification primers and probes and thermal cycling conditions for all Candida spp. and C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei and fluorogenic probes were designed by Shin and coworker [17]. In the first step, a blood sample was tested with all Candida spp. probes (universal). If it was positive, testing was continued in order to identify the Candida species. The probe and primers for Aspergillus used in this study and the setup, which could identify all Aspergillus spp., were described previously [18]. All primers and TaqMan probes were from Metabion (Martinsried, Germany). DNA from 200 µl of the patients’ sera was extracted using the QIAmp DNA Minikit (Qiagen); the elution volume was 50 µl. The Gene Amp 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) with TaqMan universal PCR master mix (Roche, Branchburg, NJ, USA) and 0.2 μmol/l of each primer and 0.2 μmol/l of
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sterile sites, as well as positive histopathological findings. Aspergillus spp. and Candida spp. are the most frequent etiologic agents of FE. Unfortunately, recovery of Aspergillus spp. from blood cultures is rare [3], and tests for Candida spp. detect only 45%–75% of all cases of systemic candidiasis [4,5]. Efforts have been made to develop a simple, objective, more sensitive and specific method that can serve as a better diagnostic assay than routine microbiological methods. This is of particular importance in situations when culture fails or when the sensitivity of histopathological identification is low [6]. Serological diagnostic methods such as the detection of mannan and galactomannan (GM) can be useful for the detection of fungal infections [7–10]. Mannan is the major component secreted by Candida spp., especially C. albicans. GM is a specific Aspergillus cell wall polysaccharide that is released during aspergillosis. The GM enzyme immunoassay (GM EIA) kit, which uses the rat monoclonal antibody EBA-2 against the Aspergillus GM antigen [8], can serve as a noninvasive method for the diagnosis of Aspergillus infections, but false positive results have been reported [11]. Molecular diagnostic tools for detecting fungal nucleic acid have resulted in high detection rates and can be used to identify specific species [12,13], but to date few studies of molecular tools for the diagnosis of FE have been conducted. The aim of this study was to evaluate GM EIA, mannan EIA, real-time polymerase chain reaction (PCR), and culture of blood and tissue samples for the diagnosis of FE in patients with suspected infective endocarditis (IE).
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Ethical considerations The Clinical Microbiology Research Center Ethics Committee, Shiraz University of Medical Sciences, approved this study, which was carried out in accordance with the 1975 Declaration of Helsinki, as revised in 1983. Written informed consents were obtained from all patients prior to the study.
Results During this 19-month study, 25 patients with large vegetations in echocardiography and clinical signs such as fever (38◦ C–38.5◦ C), leg and arm pain, finger gangrene, tachycardia, left pleural effusion, history of previous surgery (one to four), previous mitral valve regurgitation, or aortic valve regurgitation were entered into the study. Mean age of the patients was 35.5 years, the male to female ratio was 15:10, and the mean number of previous operations was two. Of the 25 patients with suspected IE, 8 had proven FE based on the tissue culture results. The etiologic agents were Aspergillus spp.in six cases (A. niger, three cases; A. flavus, two cases; A. fumigatus, one case) and C. albicans in two cases. Blood culture was positive in only one case with C. albicans (case 5; Table 1), and the Candida species isolated from the blood and tissue samples was the same. The GM antigen test was positive in 5 of 6 patients with documented aspergillosis and in 3 of 19 patients without signs or symptoms of Aspergillus endocarditis. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios for the GM test were 83.3%, 84.2%, 62.5%, 94.1%, 5.3, and 0.2, respectively.
The PCR assay for the diagnosis of Aspergillus and Candida was positive for seven of eight patients with FE; one patient with a negative culture was positive with molecular methods. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios for the PCR test were 87.5%, 94.4%, 87.5%, 94.4%, 15.6, and 0.14, respectively. Because mannan antigen was positive in only one patient, we opted not to calculate the sensitivity, but the specificity value in 23 cases without IE caused by Candida spp. was 100%. The characteristics of the patients and their corresponding clinical and laboratory data are presented in Tables 1 and 2.
Discussion The number of fungal infections with high rates of morbidity and mortality has been increasing in recent years and are difficult to diagnose in some patients. In this study, the rate of FE was found to be 32% because the sample collection criteria included only those patients with suspected IE who were not responsive to antibacterial or antifungal agents and had undergone surgical procedures. The purpose of this study was to compare various diagnostic methods with culture. Consequently, we needed to surgically obtain tissue specimens from the infected sites of patients. Since medical therapy and surgical intervention are needed in cases of Aspergillus FE, its rate was high in this study because we were able to obtain the appropriate clinical samples. FE accounts for 1.3%–6% of all IE cases [19], and 1%–10% of these patients have a prosthetic valve [20]. Aspergillus FE has increased during the last two decades [21], and the incidence rates of infection, as assessed by Rubinstein et al. using culture [22] and by Challa et al. using autopsy results [23], were reported to be 12%–20% and 37.5%, respectively. While early diagnosis and therapy can lead to good clinical outcomes, the use of blood culture to diagnose FE is not reliable, and use of culture of material from the infected site remains the “gold standard.” However, this method is invasive, requires major surgery for sample collection, and is not possible in patients in poor condition. The time between sampling and obtaining the results of culture may be as long as 7–10 days, which may be too long for the introduction of effective treatment. Blood culture is a major Duke criteria for the diagnosis of IE. Unfortunately, the sensitivity of this method for the isolation of fungi is low [3,4]. Therefore, these methods cannot identify the etiologic agent in the early stages of illness. Serological and molecular methods may be more accurate and reliable for the diagnosis of these infections. Candida spp. are most frequently responsible for FE. The detection of Candida mannan antigen is a reliable, sensitive, and noninvasive serological method for the diagnosis
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each species probe were used for samples analysis (5 µl of template volume in a final PCR volume of 20 µl). Thermal cycling conditions consisted of heating at 94◦ C for 10 min prior to a two-stage temperature profile of 30 s at 95◦ C and 90 s at 60◦ C for 40 cycles. The DNA extracted from A. fumigatus and C. albicans isolates was used as the positive control, and distilled water was used as the negative control in the real-time PCR assay. To determine the sensitivity of the real-time PCR assay, 100 copies/well of each A. fumigatus and C. albicans DNA was serially diluted and measured. The limitation of PCR assay was 10 copies/well. Statistical analyses were performed using SPSS software for Windows (version 15.0; SSPS Inc., Chicago, IL, USA). Negative control participants were selected from patients with the same conditions. Hence, patients with negative results for fungal elements in the microscopic examination or negative results in fungal cultures were considered to be the negative control group for the present study.
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17/F
4/M
50/M
27/F
31/M
67/F
19/M
21/M
1
2
3
4
5
6
7
8
IV drug abuser
Previous operation (3)
Previous operation (1)
IV drug abuser
Previous operation (2)
Previous operation (2)
Previous pacemaker insertion
Previous operation (3)
Risk factor
F, female; IV, intravenous; M, male.
Age (y)/sex
Large vegetation
Large mobile vegetation, Para, new valvular regurgitation
Large vegetation
Large mobile vegetation, para valvular abscess
Large vegetation
Large vegetation, new valvular regurgitation
Large vegetation, new valvular regurgitation
Large vegetation, para valvular abscess
Major Duke criteria
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Fever >38 C, septic pulmonary infarct Fever >38 C
Fever >38 C
Fever >38 C, conjuctival hemorrhage, glomerunephritis Fever >38 C, IV drug abuser Fever >38 C, prostatic heart valve, conjuctival hemorrhage Fever >38 C, conjuctival hemorrhage, septic pulmonary infarct Fever >38 C, IV drug abuser, septic pulmonary infarct
◦
◦
◦
◦
◦
◦
◦
◦
Potassium hydroxide solution
Minor Duke criteria
0.1
0.85
0.35
0.2
2.0
0.65
0.8
5.1
Galacto mannan
0.65
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Mannan test
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Case number
Table 1. Characteristics of patients with corresponding data for fungal endocarditis diagnosis. Tissue culture
Aspergillus flavus
A. flavus
A. fumigatus
A. niger
Candida albicans
A. niger
A. niger
C. albicans
Polymerase chain reaction result A+ C–
A+ C–
A+ C–
A+ C–
A– C–
A+ C–
A+ C–
A– C+
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Blood culture
Badiee et al. 531
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Medical Mycology, 2014, Vol. 52, No. 5
Table 2. Characteristics of patients with negative results for fungal endocarditis. Case number
Age (y)/sex
Major Duke criteria
Minor Duke criteria ◦
Fever >38 C, septic pulmonary infarct ◦ Fever >38 C, IV drug abuser
30/M
Large mobile vegetation
Fever >38 C, IV drug abuser, septic pulmonary infarct
5
29/M
Large vegetation
Fever >38 C, prostatic heart valve, conjuctival hemorrhage
6
67/M
Fever >38 C, glomerunephritis
7
54/M
Large vegetation, new valvular regurgitation Large vegetation, para valvular abscess
8
23/M
Fever >38 C, IV drug abuser
9
31/M
10
54/M
11
21/M
12
31/M
13
48/M
14
23/M
Small mobile vegetation, para valvular abscess Large vegetation, para valvular abscess Large vegetation, new valvular regurgitation Para valvular abscess, new valvular regurgitation Large mobile vegetation, para valvular abscess Small vegetation, para valvular abscess Large vegetation
15
32/M
Large mobile vegetation
16
18/F
17
52/M
Large mobile vegetation, new valvular regurgitation Large mobile vegetation
40/F
2
45/F
3
58/M
4
◦
Fever >38 C
◦
◦
◦
◦
Fever >38 C
◦
◦
Fever >38 C, IV drug abuser ◦
Fever >38 C, septic pulmonary infarct ◦ Fever >38 C ◦
Fever >38 C, IV drug abuser ◦
Fever >38 C ◦
Fever >38 C, prostatic heart valve, septic pulmonary infarct ◦ Fever >38 C, IV drug abuser, conjuctival hemorrhage ◦ Fever >38 C, prostatic heart valve ◦
Fever >38 C, prostatic heart valve, conjuctival hemorrhage
Chest pain, malaise, dry cough Malaise, anorexia, changing murmur, positive CRP test Malaise, chill, pacemaker endocarditis, repair of tricuspid valve Distraction of tricuspid valve, chest pain, left-side pleural effusion, multiple embolic lesions in lung Distraction of tricuspid wall, perforation of septum below the valve, cardiomegaly Elevated ESR, splenomegaly, dyspnea Diabetic mellitus, general body pain, positive CRP test, elevated ESR Edema, malaise, weight loss Chill, cough, malaise, cardiomegaly Diabetic mellitus, cardiomegaly, bilateral severe pleural effusion Malaise, weight loss Cardiomegaly, elevated ESR, positive CRP test Diabetic mellitus, chill, cardiomegaly Chest pain, dyspnea, cough, night sweats Night sweats, malaise, weight loss Edema, malaise, weight loss Malaise, weight loss
CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; F, female; IV, intravenous; M, male.
of systemic candidiasis [7,9,24], with an overall sensitivity of 90.9% and specificity of 46.2% [10]. In one study it was noted that Candida mannan antigen is rapidly cleared from the patient’s serum [25]. In the present study, the mannan antigen test was positive only with samples from one patient with Candida endocarditis and negative in all other patients (the specificity of this test was 100%). In this investigation, only two patients had Candida infection, and the result was positive in one of them. These low numbers prevent a discussion of the implications of this single positive result. However, based on specificity, in the patients
with suspected systemic candidiasis and negative results for the infection, systemic candidiasis could be ruled out. The detection of Aspergillus GM in serum has been reported to be a valuable tool for the diagnosis of invasive aspergillosis. Several factors affect the accuracy of this test, as high rates of false-positive results were reported in patients treated with amoxicillin–clavulanate [26], piperacillin–tazobactam, amoxicillin, and all fermentation products of Penicillium spp. [27], as well as in patients who received the intravenous electrolyte solution Plasmalyte [28,29]. In some instances, fungi other than
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Large mobile vegetation, para valvular abscess Large mobile vegetation, new valvular regurgitation Large vegetation, new valvular regurgitation
1
Other clinical signs and symptoms
Badiee et al.
Conclusion The rate of FE is low in clinical practice. Consequently, additional studies that involve different samples and different populations are needed in order to improve the accuracy of diagnostic methods and make the prompt diagnosis of this potentially life-threatening problem possible. Until these studies are completed, we suggest that for patients with a history of surgery, heart valve vegetation be sampled
and noninvasive methods that incorporate the GM antigen and PCR tests be used to improve the chances of detecting and identifying the etiologic agent in a timely manner. Despite some limitations, the combination of these two noninvasive methods can increase sensitivity and specificity and, in turn, facilitate accurate diagnosis and earlier initiation of specific therapy.
Acknowledgments Our thanks are due to Hassan Khajehei for his help with linguistic copyediting and to K. Shashok (AuthorAID in the Eastern Mediterranean) for improving the use of English in the manuscript.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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Aspergillus caused the infections [30]. In the current study, three patients with negative findings in both culture and molecular assays were GM antigen positive, probably due to use of antibacterial agents. The usefulness of the GM antigen test is reportedly related to population characteristics. For instance, there are reports of high sensitivity and specificity in patients with neutropenia and in stem cell transplant recipients [31,32]. There have been few investigations of this method for the diagnosis of FE. In one study of the use of the GM test to establish a diagnosis of aspergillosis FE, the result was positive (≥1 ng/ml) in only two of seven patients with endocarditis and mediastinitis [33]. In another study, only four of nine patients with Aspergillus endocarditis had positive GM test results [34]. Racil et al. found the sensitivity, specificity, and positive and negative predictive values of GM for invasive aspergillosis to be 88.2%, 95.8%, 75%, and 98.3%, respectively [28]. These values are close to the ones we obtained in our sample of patients Several researchers have reported the results obtained with molecular assays to detect invasive fungal infection [12,35] and Aspergillus endocarditis [36]. The sensitivity and specificity of molecular assays vary depending on the type of test (real-time PCR, nested PCR, PCR–enzymelinked immunosorbent assay). As with other diagnostic methods, molecular methods have limitations such as cost, lack of standardization at all laboratories, and potential contamination from environmental sources. The signs and symptoms of fungal infections are nonspecific, and the interpretation of radiological findings to distinguish between fungal and bacterial endocarditis can be problematic. These potential obstacles to a prompt, accurate diagnosis make new methods for diagnosing FE advisable. Based on the results our study, both the GM antigen test and real-time PCR can serve as reliable noninvasive tests for this purpose, compared with culture, which is considered to be the gold standard. However, because the positive likelihood ratio for real-time PCR is higher and the negative likelihood ratio is lower than with the GM antigen test, PCR-based methods are more valuable for the diagnosis of FE.
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