Technical advances in laboratory diagnosis of fungalinfection

Qinfang Qian, James E. Kirby

Division of Laboratory and Transfusion Medicine, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA

Rapid detection and identification of fungi has become increasingly important for several reasons: (1) the increasing number of immunocompromised patients at risk for fungal infection; (2) the broader spectrum of fungal agents causing disease; and (3) emerging resistance to anti-fungal agents among medically important fungi. This review will therefore focus on technical advances in laboratory diagnosis that address these new challenges. In this analysis of the current state of the art in fungal diagnosis, we will especially emphasize methods that aid early diagnosis and appropriate empiric anti-fungal therapy and that can be practically applied in modern diagnostics laboratories.

1 Direct microscopic examination

Direct microscopic observation of clinical specimens is a simple and low cost procedure, yet remains a very helpful starting point in detecting the presence of fungal infection. It is also advantageous in that it can be performed rapidly and may yield initial clues to the type of fungal infection or at times even allows definitive speciation for fungi with distinct morphologies. Although Gram stain is generally used as the workhorse direct stain to detect the presence of bacteria in clinical samples, fungal forms including both yeast and hyphae can also be seen although with different staining characteristics (none to strong staining) depending on the type of fungus. For example,Candidastains extremely well, while dimorphic fungi may stain variably. Fungal detection is improved by using KOH and calcofluor white. The former digests tissue to make fungal forms more obvious, while the latter binds specifically to fungal cell wall polysaccharides and thereby increases sensitivity and specificity of detection[1,2]. The rapidity of the KOH/calcofluor stain is extremely helpful when a fungus ofZygomycetesgroup such asMucoris potentially present asZygomycetesinfections progress rapidly and often require timely surgical intervention to spare the patients’ life or limb. Using this stain a number of additional fungal types can readily be distinguished based on their specific morphology characteristics in tissue samples, for example, dimorphic fungi such asPenicilliummarneffei(P.marneffei),Histoplasmacapsulatum(H.capsulatum),Coccidioidesimmitis(C.immitis),Paracoccidioidesbrasiliensis(P.brasiliensis),Blastomycesdermatiditis(B.dermatiditis), andSporothrixschenckii(S.schenckii)[3]. These fungi often grow slowly and many require complex procedures for identification of culture organisms where morphology is less specific. For example, identification ofP.marneffeiin culture requires conversion to its yeast phase through special culture techniques and absence of molecular probe and antigen assays available for this dimorphic fungus. Therefore, morphological identification of organisms directly in tissue through use of the KOH/calcofluor stain can greatly expedite diagnosis.

Direct fluorescent-conjugated monoclonal antibody (DFA) assay is still considered the gold standard for identification ofPneumocystisjirovecii(P.jirovecii) in respiratory samples in clinical laboratories. It has excellent sensitivity and specificity[4,5]in appropriate specimens, although some polymerase chain reaction (PCR) methods have been developed with higher sensitivity[6,7]. For induced sputa in human immunodeficiency virus (HIV)-positive patients, the sensitivity of DFA and PCR was previously found to be 82% and 95%, respectively. The specificity of PCR for induced sputum specimens was 94%, the lower specificity related to potential low level colonization in patients without disease, not detectable by traditional DFA methods. For bronchoalveolar lavage (BAL) specimens from HIV-positive patients, the sensitivity and specificity of PCR using DFA as a diagnostic gold standard were 100% and 98%, respectively[6]. It should be noted that organism burden is generally lower in transplant patient population. Therefore false-negative results may occur especially from induced sputum samples[8].

Recently, Wangetal.[9]retrospectively investigated diagnostic algorithms for optimal use of DFA testing. They found that repeat testing of induced sputum specimen forP.jiroveciidid not significantly increase diagnostic yield in an urban tertiary care teaching hospital. Furthermore, in HIV-negative patients, induced sputum testing was diagnostically insensitive. Therefore, they recommend that BAL testing should be performed initially in HIV-negative patients and after the first negative induced sputum in HIV-positive patients. In contrast, Paganoetal. in a retrospective study on HIV-negative patients with hematological malignancies detected organism in induced sputa from 9 out of 18 patients[10]. LaRocqueetal. also described detection of 6 positives out of 156 first induced sputa and 2 positives out of 20 follow-up induced sputa[11]and concluded that induced sputa were the specimens of initial choice forPneumocystiscarinii(P.carinii) pneumonia diagnosis in HIV-negative patients. It is unclear why these groups were able to detectPneumocystisat a higher frequency than Wang’s study. However, potential reasons include pre-selection of populations with a higher organism burden, testing at different stages of disease, differences in testing methodology or performance, and/or differences in induced sputum collection technique. Despite the conflicting recommendations on appropriate first specimens to test in the literature, the value of DFA testing as a rapid means of establishing the diagnosis ofP.cariniipneumonia is clear and now may be supplemented by additional techniques described below.

2 Detection of fungal antigen in clinical samples

Several assays are available for rapid detection of antigen for specific or a broad range of fungi directly from clinical specimens. These include but not limited to the following tests.

2.1 Cryptococcal antigen

The detection ofCryptococcusneoformans(C.neoformans) capsular polysaccharide antigen in patients’ serum and cerebrospinal fluid (CSF) by the latex agglutination assay is widely used in clinical laboratories[12]. This is a rapid and easy test. It also shows enhanced sensitivity and specificity compared to the classic identification ofCryptococcusorganisms by virtue of their negative staining of their prominent capsule in clinical samples when admixed with India ink. Therefore the traditional India ink test is no longer used by many clinical laboratories. However, very rarely, false-positive results have been seen in patients with disseminatedTrichosporoninfection[13]andCapnocytophagasepticemia[14]. False-negatives may also occur due to prozone effect when the level of antigen in CSF is extremely high[15]. The antigen test is also advantageous as it is quantitative. Typically, positive patient samples are serially diluted to establish a titer (maximal dilution) that still yields reactivity, establishing the level of antigen and therefore organism burden in the patient[16-18]. Therefore serially titers prove useful in following response to therapy, however, it should be noted that titers are not precise[16]and antigen may not disappear completely after successful therapy[17]. Paired testing of serial specimens will increase the accuracy in assessing changes in titer, a four-fold change in titer or greater generally considered a clinically significant change.

2.2 Candida antigen

Candidaspp. is still one of the most common causes of blood stream infections. Candidemia is associated with high mortality rate (49%), longer hospital stay, and higher hospital costs relative to bacterial blood stream infection[19-21]. MostCandidablood stream infections are caused by eitherCandidaalbicans(C.albicans) orCandidaglabrata(C.glabrata)[22-24]. Mean time to yeast detection and final identification were 35 h and 86 h forC.albicansand 80 h and 154 h forC.glabrata, respectively[23,25]. Recent studies indicate that delayed treatment of candidemia greatly increased morbidity and mortality[26,27]. Therefore, rapid detection ofCandidaantigen is desirable, and antigen testing has recently been explored as a way to identify early infection in patients at risk.

Along with β-glucan (see below), mannan is a major component ofCandidacell wall and has been used in antigen-based detection ofCandidainfection. The PlateliaCandidaantigen EIA (Bio-Rad Laboratories, USA) uses a monoclonal antibody, EB-CA1, for capture and detection of a mannopentose epitope ofCandidaspp. Importantly, the assay was found to be very specific with only weak cross-reactivity with potentially cross-reacting filamentous fungi, i.e.,FusariumverticillioidesandGeotrichumcandidum[28]. Furthermore, previous studies showed a correlation between detectable mannanemia and invasiveCandidainfection in patients with candidemia, indicating that the test had clinical useful predictive value. In fact, positive mannanemia was observed days to weeks before mycological detection[16,53,55]. In addition, the test demonstrated excellent performance characteristics in detecting proven and probable candidiasis in a neonatal intensive care unit (ICU) showing a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 94.4%, 94.2%, 85%, and 98%, respectively[33]. However, this assay showed lower sensitivity in general patient population with a sensitivity of only 67%[29]. The greater sensitivity in the neonatal population was postulated to be potentially related with several factors: (1) the absence of circulating anti-mannan antibodies in the neonatal population; (2) a low concentration of circulating lectins (mannose-binding protein); and/or (3) to a different pathophysiology of candidiasis in neonates. It is also important to note that low sensitivity was found for certain species such asC.parapsilosisandC.krusei[29], organisms that might be found more frequently in adults or at least for the later in transplant populations on prophylaxis. The above studies in adults were all based on the detection of α-linked mannan. However, in a separate study examining detection of both α- and β-linked mannan, the sensitivity reached 90% in the adult population[30], suggesting additional potential for this assay in clinical practice.

2.3 P. marneffei antigen

P.marneffeiis an endemic dimorphic fungus in Southeast Asia and China, and is especially clinically problematic as an opportunistic pathogen in immunocompromised patients from this region. As mentioned above, speciation is difficult because of the time required to culture and convert the mold into its diagnostic yeast phase in the laboratory. Because of the need to rapidly identify infection with this emerging pathogen in endemic areas, an enzyme-linked immunosorbent assay (ELISA) was developed to detectP.marneffeiantigen in patient sera using an antibody specific forP.marneffeimannoprotein (Mp1p). In a clinical study, it identified the presence of penicilliosis in 17 of 26 (65%) patients[31]. Another enzyme immunoassay, a dot blot ELISA and a latex agglutination test were also developed for detection and quantification ofP.marneffeiantigen in the urine using a polyclonal anti-P.marneffeiantigen Mp1p. These tests showed excellent performance characteristics with a sensitivity of 97%, 94%, and 100% respectively, and a specificity of 98%, 97%, and 99% respectively in patients with acquired immunodeficiency syndrome (AIDS)[32,33], the major risk group for infection with this organism. Similarly, another ELISA test using monoclonal antibody which was produced against crude culture filtrate had a sensitivity of 72%, a specificity of 100%, a PPV of 100%, and a NPV of 97%. It detectedP.marneffeiantigen in 16 of 18 (89%) culture-confirmed cases[34,35]. Therefore, these rapid methods should prove extremely useful in rapid diagnosis of penicilliosis in endemic regions much as antigen tests for other dimorphic fungi in other parts of the world.

2.4 Histoplasma antigen

It is estimated that 500 000 newH.capsulatuminfections occur annually in USA. This dimorphic fungus is also found worldwide including China[36-39]. Detection ofHistoplasmaantigen in body fluids can expedite the diagnosis of histoplasmosis with the need for biopsy and culture.Histoplasmaantigen has been detected in the urine of over 90% of patients with disseminatedHistoplasmainfection, and in the sera of at least 75% of these patients[40]. As antigen levels decline with treatment[41]and increase with relapse[42], it also provides a useful tool for monitoring therapy[43]. Antigen may also be detected in BAL sample of patients with pulmonary histoplasmosis[17]. In fact, antigen was detected in BAL samples of 84% of patients with pulmonary histoplasmosis, including a few patients with a negative urine antigen test, suggesting that it might prove a preferred diagnostic test in patients with pulmonary symptoms where BAL collections are being performed during the diagnostic workup.Histoplasmaantigen has also been detected in the spinal fluid of more than 50% of patients with central nervous system infection[44]. Antigen also may be detected in pleural, pericardial, peritoneal, synovial, and ocular fluids in patients with disseminated diseases involving these sites[40]and may therefore provide useful diagnostic evidence of invasion into privileged sites where agents or duration of anti-fungal therapy might be considered.

Notably, however, false-positive results may occur due to cross-reactivity in patients infected with other dimorphic fungi[45]such asB.dermatiditis, a fungus which has been reported causing human infection throughout the world including China[46]. In addition, false-positive results were found in approximately 16% of solid organ transplant patients who received rabbit anti-thymocyte globulin to prevent allograft rejection[12,72]. It was that patients who received the rabbit anti-thymocyte globulin produced heterophile antibodies which cross-reacted with earlier versions of theHistoplasmaantigen assay. In 2004, MiraVista Diagnostics (USA) implemented its second generationHistoplasmaantigen EIA, which has increased sensitivity and reduced cross-reactivity with patient heterophile antibodies. Therefore, it is important to communicate with the clinical laboratory to understand specific assay interferences in patients treated with anti-thymocyte globulin. A third generation quantitative EIA assay has also been developed[40]. The lower limit of detection is 0.3 ng/ml, and the reportable range is 0.6 to 39 ng/ml. The sensitivity of this new assay has not been determined, but presumably is higher than that reported for previous generations.

2.5 Galactomannan (GM)

Aspergillusfumigatusis the most common invasive filamentous fungal infections in immunocom-promised patients. Infections are often deep-seated and difficult to detect in a timely fashion by standard culture techniques. Moreover, the ability to distinguish invasive infectionversuscolonization—the latter is also common in this patient population—is critical, especially in the therapy with agents such as amphotericin B, which may be especially toxic in patients with multiple comorbidities and may not be required in the absence of invasive infection. The presence of fungi or fungal antigen in the blood is extremely specific for invasive infection and therefore serves as an important diagnostic criterion. Culture diagnosis of filamentous fungi from the blood is extremely insensitive. In contrast, detection ofAspergillusantigen has shown great promise.

Most clinical assays for detection ofAspergillusantigen are based on detection ofAspergillusGM, a fungal wall constituent produced in large amounts and relatively specific for this fungus. The PlateliaAspergillusEIA assay (Bio-Rad) detectsAspergillusGM in serum samples and was recently approved by the US Food and Drug Administration (FDA) for detection of deep-seated, invasiveAspergillusinfection. There have been multiple clinical studies investigating the performance characteristics of this assay in various patient populations. Notably, the assay includes a high- and mid-range “cutoff” control. The results are reported as GM index determined by dividing the optical density (OD) of the sample by the mean OD of the cutoff control. In US, an index of 0.5 is reported as positive, while in Europe, an index of 1.5 is reported as positive, thereby reducing sensitivity but increasing specificity. The different cutoffs for positivity used in different locals have made it difficult to compare literature. Nevertheless, there have been several general observations. In patients on chemotherapy or who have had hematopoietic stem cell transplanta-tions, the sensitivity of the assay varies from 67% to 93% with specificity of 86%-95% using cutoffs from 1 to 1.5[47-49]. In contrast, lower sensitivity (30%) has been reported in lung transplant patients, even using cutoff of 0.5[50].

Although historical positivity was more generally defined as an index greater than 1.0, it was found that using a lower index cutoff would allow earlier disease detection[51]. However, for specimens with an index between 0.5 and 0.9, the positive results were only reproducible in 87% of the samples[52]versusmore consistent reproducibility with higher indexes. Subsequently, Maertensetal. examined the performance characteristics of the assay when requiring two consecutive positive assays to be interpreted as a true positive result[53,54]. They found using a cutoff of 1.5, the sensitivity of dual positivity was 94% and the specificity was 99%, while using a cutoff of 0.5, the sensitivity increased to 96.5% with only a marginal decrease in specificity to 98.6%. Note, the generally higher sensitivity observed in these studies were likely due to limitation of testing to those with a high pre-test probability of disease. Therefore, it has become standard practice to confirm a positive result with a second assay, especially when the lower cutoff is used[53,55,56]. Moreover, duplicate testing also mitigates against false-positives due to contamination of samples with antigen from other fungi described below.

Notably, Aspergillus GM EIA test also reacts with GM of several other molds includingPenicillium,Trichophyton,Cladosporium,Acremonium,Alter-naria,Fusarium,Wangiella,Rhodotorula, andPaecilomyces[57-59]. Therefore false-positiveAspergillusGM test results may occur via several mechanisms:

(1) Patients may be infected with one of other fungi. In fact, this assay has been shown to be promising in a study for earlier diagnosis of the dimorphic fungusP.marneffeiinfection which is endemic in Southeast Asia including China[60-63]. Sera from 11 out of 15 confirmed penicilliosis patients were positive by this assay (cutoff >0.5)[64]. Nevertheless specific detection ofP.marneffeiantigen described above is probably a preferable strategy if test is available. False-positive result has also been shown in a patient with Cryptococcal pulmonary infection and Cryptococcal fungemia. Further study found thatC.neoformansgalactoxylomannan cross-reacted with the GM test[65].

(2) Serum samples used for the GM assay may be contaminated with fungal organisms if not handled under sterile fashion. Therefore, it is recommended that a separate tube of blood is used for the GM test and not used for multiple tests where the tube may be contaminated during routing through the clinical laboratory.

(3) False-positive results have also been reported in patients treated with the antibiotics piperacillin/tazobactam (Zosyn)[66,67]and amoxicillin-clavulanate (Augmentin)[68,69]. Piperacillin is produced byPenicilliumspp., which contains GM in its cell wall. Carryover of GM through production was proposed as the cause for the false-positive results[2,65].

(4) A higher frequency of false-positive results has been observed in pediatric patients[49,51]. Further investigation suggests that these findings may be related to the presence of cross-reacting bacterial antigen found at higher concentration in children and neonates. Specifically,Bifidobacteriumspp. was found to be present at high concentration in the intestine of neonates. The lipoteichoic acid found in the cell wall of this organism was shown to cause false-positive results in the PlateliaAspergillusassay[70,71].

Debate continues about the ability of the antigen test to predict the presence of invasive infection prior to the development of observable radiological findings. Previously GM antigenemia was shown to predate radiological and clinical symptoms in patients with hematological malignan-cies[48,49]. However, Weisseretal.[72]found that GM did not precede major signs on invasive aspergillosis on pulmonary computerized tomographic scans. Therefore, different strategies for using the test have been proposed including serial testing in patients at riskversustesting only in the presence of compatible radiographic findings.

The GM test has also been used to detect the presence ofAspergillusantigen in other body fluids. Detection of GM in BAL was found helpful for diagnosis of invasive pulmonary aspergillosis. For example, in haematological patients with neutropenia for more than 10 days, and on anti-fungal prophylaxis with either fluconazole or itraconazole, the sensitivity of the test was 100% on proven, 89% on probable, and 42% on possible cases of invasive aspergillosis. The specificity was also 100% in this relatively small patient cohort[55]. Similarly, in solid organ transplant patients, the sensitivity was 100% and the specificity was 84.2%[73]. In contrast, Musheretal. found that the sensitivity was only 76% and the specificity was 94% in a larger study on hematopoietic stem cell transplant patients[74]. There is significant concern that the use of BAL testing may over-diagnose invasive infection, as colonization or presence of a non-invasive fungus ball may lead to false-positive results. Therefore, the authors recommend only testing serum to increase the specificity for detecting invasive infection.

The role of testing fluids for diagnosis of infections in specific anatomic compartments is not clear and at this point also cannot be recommended. GM was detected in CSF from antigenemic patients. However, in the absence of obvious central nervous system involvement, the presence of intrathecal antigen was probably due to contamination of CSF by traumatic lumber puncture or penetration of GM through the blood-brain barrier[75]. The accuracy of GM detection in other tissues and fluids has not been determined[76,77].

2.6 β-D-glucan

An exciting advance in detection of occult fungal infection has been the development of a diagnostic test for the fungal antigen (1,3) β-D-glucan (BDG). BDG is a cell wall polysaccharide component of most fungi. Notable exceptions include theZygomycetes(e.g.,MucorandRhizopus) which lack BDG, andCryptococcuswhich produces little BDG. Fungitell (Associates of Cape Cod, USA), a chromogenic assay for BDG was approved by US FDA in 2004 for diagnosis of fungal infection[78]. This assay is rapid, simple and straightforward. The principle of this test is similar to the Limulus amebocyte reaction for endotoxin detection. Clotting factors in horseshoe crab hemolymph are exquisitely sensitive to activation by BDG and endotoxin. BDG and endotoxin activate separate arms in the clotting cascade which converge in a final common pathway leading to coagulation. In the Fungitell assay, the BDG-specific factors and common factors are purified to create a sensitive and specific assay for BDG.

The presence of BDG in serum sample has been used to predict the presence of systemic fungal infection. Normal human serum contains low levels of BDG, usually 10-40 pg/ml. Therefore, a positive result is defined as a BDG level above 80 pg/ml in patients at risk. Rising BDG levels has been shown proceeding clinical presentation of invasive aspergillosis and decreasing levels correlate with therapy. The sensitivity of the BDG assay for the diagnosis of invasive aspergillosis was 80% with a specificity of 81%. Overall, the sensitivity of the assay ranges from 60% to 100% and specificity ranges from 64% to 99%[3]. Elevated BDG levels are also associated withCandidainfections. Interestingly, very high levels of BDG are observed inP.jiroveciiinfection and therefore may be used to support the diagnosis ofPneumocystispneumonia in concert with compatible radiological findings. Current availability of the assay is limited by the need to prepare test reagents from a limited supply of horseshoe crabs. However, recombinant DNA technology will undoubtedly replace this more primitive process and allow widespread and economical implementation of this very useful assay. It should be noted that this assay is subject to some peculiar and specific false-positive reactions. For example, cellulose contamination from blood product filters and hemodialysis membranes can falsely elevate BDG results[79]. Bacteremia has also been shown to be associated with rare false-positive reactions for unclear reasons[80].

3 Detection of fungal antibodies in serumsamples

Detection of specific antibodies in a patient’s serum has long been used to aid the diagnosis of fungal infections caused by dimorphic fungi such asH.capsulatum,B.dermatiditis,C.immitis,andP.brasiliensis[3].H.capsulatumandB.dermatiditisare found in China and cause human infections, making these assays useful in Chinese medical practice.

P.marneffeiis another endemic dimorphic fungus in Southeast Asia and China[60-63]. In the last decade, several studies have therefore evaluated the potential of serological assays for the detection ofP.marneffeiinfection using several techniques including immunodiffusion[81, 82], indirect fluorescent antibody[83], and immunoblot[84]. Assays using antigens produced from the yeast form ofP.marneffeihad higher sensitivity than those using antigens from mycelial phase. This is not surprising as the yeast phase is found in the human host and would therefore seem a more likely target for antibodies produced in the infection. The 61-kDa, 54-kDa, 50-kDa, 38-kDa yeast phase proteins have been found to be especially promising as recombinant targets[84-86]. In one study, sera from 86% ofP.marneffei-infected patients reacted with the 61-kDa antigen, 71% reacted with the 54-kDa antigen, and 48% reacted with the 50-kDa antigen[86]. Of note, these tests were either evaluated only in a small number of clinical samples or had low sensitivity. In addition, it was found that 25% (17/67) of HIV patients infected withCryptococcusorCandidaalso had weak positive reaction by the immunoblot when using the 38-kDa antigen, as were 17% (45/262) of asymptomatic HIV patients[85]. Since these patients were from endemic area, it is unknown if these patients had subclinical or prior infection, or non-specific reaction. Therefore the performance characteristics of these serological assays deserve further investigation to evaluate their usefulness and application. Recently, an ELISA test was developed to detect antibodies to recombinant Mp1p and showed a promising sensitivity of 80% and a specificity of 100%[87].

4 Detection and phenotypic identification of fungi by culture

Currently, there are no formal guidelines for the appropriate use of fungal blood cultures. However, a general observation is that the detection of filamentous fungi in blood cultures has a very low yield and is often associated with overwhelming systemic infection where fungi may be more easily isolated from other sites. The manual lysis-centrifu-gation tube assay (Isolator, Wampole Laboratories, USA) was one of the earliest methods for fungal blood culture. However, the method is labor-intensive and prone to contamination[88]. Automated fungal blood culture systems such as BACTEC Myco/F Lytic blood culture (Becton Dickinson, USA) are commercially available. However, these automated fungal blood culture bottles are more expensive than manual methods. They also take a dispropor-tionate amount of potentially limited incubator space since they require a six-week incubation period in contrast to five days for standard blood culture. Notably, recent studies suggest substantial clinical equivalence of standard blood culture (aerobic and anaerobic) and Myco/F Lytic culture for detection of yeast includingCandidaspp. andC.neoformans[25,89]. Therefore, the isolator tube assay and commercial fungal blood culture bottles should be reserved for detection of fastidious fungi such asH.capsulatumrather than for common yeasts includingCandidaspp. andC.neoformans[90,91]. Due to limited number of dimorphic fungi and filamentous fungal infections identified in these studies, further investigation will be helpful in determining the usefulness of current and new blood fungal culture systems for the detection of these classes of fungal pathogens.

Once fungi are grown in culture several rapid methods are now available for speciation. These include the germ tube test, trehalose assimilation test, and the peptide nucleic acid fluorescentinsituhybridization (PNA-FISH) (see the molecular section below). The germ tube test is a simple test for presumptive identification ofC.albicans[92,93]. Although this test can not differentiateC.albicansfrom the rarely isolatedC.dubliniensis, it is very inexpensive and simple. It is still widely used for presumptive identification ofC.albicansisolated from non-sterile sources, where definitive identification is not necessary because both species are usually sensitive to anti-fungal agents[94,95]. The trehalose assimilation test[96]is an additional rapid method (within 3 h) for presumptive identification ofC.glabrata[96-98], the second most common yeast isolated from blood stream infections.

Both manual and automated biochemical test panels are also available for identification of yeast isolated from clinical cultures. The traditional manual API 20C AUX yeast identification panel is labor intensive and can take up to 72 h for identification of some species. Automated yeast identification systems not only save labor, but also have faster turn around time, e.g., 18 h for the VITEK 2 system and 4 h for the MicroScan system[99]. The use of the ID-YST card of automated VITEK 2 system (BioMerieux, France) for identification of commonly isolated yeast species has become a well established method in clinical laboratories[100-102]. It was found that the earlier VITEK 2 ID-YST card had some problem with identification of certain species includingC.dubliniensis,C.inconspicua,C.norvenensis, andCryptococcusspp.[102,103]. However, a new colorimetric VITEK YST card, designed to replace the fluorometric VITEK ID-YST, was shown to perform well and demonstrated excellent reprodu-cibility. It correctly identified 98.2%-98.5% of clinical isolates[104,105]with 1.0% of isolates incorrectly identified and 0.5% unidentified[105]. Low-discrimination results were reduced from 30.0% with API to 18.9% with YST requiring less confirmative or supplement assays. For both YST and API, low-discrimination results were always observed forC.inconspicua,C.krusei,C.lambica, andGeotrichumcapitatum. This is due to the fact that these species are fairly un-reactive and very similar in their carbohydrate assimilation profiles. However, these species are isolated only rarely from clinical samples. YST has shown definite improvement in identification ofCryptococcusspp.

ChromogenicCandidaagar such as CHRO-Magar[106-109]and Oxoid BrillianceTMCandidaAgar (formerly Oxoid Chromogenic Candida Agar agar)[110]have been widely used forCandidaspp. speciation by color in primary culture media. The media incorporates color generating substrates that are differentially metabolized by different species, leading to a specific color signature. This allows rapid speciation immediately on isolation of certain species and also permits the identification of mixedCandidainfections that might be missed because of indistinguishable colony morphologies. Speciation ofC.albicans/C.dubliniensis,C.tropicalis, andC.kruseiis obtained with high specificity (up to 100%)[109].

5 Molecular methods for the detection and identification of fungi from clinical specimens and positive cultures

ManyCandidaspecies show fairly consistent susceptibility profiles to anti-fungal agents. Therefore, the Infectious Diseases Society of American Guidelines recommends selection of initial anti-fungal therapy based on species identification[111]. Thus, rapid identification ofCandidaspp. is critical as it directs the appropriate choice of anti-fungal agents until definitive susceptibility testing can be performed. Therefore, the identification of fungi as early as possible is important. No FDA-approved molecular tests are available for the detection of fungi directly from a specimen. However, recently PNA-FISH Kit (AdvanDx, USA) was found to be a reliable assay for identification ofC.albicansand/orC.glabratafrom positive blood culture broth[112-116]with a sensitivity of 99%-100% and a specificity of 100% respectively. A new PNA-FISH assay (Yeast Traffic Light) was approved by FDA in 2008 for rapid detection and differentiation ofC.albicans/C.parapsilosis,C.tropicalis, andC.glabrata/C.kruseifrom positive blood culture broth, separating species groups that are most likely to be either sensitive or resistant to fluconazole. A multiplex Tandem PCR assay has also been developed for the detection ofCandidaspp. and other fungi directly from positive blood culture broth[117]. It is highly specific and sensitive with a detection limit of 10 CFU/ml blood. The advantage of this assay is that it is simple, specific, commercially available, and automated (AusDiagnos-tics Pty. Ltd., Australia), of particular advantage for laboratory staff without molecular test experience. The whole procedure from DNA extraction to PCR analysis requires <4 h. Additional assays employing real-time PCR[118]with species-specific probes and post-amplification melting temperature analysis for speciation directly from positive blood culture broth, similar to commercially available methods for identifyingStaphylococcusaureus(S.aureus) and methicillin-resistantS.aureus, are likely to make this analysis even more straightforward. Another real-time PCR assay for detection ofAspergillusandCandidaspecies in clinical respiratory samples was developed as a potential tool for early diagnosis of infections by these fungi[119].

Molecular methods have also been developed for definitive identification of dimorphic fungi isolated from clinical cultures and from histopathology slides. Commercially available DNA probes (AccuProbe Culture Identification Reagent Kits, USA) have been FDA-approved for identification ofH.capsulatum,C.immitisandB.dermatiditis, and have been widely used for years[120-122]. These chemiluminescent-based hybridization assays have been shown to have excellent sensitivity (98%-100%) and specificity (98%-100%), and in addition produce results within 2 h. Very rarely, aChrysosporiumspp.[123]and anAspergillusniger[122]were found to give false-positive results; however, attention to colony morphology and microscopic appearance should prevent misidentification. Interestingly, the AccuProbe may also be used for rapid identification ofH.capsulatumin tissue samples when histopathology suggests the presence of this organism. A previous study successfully identifiedH.capsulatumin the way in excised hart valve tissue[124]. Furthermore, 18S and 28S rRNA probes were developed to identify all dimorphic fungi mentioned above and alsoS.schenckii[125]. Although Grocott Methenamine silver staining was about 10% more sensitive in detecting these pathogens,insituhybridization studies were 100% specific and aided speciation in cases with rare organisms with non-diagnostic morphologies. Therefore, this type of molecular analysis will likely aid interpretation of diagnostically difficult cases.

Several molecular methods have been established to identifyP.marneffeieither in cultures or directly from clinical specimens. Both single and a nested PCR were developed for rapid identification ofP.marneffeifrom clinical cultures using primers specific for the 18S rRNA gene sequence of this organism[126]. The limit of detection of these assays was 1.0 pg/μl and 1.8 fg/μl of purified DNA respectively. Another one-tube semi-nested PCR assay based on 18S rRNA gene sequences identifiedP.marneffeiDNA both from pure cultures and from 2/2 clinical samples from patients with establishedP.marneffeiinfection[127]. Lastly, a real-time PCR-based assay using primers for 5.8S rRNA gene sequence for identification ofP.marneffeiin blood cultures and directly from peripheral blood and positive blood culture broth has recently been published[128]. The limit of detection was 10 yeast cells/ml of seeded blood. The utility of these PCR methods for early diagnosis of the disease remains to be established through additional clinical studies. However, as with molecular techniques to identify other fungal pathogens, we expect that PCR methods such as these will likely be used with greater frequency in the future.

PCR has been shown to be useful for the diagnosis of invasiveAspergillusinfection directly from whole blood[129], serum[4,29], BAL[37,49]and biopsy tissues[130]. It has been found comparable toAspergillusGM testing and commercially available assays in real-time PCR format being available in Europe[131]. In fact, it showed enhanced sensitivity compared to GM testing when larger volumes (1 ml) of serum were analyzed[132]. Of note, such real-time PCR assays do not suffer from cross-reactivity issues found with the GM assay, approaching 100% analytic specificity.

PCR in combination with DNA sequencing is an emerging method for diagnosis of fungal infection in both clinical specimens and positive fungal cultures[133,134]. The MicroSeq D2 28S rRNA Gene Fungal-sequencing Kit (Applied Biosystems, USA) is commercially available which includes PCR and sequencing reagent, software, and fungal-sequence library. However, it is not FDA-approved and currently labeled for research use only in US. This MicroSeq Fungal-sequencing Kit demonstrated accurate identification of filamentous fungi and yeasts from clinical specimens[133,135]. However, identification of dermatophytes was limited due to the lack of relevant sequences in the library[136]. ViroDec (Roche Diagnostics), a web-based sequence analysis software, offers an alternative database to MicroSeq for fungal identification[137].

Multiplex probe assays also appear to hold promise. A recent study also reported a DNA microarray-based system for rapid identification of a large number of common fungal pathogens isolated from clinical samples includingCandidaspp.,C.neoformans,Aspergillusspp., dermato-phytes,Fusarium, andP.marneffei[138]. The universal fungal primers ITS1 and ITS4 were used to amplify the non-coding ITS regions (ITS1 and ITS2) as well as the 5.8S rRNA gene positioned between the ITS regions. Although only a small number of clinical isolates were studied, it showed to be quite discriminative with specificity of 100% to genus level and of 85% to species level identification. There were no clinical isolates ofP.marneffeitested in this study. Further evaluation is necessary for this test to be used in clinical laboratories. Luminex-based probe assays have also been described and are likely to be successfully applied as they have been for diagnosis of respiratory viruses in several commercially available methods[139].

6 Conclusions

Many new rapid assays have been developed to aid early diagnosis of fungal infections. These make use of several methodological approaches including antigen detection in clinical specimens; rapid, culture-based identification assays; and molecular diagnostic assays. Because of rapid advances in this field, the relative merits of these different approaches are still being defined. However, several conclusions and predictions can be made.

Firstly, simple antigen detection-based methods, are likely to widespread use. They require minimal investment in equipment and personnel for implemen-tation, and are potentially very rapid. Their use can also be target to specific clinical situations. For example, in appropriate geographic areas and patient populations, antigen tests forP.marneffeican rapidly establish diagnosis and allow clinicians to initiate appropriate therapy. They may even be developed as lateral flow-based sandwich enzyme immunoassays in a card format that will allow point of care testing in a doctor’s office, similar to currently used assays for rapid malaria diagnosis.

Secondly, there has been rapid development in the molecular diagnostics field. The development of automated extraction and real-time PCR technology will make more robust molecular diagnostic assays increasingly available to the diagnostic laboratories. The cost of molecular assays will continue to decrease, allowing the power of molecular diagnostic techniques to find an increasing presence in resource-poor areas. Once investment in a molecular diagnostics laboratory has been made, multiple different assays can be developed for both fungal and non-fungal pathogens. Therefore, we expect molecular methods will find increasing use in diagnosis of fungal infections. This advance will affect several aspects of diagnosis. For example, direct detection of fungi in clinical samples (e.g., invasiveAspergillusdetection in blood) will eventually replace antigen-based assays because of diagnostic specificity. Furthermore, molecular techniques will allow rapid speciation of cultured organisms, currently a laborious and slow process, depending on expert recognition of colonial and microscopic fungal morphology.

Taken together, the diagnostic advances described in this review will allow more rapid and specific diagnosis of fungal diseases. We expect that they will contribute to improved patient care.

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