Invasive Fungal Infection — Laboratory Diagnosis And .
2. Shao2006 olInfect2006;39:178-188Review ArticleInvasive fungal infection — laboratory diagnosis andantifungal treatmentPei-Lan Shao1,2, Li-Min Huang2, Po-Ren Hsueh1,3Departments of 1Laboratory Medicine, 2Pediatrics and 3Internal Medicine, National Taiwan UniversityHospital, National Taiwan University College of Medicine, Taipei, TaiwanReceived: February 1, 2006 Accepted: February 15, 2006Invasive fungal infections (IFIs) have become increasingly prevalent in the recent decade along with the increasingpopulations of immunocompromised patients and widespread use of the broad-spectrum antibiotics. The morbidityand the mortality of IFIs remain high while the diagnosis and treatment of IFIs are highly challenging. Recentadvances in diagnostic methods and antifungal agents provide the potential to improve the outcomes of theseinfections. Conventional diagnostic methods including microbiological cultures and histopathological diagnosishave the disadvantages of either insensitivity or requiring invasive procedures. The innovative techniques of detectingcirculating fungal antigens and detecting fungal genomic DNA represent improvements in the diagnosis of invasiveaspergillosis. Several antifungal agents have been developed in recent years, such as lipid formulations ofamphotericin B, newer azoles, and echinocandins. These agents have either lower toxicities or greater activitiesagainst certain fungi compared with older treatments. With the availability of diverse antifungal agents, their use incombination has the potential to produce additive or synergistic effects, leading to better treatment outcomes.Large-scale randomized clinical trials are needed to confirm the efficacy of combination strategies.Key words: Antifungal agents, diagnosis, laboratory techniques and procedures, mycoses, reviewIntroductionInfection is a constant threat to human health. Remarkableprogress has been made in the prevention and treatmentof infectious diseases. At the same time, patterns of humaninfections have undergone significant changes duringthe past several decades. Bacterial infection was thepredominant issue before the availability of antibiotics.The use of penicillins and cephalosporins led to anincrease in Gram-negative bacterial infections in the 1960sand 1970s [1]. Thereafter, fungal infection started toemerge as a major clinical issue [2-4]. Two main factorscontributed to the steady increase of fungal infection,namely, the widespread use of antibacterial agents andrapid increase in numbers of immunocompromisedpopulations [3,4].Clinically important fungi consist of yeasts, molds,and dimorphic species. Each poses a different challengeCorresponding author: Dr. Po-Ren Hsueh, Departments ofLaboratory Medicine and Internal Medicine, National TaiwanUniversity Hospital, No 7, Chung-Shan South Road, Taipei, Taiwan.E-mail: hsporen@ha.mc.ntu.edu.tw178to clinicians. Overall, diagnosis of fungal infectionis more difficult compared with bacterial infections byconventional culture, and treatment is also difficultbecause of the limited range of antifungal agents.Modern therapeutic modalities such as cancerchemotherapy and organ transplantation have a greatlyincreased risk of invasive fungal infections (IFIs) [2,3].Currently, morbidity and mortality from IFIs remainhigh in patients with hematologic malignancies whoreceive intensive myelosuppressive chemotherapy orundergo bone marrow transplantation [2,5-11]. Thus,there is considerable scope for improvement both inthe diagnosis and management of fungal infection.Advances in Laboratory DiagnosisEarly diagnosis of IFIs remains a great challenge.The symptoms and signs are often nonspecific andmicrobiological cultures are usually negative. Histopathological diagnosis, which requires invasiveprocedures to obtain the specimens, is often hinderedby the grave conditions in these patients. The high 2006 Journal of Microbiology, Immunology and Infection
Shao et almortality of IFI has contributed to the difficulties inestablishing timely diagnosis and initiating promptantifungal therapy. Recently, more rapid diagnosis hasbeen achieved by use of detection of the circulatingmarkers, including fungal cell wall components andfungal genomic DNA. These techniques have advancedthe diagnosis of aspergillosis. Non-culture-baseddiagnosis for candidiasis and noninvasive diagnosisfor molds other than Aspergillus are still mainlyinvestigational [12].Detection of Fungal AntigensGalactomannan detectionGalactomannan (GM) is a polysaccharide cell wallcomponent that is released by the fungus into the serumduring its growth in tissues [13]. A sandwich enzymelinked immunosorbent assay (ELISA) for the detectionof GM antigen of Aspergillus was commercializedand has been applied in the diagnosis of invasiveaspergillosis (IA). The ELISA test (Platelia Aspergillus;Biorad, Marnes-La-Coquette, France) uses the EB-A2monoclonal antibody to recognize the galactofuranepitopes of the GM molecules [14]. The number ofepitopes on the GM antigen released by the fungi mayvary between strains, between species, and over time.Angioinvasion is assumed to be required for the GMantigens released from fungal hyphae to reach thecirculation [14]. The degree of angioinvasion varies inrelation to the underlying conditions and toxic damagecaused by cytotoxic drugs or irradiation. In certainunderlying diseases, such as chronic granulomatousdisease, in which the formation of abscess predominatesand may hamper the leakage of GM antigens into thecirculation, IA may occur with the absence of theantigenemia [14]. In contrast, antigen detection hasexcellent sensitivity and specificity (up to 89.7% and98.4%, respectively) in stem cell transplant recipientsand in patients with prolonged neutropenia [15]. Thisresult might indicate rapid progression of disease causedby the angioinvasive growth of Aspergillus in damagedlung epithelia [14].Once the GM reaches the circulation, it might bindto substances present in the blood, including Aspergillusantibodies and other human proteins. Such binding couldinterfere with the performance of the ELISA test andcause false-negative results [14]. Clearance of the GMfrom the blood due to renal excretion and uptake bymacrophage was demonstrated in an animal model [14].Renal clearance depends on the renal function of thepatient and the size of the GM antigen.The performance of the GM ELISA test wasreported to range from 50% to 100% in sensitivity andfrom 92% to 100% in specificity (Table 1) [13-20].In these reports, sensitivity varied considerably, whilespecificity remained more consistent and was usuallygreater than 85%. When the test is applied to clinicaldiagnosis, double-checking of positive samples mightbe necessary because of the possible lack of reproducibility [17]. As mentioned by the manufacturer, IATable 1. Summary of studies on evaluation of the Platelia Aspergillus galactomannan (GM) enzyme-linked immunosorbentassay for diagnosis of invasive aspergillosis (IA)No. ofpatients(episodes)135 (193)a191 (362)74807a67797a71aUnderlying conditionsHeM with neutropenia SCTHeM with neutropenia SCTSCTHeM or admitted to anintensive care unitsSCTHeM, SCTHeM with neutropenia and/orsteroid treatment CGD, SCTIApatientsGM cut-offindex 290.692.61009495.49395181920Abbreviations: PPV positive predictive value; NPV negative predictive value; HeM hematological malignancies; SCT stem celltransplant; CGD chronic granulomatous diseaseaIncludes pediatric patients.bIncludes proven, probable, and possible IA.cIncludes proven and probable IA.dIncludes proven IA. 2006 Journal of Microbiology, Immunology and Infection179
Invasive fungal infectionshould be considered when 2 consecutive positivesamples from a patient have been obtained. In patientswith only single positive samples, the sensitivity is low,perhaps around 40% or less [14]. The cut-off index valuefor a positive result suggested by the manufacturerwas above 1.5. Some studies suggested that a cut-offvalue of 1.5 was too high and that lowering the cut-offto 1.0 could improve sensitivity without compromisingspecificity [13,20]. However, it also had been reportedelsewhere that lowering the cut-off value to 1.0 did notimprove sensitivity [19]. In patients with IA, the ELISAtest usually gives positive results before the clinicalsymptoms and signs become detectable [18,19]. Whileserving as an early diagnostic tool, some study suggesteddecreasing the cut-off value further to 0.5 in order toincrease the duration of test positivity before diagnosiscould be established by clinical means [18]. Moreover,serial determination of serum GM index values isuseful in order to evaluate the prognosis of IA. A 1.0increase in GM level above baseline was a markerof disease progression and predictive of treatmentfailure in allogeneic stem cell transplant recipients [21].Nevertheless, it is important to recognize the causes offalse-positives and false-negatives when the ELISA testis utilized in the clinical setting.The false-positive rate ranged from 5% [13] to 14%[15]. The occurrence of false positivity frequentlycoincided with mucositis, cytotoxic chemotherapy,and/or graft-versus-host disease [13,15]. GM wasdetected in various kinds of foods [14,17,19]. It ispostulated that translocation of dietary GM via damagedor immature intestinal mucosa could result in falsepositive results [15,17]. The false-positive rate wasreported to be high in up to 83% of newborn babies[14]. Besides GM of food origin, lipoteichoic acid ofBifidobacterium spp., which heavily colonize theneonatal gut, might cause ELISA reactivity in infantsafter translocation through immature intestinal mucosa[22]. Cross-reactivity to other fungi or bacteria wasnot reported, with the exceptions of Penicilliumchrysogenum, Penicillium digitatum, and Paecilomycesvariotii, fungi that rarely cause human infections [23].Some drugs of fungal origin, such as antibiotics, couldalso cause persistent or transient antigenemia [17].False-positive results had been described in somepatients receiving piperacillin-tazobactam [24,25] andin a patient receiving amoxicillin-clavulanic acid froma case report [26]. However, the nature of false-positivesremains undetermined in many cases and the causes maybe multifactorial.180False-negatives may result from low-level releaseof the GM of the growing fungi, the use of prophylacticantifungal agents, and limited angioinvasion. Exposureto antifungal agents such as amphotericin B (AmB)might reduce the mycelial growth and/or alter the hyphalrelease of GM [18], causing the false-negative results.Because of the risk of false-negative results, GM antigendetection does not replace other diagnostic tools, suchas computed tomography imaging, in the explorationof IFIs in high-risk patients [16].The utility of the GM antigen ELISA in specimensother than serum has been evaluated [27]. Detection ofGM antigen in bronchoalveolar lavage (BAL) fluid hadgood sensitivity and was more sensitive than antigendetection in serum. Increased GM index in cerebrospinalfluid indicated IA of the central nervous system. Theutility of the ELISA in urine remains controversial andneeds further validation.Noninvasive testing of GM ELISA has advancedthe diagnosis of IA. In practice, routine follow-up ofpatients at high risk for aspergillosis should includeserum GM determination twice per week duringneutropenia and when patients have additional riskfactors, such as graft-versus-host-disease, and/orprolonged corticosteroid therapy. GM detection remainsone of the major criteria to establish the IA diagnosiseven when mycological detection is negative, accordingto the consensus group of the European Organizationfor Research and Treatment of Cancer Invasive FungalInfections Cooperative Group and the National Instituteof Allergy and Infectious Diseases Mycoses StudyGroup [28]. Because of the overall good performanceof the ELISA test, a positive result should trigger furtherevaluation for disease by radiography, and lead toprompt treatment if indicated, while a negative resultshould lead to the aggressive search for other etiologies.Glucan detection(1-3)-Beta (β)-D-glucan ([1-3]-BDG) is a componentof the cell wall of a variety of fungi [29] and can beutilized as a nonspecific marker for IFIs. It can bedetected by its ability to activate factor G of thehorseshoe crab coagulation cascade [29]. Commerciallyavailable BDG assays (e.g., Fungitec G; Seikagaku,Tokyo, Japan) allow determination of serum BDG bycolorimetric or kinetic assay [30]. The BDG assay hadgood performance in patients infected with Candida,Aspergillus, and Fusarium spp., but failed to detectpatients infected with zygomycetes and Cryptococcus,which contain little or no BDG [31]. Sensitivity and 2006 Journal of Microbiology, Immunology and Infection
Invasive fungal infectionshould be considered when 2 consecutive positivesamples from a patient have been obtained. In patientswith only single positive samples, the sensitivity is low,perhaps around 40% or less [14]. The cut-off index valuefor a positive result suggested by the manufacturerwas above 1.5. Some studies suggested that a cut-offvalue of 1.5 was too high and that lowering the cut-offto 1.0 could improve sensitivity without compromisingspecificity [13,20]. However, it also had been reportedelsewhere that lowering the cut-off value to 1.0 did notimprove sensitivity [19]. In patients with IA, the ELISAtest usually gives positive results before the clinicalsymptoms and signs become detectable [18,19]. Whileserving as an early diagnostic tool, some study suggesteddecreasing the cut-off value further to 0.5 in order toincrease the duration of test positivity before diagnosiscould be established by clinical means [18]. Moreover,serial determination of serum GM index values isuseful in order to evaluate the prognosis of IA. A 1.0increase in GM level above baseline was a markerof disease progression and predictive of treatmentfailure in allogeneic stem cell transplant recipients [21].Nevertheless, it is important to recognize the causes offalse-positives and false-negatives when the ELISA testis utilized in the clinical setting.The false-positive rate ranged from 5% [13] to 14%[15]. The occurrence of false positivity frequentlycoincided with mucositis, cytotoxic chemotherapy,and/or graft-versus-host disease [13,15]. GM wasdetected in various kinds of foods [14,17,19]. It ispostulated that translocation of dietary GM via damagedor immature intestinal mucosa could result in falsepositive results [15,17]. The false-positive rate wasreported to be high in up to 83% of newborn babies[14]. Besides GM of food origin, lipoteichoic acid ofBifidobacterium spp., which heavily colonize theneonatal gut, might cause ELISA reactivity in infantsafter translocation through immature intestinal mucosa[22]. Cross-reactivity to other fungi or bacteria wasnot reported, with the exceptions of Penicilliumchrysogenum, Penicillium digitatum, and Paecilomycesvariotii, fungi that rarely cause human infections [23].Some drugs of fungal origin, such as antibiotics, couldalso cause persistent or transient antigenemia [17].False-positive results had been described in somepatients receiving piperacillin-tazobactam [24,25] andin a patient receiving amoxicillin-clavulanic acid froma case report [26]. However, the nature of false-positivesremains undetermined in many cases and the causes maybe multifactorial.180False-negatives may result from low-level releaseof the GM of the growing fungi, the use of prophylacticantifungal agents, and limited angioinvasion. Exposureto antifungal agents such as amphotericin B (AmB)might reduce the mycelial growth and/or alter the hyphalrelease of GM [18], causing the false-negative results.Because of the risk of false-negative results, GM antigendetection does not replace other diagnostic tools, suchas computed tomography imaging, in the explorationof IFIs in high-risk patients [16].The utility of the GM antigen ELISA in specimensother than serum has been evaluated [27]. Detection ofGM antigen in bronchoalveolar lavage (BAL) fluid hadgood sensitivity and was more sensitive than antigendetection in serum. Increased GM index in cerebrospinalfluid indicated IA of the central nervous system. Theutility of the ELISA in urine remains controversial andneeds further validation.Noninvasive testing of GM ELISA has advancedthe diagnosis of IA. In practice, routine follow-up ofpatients at high risk for aspergillosis should includeserum GM determination twice per week duringneutropenia and when patients have additional riskfactors, such as graft-versus-host-disease, and/orprolonged corticosteroid therapy. GM detection remainsone of the major criteria to establish the IA diagnosiseven when mycological detection is negative, accordingto the consensus group of the European Organizationfor Research and Treatment of Cancer Invasive FungalInfections Cooperative Group and the National Instituteof Allergy and Infectious Diseases Mycoses StudyGroup [28]. Because of the overall good performanceof the ELISA test, a positive result should trigger furtherevaluation for disease by radiography, and lead toprompt treatment if indicated, while a negative resultshould lead to the aggressive search for other etiologies.Glucan detection(1-3)-Beta (β)-D-glucan ([1-3]-BDG) is a componentof the cell wall of a variety of fungi [29] and can beutilized as a nonspecific marker for IFIs. It can bedetected by its ability to activate factor G of thehorseshoe crab coagulation cascade [29]. Commerciallyavailable BDG assays (e.g., Fungitec G; Seikagaku,Tokyo, Japan) allow determination of serum BDG bycolorimetric or kinetic assay [30]. The BDG assay hadgood performance in patients infected with Candida,Aspergillus, and Fusarium spp., but failed to detectpatients infected with zygomycetes and Cryptococcus,which contain little or no BDG [31]. Sensitivity and 2006 Journal of Microbiology, Immunology and Infection
Shao et alConclusionsLaboratory diagnosis of IFIs has progressed in recentyears, with advances mainly occurring in the area ofdiagnosis of IA. In the evaluation of the diagnosticmethods for IA, the determination of sensitivity andspecificity has been confounded by the uncertainty ofthe disease status; inclusion of probable cases of IA inthe evaluation would affect performances of diagnostictests. Non-culture-based diagnosis for candidiasis andnoninvasive diagnosis for molds other than Aspergillusmostly remain investigational. New methods or betterstrategies are needed to improve diagnostic methodsfor IFIs.There have been many advances in antifungaltreatment in the last decade. The availability of morepotent and less toxic antifungal agents, such as secondgeneration triazoles and echinocandins, has greatlyimproved the treatment of IFIs. However, the mortalityof IFIs remains high. Combination therapy is promisingconceptually as a means to increase the success rate oftreatment, but more controlled clinical trials are neededto verify the efficacy of this approach.References1. Waterer GW, Wunderink RG. Increasing threat of Gramnegative bacteria. Crit Care Med 2001;29(Suppl 4):N75-81.2. Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiologyand outcome of mold infections in hematopoietic stem celltransplant recipients. Clin Infect Dis 2002;34:909-17.3. Baddley JW, Stroud TP, Salzman D, Pappas PG. Invasive moldinfections in allogeneic bone marrow transplant recipients. ClinInfect Dis 2001;32:1319-24.4. Clark TA, Hajjeh RA. Recent trends in the epidemiology ofinvasive mycoses. Curr Opin Infect Dis 2002;15:569-74.5. Husain S, Alexander BD, Munoz P, Avery RK, Houston S,Pruett T, et al. Opportunistic mycelial fungal infections in organtransplant recipients: emerging importance of non-Aspergi
the diagnosis and management of fungal infection. Advances in Laboratory Diagnosis Early diagnosis of IFIs remains a great challenge. The symptoms and signs are often nonspecific and microbiological cultures are usually negative. Histo-pathological diagnosis, which requires invasive procedures to obtain the specimens, is often hindered
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