Immunocompromised hosts are at special risk for invasive fungal infections (IFI). Traditional methods of diagnosis (e.g. culture and molecular methods) largely suffer from poor sensitivity, thus limiting their clinical utility. Enzyme immunoassay-based detection of fungal antigens represents an attractive, supplementary method for IFI identification and is the focus of this review.
by Phillip R. Heaton and Elitza S. Theel
Background
Invasive fungal infections (IFI) are a significant cause of morbidity and mortality in patients with hematologic malignancies and in hematopoietic stem cell transplant (HSCT) recipients. Although Aspergillus species and Candida albicans are among the most common agents of IFI, an increasing incidence of IFI due to other filamentous fungi (e.g. Fusarium, Zygomycetes) and non-albicans Candida (e.g. C. tropicalis, C. krusei, C. glabrata) has been reported. Currently, IFI diagnosis is based on clinical evaluation, radiologic imaging (both of which may lack clinical specificity) and culture-based laboratory findings. Unfortunately, culture of the aforementioned fungi from bronchoalveolar lavage fluid (BAL) and blood, the most commonly collected specimens in suspected IFI cases, suffers from poor sensitivity: only 45 to 60% of BAL specimens and up to 50% of blood cultures yield fungal growth [1]. Additionally, as some fungi are common in the environment (e.g. Aspergillus), providers must determine whether growth from BAL cultures is indicative of invasive disease or colonization of the respiratory tract. Finally, procedures to collect alternative specimens, including lung tissue, are often contra-indicated due to the critical state of the patient. These limitations have led to a clinical need for alternative methods to identify IFI – techniques independent of culture, which are both sensitive and specific. This demand has driven the development of novel assays to detect fungal biomarkers including the Aspergillus galactomannan (GM) antigen, the (1→3) β-D-glucan (BDG) polysaccharide common to many fungi, and the Candida mannan antigen (Mn-A). This brief review will discuss the clinical utility, advantages and limitations of GM, BDG and Mn-A detection assays in patients at risk for IFI.
Detection of the Aspergillus galactomannan antigen
Galactomannan, composed of a mannan core and highly immunogenic galactofuranosyl side chains, is a dominant cell wall component present in the majority of clinically relevant Aspergillus species and is released during hyphal growth into surrounding tissue (Fig. 1). Currently, the Platelia Aspergillus antigen (Bio-Rad, Marnes-la-Coquette, France) enzyme immunoassay (EIA) is the only FDA approved assay for GM detection of in serum and BAL fluid, though other kits are also available (e.g. Pastorex kit, Sanofi Diagnostics, Pasteur, Marnes-La-Coquette, France). The Platelia EIA is a quantitative assay with GM levels ≥0.5 ng/mL considered as positive. The presence of GM in patient specimens can be used as an aid, alongside other clinical studies, to specifically detect invasive aspergillosis (IA), a potentially devastating condition encountered in 5–20% of HSCT patients [2]. The performance characteristics of the Platelia GM assay have been widely evaluated with overall favorable outcomes. Briefly, one study reported a clinical sensitivity and specificity of 94.4% and 98.8% respectively, from serum of HSCT patients with proven or probable IA [as defined by the European Organization for Research and Treatment of Cancer (EORTC) criteria], with similar positive and negative predictive values [3]. While a subsequent meta-analysis of GM studies found a significantly lower sensitivity in this patient population (58%), specificity remained comparable at 95% [4]. Notably, these results are in stark contrast to the sensitivity of this assay in other immunocompromised (ICH) patient populations, specifically in solid organ transplant (SOT) recipients, where sensitivity can be as low as 22–41% [4]. Additionally, while the kinetics of GM clearance are not yet well defined, serial testing and trending of GM levels following initiation of antifungal therapy has been shown to correlate well with patient outcome. Specifically, while persistently elevated GM levels were associated with treatment failure, a decrease of GM levels by ≥35% between baseline and week one of antifungal treatment was associated with clinical improvement [5].
Despite the advantage of rapid GM testing in serum, a readily available specimen source, and the potential to monitor response to therapy, a number of limitations affecting assay specificity have been described. First, false-positive GM reactions have been associated with prior (<12 hours) administration of piperacillin/tazobactam, a fungal-derived antibiotic [6]. Recently, however, Mikulska and colleagues demonstrated negligible GM levels in patients on piperacillin/tazobactam therapy, suggesting that modern day manufacturing practices may have improved antibiotic purity [7]. Nonspecific reactions have also been noted in patients with non-Aspergillus IFI, including Fusarium and Penicillium (an exceedingly rare agent of IFI which also expresses GM) species infections and in individuals with either graft versus host disease (GVHD) or a damaged intestinal wall through which GM from food products can translocate [8].
Detection of β-D-glucan, a pan-fungal biomarker
(1→3)-β-D-glucan (BDG) is an abundant cell wall polysaccharide found in most fungi with the exception of Cryptococcus species, the Zygomycetes and Blastomyces dermatitidis (Fig. 2). The most commonly used BDG detection method, the Fungitell assay (Associates of Cape Cod, East Falmouth, MA), is a quantitative EIA (values ≥60 pg/mL considered positive) which detects BDG in serum using a modified version of the Limulus (horseshoe crab) clotting cascade. As a pan-fungal biomarker, BDG detection in patients with hematologic malignancies and HSCT recipients has been associated with high clinical specificity (76–99%) and negative predictive values (87–96%) for the presence of proven or probable IFI [9]. Similar to the GM assay however, inclusion of other ICH groups (e.g. SOT patients) dramatically lowers the performance characteristics of this assay. Interestingly, regardless of the patient population, the associated clinical sensitivity and positive predictive value of the BDG assay are generally poor (range 38 – 80%), collectively indicating that a single, negative BDG result should not be used to exclude the diagnosis of IFI [9]. Serial BDG testing, however, can significantly improve the clinical sensitivity of this assay and trending BDG levels during antifungal therapy has some prognostic value with respect to treatment failure or response, particularly in patients with disseminated candidiasis [9, 10]. Furthermore, BDG was shown to be detectable in critically ill patients prior to development of clinical symptoms, radiological findings or culture positivity, suggesting that in patients at increased risk for IFI, the presence of BDG should warrant further evaluation to identify an infectious process [11]. Finally, in patients with Pneumocystis jirovecii pneumonia (PjP), for whom invasive BAL or biopsy procedures are often precluded due to safety concerns, the demonstration of elevated BDG levels has been associated with high clinical sensitivity (>95%) [12]. Though this data is encouraging, especially in light of the limited sensitivity of current diagnostic methods to detect P. jirovecii, due to the pan-fungal nature of BDG, a positive result cannot be used to diagnose PjP pneumonia; a negative BDG finding can, however, be used to potentially exclude P. jirovecii as the causative agent.
The greatest limitation of BDG assays is their poor specificity. Many studies have now documented the generation of false-positive results in patients who received or have been exposed to albumin, intravenous immunoglobulin, amoxicillin-clavulanic acid, gauze during surgery, or cellulose based filters during dialysis. Additionally, infection with certain bacterial agents, including Alcaligenes faecalis, can also lead to false-positive results. Therefore, providers using these assays must confidently exclude these confounding factors prior to interpreting BDG results.
Detection of the Candida mannan antigen
The Candida Mn-A is an oligomannan cell wall component which can be detected by multiple quantitative EIAs (Fig. 3). Currently, the Platelia Candida Ag Plus quantitative EIA (Bio-Rad) is most commonly used. A recent meta-analysis of 14 studies evaluating the utility of Mn-A detection found significant heterogeneity in clinical sensitivity for detection of invasive candidiasis (IC), which, interestingly, appeared to be species dependent. For example, among patients with disseminated C. albicans, C. glabrata or C. tropicalis, the sensitivities ranged from 58–70%, whereas for patients with invasive C. parapsilossis and C. krusei, sensitivity of the assay ranged between 25–30% [13]. Importantly, however, despite the low sensitivity of the assay, the majority of Mn-A positive patients were subsequently confirmed as culture positive, suggesting the utility of this assay as an early diagnostic marker in at risk patients. Notably, the specificity of this assay is high (>90%) with cross-reactivity reported in patients with Geotrichum or Fusarium species infections, both fairly uncommon [14]. Due to the described performance variability and the short duration of Mn-A circulation, many authors have suggested combination testing with anti-Mn antibodies (Platelia Candida Ab Plus, Bio-Rad), which are detectable in at risk patients >10 days prior to proven candidemia [15]. One study evaluated neutropenic patients using combination testing and found that Mn-A/anti-Mn outperformed traditional diagnostic methods (cultures, radiology, and histopathology) for detection of IC with a sensitivity and specificity of 89% and 84%, respectively [15]. Based on these and other studies, current ECIL-3 recommendations support using a combination of Mn-A and antibody testing as an aid to detect IC [13].
Conclusions
The diagnosis of IFI in ICHs remains a challenge, and despite the limited sensitivity and specificity of the various fungal antigen detection assays, in 2008 the EORTC included detection of GM and BDG as supportive evidence for proven or probable IFI in specific patient populations [3]. When used appropriately (i.e. serial testing of high risk patients) and by providers knowledgeable of the associated limitations, antigen detection can be crucial marker for the identification of IFI. Future advancement of IFI diagnostics lies in the molecular arena and real-time polymerase chain reaction (RT-PCR) assays to detect fungal nucleic acid.
References
1. Singh N, Paterson DL. Aspergillus infections in transplant recipients. Clin Microbiol Rev. 2005; 18: 44–69.
2. Tamma P. The Galactomannan antigen assay. Pediatr Infect Dis J. 2007; 26: 641-642 610.1097/INF.1090b1013e318070c318525.
3. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo A, Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T, Bennett JE. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis. 2008; 46: 1813–1821.
4. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin Infect Dis. 2006; 42: 1417–1727.
5. Chai LY, Kullberg BJ, Johnson EM, Teerenstra S, Khin LW, Vonk AG, Maertens J, Lortholary O, Donnelly PJ, Schlamm HT, Troke PF, Netea MG, Herbrecht R. Early serum galactomannan trend as a predictor of outcome of invasive aspergillosis. J Clin Microbiol. 2012; 50: 2330–2336.
6. Machetti M, Majabo MJ, Furfaro E, Solari N, Novelli A, Cafiero F, Viscoli C. Kinetics of galactomannan in surgical patients receiving perioperative piperacillin/tazobactam prophylaxis. J Antimicrob Chemother. 2006; 58: 806–810.
7. Mikulska M, Furfaro E, Del Bono V, Raiola AM, Ratto S, Bacigalupo A, Viscoli C. Piperacillin/tazobactam (TazocinTM) seems to be no longer responsible for false-positive results of the galactomannan assay.
J Antimicrob Chemother. 2012; 67: 1746–1748.
8. Mennink-Kersten MASH, Donnelly JP, Verweij PE. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis. 2004; 4: 349–357.
9. Lamoth F, Cruciani M, Mengoli C, Castagnola E, Lortholary O, Richardson M, Marchetti O. Beta-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3). Clin Infect Dis. 2012; 54: 633–643.
10. Jaijakul S, Vazquez JA, Swanson RN, Ostrosky-Zeichner L. (1,3)-β-D-Glucan as a prognostic marker of treatment response in invasive candidiasis. Clin Infect Dis. 2012; 55: 521–526.
11. Odabasi Z, Mattiuzzi G, Estey E, Kantarjian H, Saeki F, Ridge RJ, Ketchum PA, Finkelman MA, Rex JH, Ostrosky-Zeichner L. Beta-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome. Clin Infect Dis. 2004; 39: 199–205.
12. Karageorgopoulos DE, Qu JM, Korbila IP, Zhu YG, Vasileiou VA, Falagas ME. Accuracy of β-D-glucan for the diagnosis of Pneumocystis jirovecii pneumonia: a meta-analysis. Clin Microbiol Infect. 2013; 19: 39–49.
13. Marchetti O, Lamoth F, Mikulska M, Viscoli C, Verweij P, Bretagne S. ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients. Bone Marrow Transplant. 2012; 47: 846–854.
14. Rimek D, Singh J, Kappe R. Cross-reactivity of the PLATELIA CANDIDA antigen detection enzyme immunoassay with fungal antigen extracts. J Clin Microbiol. 2003; 41: 3395–3398.
15. Prella M, Bille J, Pugnale M, Duvoisin B, Cavassini M, Calandra T, Marchetti O. Early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies. Diagn Microbiol Infect Dis. 2005; 51: 95–101.
The authors
Phillip R. Heaton PhD and Elitza S. Theel PhD*
Division of Clinical Microbiology,
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota, USA
*Corresponding author
E-mail: theel.elitza@mayo.edu
Diagnostics. It’s in our blood.
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, /in Featured Articles /by 3wmediaOlder fathers and morbidity in their offspring: what’s new ?
, /in Featured Articles /by 3wmediaAround sixty years ago an article was published in the Lancet demonstrating, for the first time, a link between advancing paternal age and the increased risk of a birth defect in their offspring, namely achondroplasia. In spite of the fact that in the six decades since then there have been numerous studies showing similar associations between the age of fathers and a variety of birth defects, relevant textbooks usually fail to mention these findings and until recently the popular press has resolutely ignored the topic. So while healthcare workers and indeed prospective parents are very much aware of the increased risk of morbidity in the infants of older expectant mothers, most remain blissfully ignorant of potential problems linked to advanced paternal age.
The most robust studies in the field have been carried out in the Nordic countries, where the age of prospective fathers has long been included in antenatal records. Advanced paternal age has been correlated with Down syndrome, as well as with many conditions resulting from autosomal dominant mutations, such as Apert syndrome, Marfan’s syndrome, osteogenesis imperfecta and neurofibromatosis. The risk of several common conditions with less straightforward inheritance patterns, such as cleft palate, increases with paternal age. Advanced paternal age has been correlated with an increased risk of recessive mutations on the X chromosome of female offspring as well. Data on sex-linked conditions such as Duchenne muscular dystrophy and hemophilia in male grandchildren have allowed this association to be demonstrated; presumably recessive autosomal mutations are also more common in the children of older fathers. Several recent studies have now linked increased paternal age at their children’s births with risk of neuropsychiatric disorders in the offspring. A very robust Danish population-based study that followed the health records of nearly 3 million individuals from birth found an increased risk of schizophrenia, mental retardation and autism in the offspring of older fathers. A Swedish/American population study of 2.6 million individuals concluded that increased genetic mutations during spermatogenesis in older men increased the risk of attention-deficit hyperactivity disorder, psychosis and bipolar disorder as well as autism in their offspring.
Such population studies are complex and their analysis can always be criticised, but surely we should not, yet again, try to sweep these results under the carpet, particularly since the trend in the West is towards delayed parenthood. Healthcare workers should be made aware of the increased risks to the offspring of older fathers as well as mothers, and relevant information should be disseminated so that older men are not lulled into reproductive irresponsibility.
Fungal antigen detection as an aid for diagnosis of invasive fungal infections
, /in Featured Articles /by 3wmediaImmunocompromised hosts are at special risk for invasive fungal infections (IFI). Traditional methods of diagnosis (e.g. culture and molecular methods) largely suffer from poor sensitivity, thus limiting their clinical utility. Enzyme immunoassay-based detection of fungal antigens represents an attractive, supplementary method for IFI identification and is the focus of this review.
by Phillip R. Heaton and Elitza S. Theel
Background
Invasive fungal infections (IFI) are a significant cause of morbidity and mortality in patients with hematologic malignancies and in hematopoietic stem cell transplant (HSCT) recipients. Although Aspergillus species and Candida albicans are among the most common agents of IFI, an increasing incidence of IFI due to other filamentous fungi (e.g. Fusarium, Zygomycetes) and non-albicans Candida (e.g. C. tropicalis, C. krusei, C. glabrata) has been reported. Currently, IFI diagnosis is based on clinical evaluation, radiologic imaging (both of which may lack clinical specificity) and culture-based laboratory findings. Unfortunately, culture of the aforementioned fungi from bronchoalveolar lavage fluid (BAL) and blood, the most commonly collected specimens in suspected IFI cases, suffers from poor sensitivity: only 45 to 60% of BAL specimens and up to 50% of blood cultures yield fungal growth [1]. Additionally, as some fungi are common in the environment (e.g. Aspergillus), providers must determine whether growth from BAL cultures is indicative of invasive disease or colonization of the respiratory tract. Finally, procedures to collect alternative specimens, including lung tissue, are often contra-indicated due to the critical state of the patient. These limitations have led to a clinical need for alternative methods to identify IFI – techniques independent of culture, which are both sensitive and specific. This demand has driven the development of novel assays to detect fungal biomarkers including the Aspergillus galactomannan (GM) antigen, the (1→3) β-D-glucan (BDG) polysaccharide common to many fungi, and the Candida mannan antigen (Mn-A). This brief review will discuss the clinical utility, advantages and limitations of GM, BDG and Mn-A detection assays in patients at risk for IFI.
Detection of the Aspergillus galactomannan antigen
Galactomannan, composed of a mannan core and highly immunogenic galactofuranosyl side chains, is a dominant cell wall component present in the majority of clinically relevant Aspergillus species and is released during hyphal growth into surrounding tissue (Fig. 1). Currently, the Platelia Aspergillus antigen (Bio-Rad, Marnes-la-Coquette, France) enzyme immunoassay (EIA) is the only FDA approved assay for GM detection of in serum and BAL fluid, though other kits are also available (e.g. Pastorex kit, Sanofi Diagnostics, Pasteur, Marnes-La-Coquette, France). The Platelia EIA is a quantitative assay with GM levels ≥0.5 ng/mL considered as positive. The presence of GM in patient specimens can be used as an aid, alongside other clinical studies, to specifically detect invasive aspergillosis (IA), a potentially devastating condition encountered in 5–20% of HSCT patients [2]. The performance characteristics of the Platelia GM assay have been widely evaluated with overall favorable outcomes. Briefly, one study reported a clinical sensitivity and specificity of 94.4% and 98.8% respectively, from serum of HSCT patients with proven or probable IA [as defined by the European Organization for Research and Treatment of Cancer (EORTC) criteria], with similar positive and negative predictive values [3]. While a subsequent meta-analysis of GM studies found a significantly lower sensitivity in this patient population (58%), specificity remained comparable at 95% [4]. Notably, these results are in stark contrast to the sensitivity of this assay in other immunocompromised (ICH) patient populations, specifically in solid organ transplant (SOT) recipients, where sensitivity can be as low as 22–41% [4]. Additionally, while the kinetics of GM clearance are not yet well defined, serial testing and trending of GM levels following initiation of antifungal therapy has been shown to correlate well with patient outcome. Specifically, while persistently elevated GM levels were associated with treatment failure, a decrease of GM levels by ≥35% between baseline and week one of antifungal treatment was associated with clinical improvement [5].
Despite the advantage of rapid GM testing in serum, a readily available specimen source, and the potential to monitor response to therapy, a number of limitations affecting assay specificity have been described. First, false-positive GM reactions have been associated with prior (<12 hours) administration of piperacillin/tazobactam, a fungal-derived antibiotic [6]. Recently, however, Mikulska and colleagues demonstrated negligible GM levels in patients on piperacillin/tazobactam therapy, suggesting that modern day manufacturing practices may have improved antibiotic purity [7]. Nonspecific reactions have also been noted in patients with non-Aspergillus IFI, including Fusarium and Penicillium (an exceedingly rare agent of IFI which also expresses GM) species infections and in individuals with either graft versus host disease (GVHD) or a damaged intestinal wall through which GM from food products can translocate [8].
Detection of β-D-glucan, a pan-fungal biomarker
(1→3)-β-D-glucan (BDG) is an abundant cell wall polysaccharide found in most fungi with the exception of Cryptococcus species, the Zygomycetes and Blastomyces dermatitidis (Fig. 2). The most commonly used BDG detection method, the Fungitell assay (Associates of Cape Cod, East Falmouth, MA), is a quantitative EIA (values ≥60 pg/mL considered positive) which detects BDG in serum using a modified version of the Limulus (horseshoe crab) clotting cascade. As a pan-fungal biomarker, BDG detection in patients with hematologic malignancies and HSCT recipients has been associated with high clinical specificity (76–99%) and negative predictive values (87–96%) for the presence of proven or probable IFI [9]. Similar to the GM assay however, inclusion of other ICH groups (e.g. SOT patients) dramatically lowers the performance characteristics of this assay. Interestingly, regardless of the patient population, the associated clinical sensitivity and positive predictive value of the BDG assay are generally poor (range 38 – 80%), collectively indicating that a single, negative BDG result should not be used to exclude the diagnosis of IFI [9]. Serial BDG testing, however, can significantly improve the clinical sensitivity of this assay and trending BDG levels during antifungal therapy has some prognostic value with respect to treatment failure or response, particularly in patients with disseminated candidiasis [9, 10]. Furthermore, BDG was shown to be detectable in critically ill patients prior to development of clinical symptoms, radiological findings or culture positivity, suggesting that in patients at increased risk for IFI, the presence of BDG should warrant further evaluation to identify an infectious process [11]. Finally, in patients with Pneumocystis jirovecii pneumonia (PjP), for whom invasive BAL or biopsy procedures are often precluded due to safety concerns, the demonstration of elevated BDG levels has been associated with high clinical sensitivity (>95%) [12]. Though this data is encouraging, especially in light of the limited sensitivity of current diagnostic methods to detect P. jirovecii, due to the pan-fungal nature of BDG, a positive result cannot be used to diagnose PjP pneumonia; a negative BDG finding can, however, be used to potentially exclude P. jirovecii as the causative agent.
The greatest limitation of BDG assays is their poor specificity. Many studies have now documented the generation of false-positive results in patients who received or have been exposed to albumin, intravenous immunoglobulin, amoxicillin-clavulanic acid, gauze during surgery, or cellulose based filters during dialysis. Additionally, infection with certain bacterial agents, including Alcaligenes faecalis, can also lead to false-positive results. Therefore, providers using these assays must confidently exclude these confounding factors prior to interpreting BDG results.
Detection of the Candida mannan antigen
The Candida Mn-A is an oligomannan cell wall component which can be detected by multiple quantitative EIAs (Fig. 3). Currently, the Platelia Candida Ag Plus quantitative EIA (Bio-Rad) is most commonly used. A recent meta-analysis of 14 studies evaluating the utility of Mn-A detection found significant heterogeneity in clinical sensitivity for detection of invasive candidiasis (IC), which, interestingly, appeared to be species dependent. For example, among patients with disseminated C. albicans, C. glabrata or C. tropicalis, the sensitivities ranged from 58–70%, whereas for patients with invasive C. parapsilossis and C. krusei, sensitivity of the assay ranged between 25–30% [13]. Importantly, however, despite the low sensitivity of the assay, the majority of Mn-A positive patients were subsequently confirmed as culture positive, suggesting the utility of this assay as an early diagnostic marker in at risk patients. Notably, the specificity of this assay is high (>90%) with cross-reactivity reported in patients with Geotrichum or Fusarium species infections, both fairly uncommon [14]. Due to the described performance variability and the short duration of Mn-A circulation, many authors have suggested combination testing with anti-Mn antibodies (Platelia Candida Ab Plus, Bio-Rad), which are detectable in at risk patients >10 days prior to proven candidemia [15]. One study evaluated neutropenic patients using combination testing and found that Mn-A/anti-Mn outperformed traditional diagnostic methods (cultures, radiology, and histopathology) for detection of IC with a sensitivity and specificity of 89% and 84%, respectively [15]. Based on these and other studies, current ECIL-3 recommendations support using a combination of Mn-A and antibody testing as an aid to detect IC [13].
Conclusions
The diagnosis of IFI in ICHs remains a challenge, and despite the limited sensitivity and specificity of the various fungal antigen detection assays, in 2008 the EORTC included detection of GM and BDG as supportive evidence for proven or probable IFI in specific patient populations [3]. When used appropriately (i.e. serial testing of high risk patients) and by providers knowledgeable of the associated limitations, antigen detection can be crucial marker for the identification of IFI. Future advancement of IFI diagnostics lies in the molecular arena and real-time polymerase chain reaction (RT-PCR) assays to detect fungal nucleic acid.
References
1. Singh N, Paterson DL. Aspergillus infections in transplant recipients. Clin Microbiol Rev. 2005; 18: 44–69.
2. Tamma P. The Galactomannan antigen assay. Pediatr Infect Dis J. 2007; 26: 641-642 610.1097/INF.1090b1013e318070c318525.
3. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo A, Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T, Bennett JE. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis. 2008; 46: 1813–1821.
4. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin Infect Dis. 2006; 42: 1417–1727.
5. Chai LY, Kullberg BJ, Johnson EM, Teerenstra S, Khin LW, Vonk AG, Maertens J, Lortholary O, Donnelly PJ, Schlamm HT, Troke PF, Netea MG, Herbrecht R. Early serum galactomannan trend as a predictor of outcome of invasive aspergillosis. J Clin Microbiol. 2012; 50: 2330–2336.
6. Machetti M, Majabo MJ, Furfaro E, Solari N, Novelli A, Cafiero F, Viscoli C. Kinetics of galactomannan in surgical patients receiving perioperative piperacillin/tazobactam prophylaxis. J Antimicrob Chemother. 2006; 58: 806–810.
7. Mikulska M, Furfaro E, Del Bono V, Raiola AM, Ratto S, Bacigalupo A, Viscoli C. Piperacillin/tazobactam (TazocinTM) seems to be no longer responsible for false-positive results of the galactomannan assay.
J Antimicrob Chemother. 2012; 67: 1746–1748.
8. Mennink-Kersten MASH, Donnelly JP, Verweij PE. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis. 2004; 4: 349–357.
9. Lamoth F, Cruciani M, Mengoli C, Castagnola E, Lortholary O, Richardson M, Marchetti O. Beta-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3). Clin Infect Dis. 2012; 54: 633–643.
10. Jaijakul S, Vazquez JA, Swanson RN, Ostrosky-Zeichner L. (1,3)-β-D-Glucan as a prognostic marker of treatment response in invasive candidiasis. Clin Infect Dis. 2012; 55: 521–526.
11. Odabasi Z, Mattiuzzi G, Estey E, Kantarjian H, Saeki F, Ridge RJ, Ketchum PA, Finkelman MA, Rex JH, Ostrosky-Zeichner L. Beta-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome. Clin Infect Dis. 2004; 39: 199–205.
12. Karageorgopoulos DE, Qu JM, Korbila IP, Zhu YG, Vasileiou VA, Falagas ME. Accuracy of β-D-glucan for the diagnosis of Pneumocystis jirovecii pneumonia: a meta-analysis. Clin Microbiol Infect. 2013; 19: 39–49.
13. Marchetti O, Lamoth F, Mikulska M, Viscoli C, Verweij P, Bretagne S. ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients. Bone Marrow Transplant. 2012; 47: 846–854.
14. Rimek D, Singh J, Kappe R. Cross-reactivity of the PLATELIA CANDIDA antigen detection enzyme immunoassay with fungal antigen extracts. J Clin Microbiol. 2003; 41: 3395–3398.
15. Prella M, Bille J, Pugnale M, Duvoisin B, Cavassini M, Calandra T, Marchetti O. Early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies. Diagn Microbiol Infect Dis. 2005; 51: 95–101.
The authors
Phillip R. Heaton PhD and Elitza S. Theel PhD*
Division of Clinical Microbiology,
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota, USA
*Corresponding author
E-mail: theel.elitza@mayo.edu
Clinical microbiology labs – gearing up for new challenges
, /in Featured Articles, Microbiology /by 3wmediaClinical microbiology laboratories were central to the tough but successful fight against infectious diseases in the 19th and first half of the 20th centuries, and resonate in the names of now-iconic figures from Jenner, Pasteur and Lister to Koch, Gram and Fleming.
Improved diagnostics of Tropheryma whipplei
, /in Featured Articles /by 3wmediaWhipple´s disease, a systemic and ultimately fatal infection with Tropheryma whipplei, is usually easily treated if diagnosed early enough. A novel real-time PCR protocol and fluorescence in situ hybridization provide an improved diagnosis. All results, however, need careful interpretation. We recommend involving specialized centres in the initial diagnosis and patient follow-up.
by Alexandra Wießner, Dr Annette Moter and Dr Judith Kikhney
Whipple’s disease: a fatal infectious disease
Tropheryma whipplei causes a rare, but fatal, bacterial infection: Whipple’s disease. This systemic disease can usually be cured by antibiotic therapy if detected early enough. The key challenge for physicians and microbiologists is to recognize the bacterial origin of various clinical symptoms in time. Diagnosis is still not trivial owing to the rarity of the disease, diverse and unspecific clinical symptoms, the fastidious nature of T. whipplei and the absence of non-invasive serological tests [1]. Improved diagnostic assays for the detection of T. whipplei are very valuable in combination with expertise to interpret the results for fast initiation of treatment.
T. whipplei belongs to the Gram-positive class of Actinobacteria and can be detected intracellularly in vacuoles or as extracellular bacteria in the tissue [2]. It is a slender rod shape, readily visible with Periodic Acid–Schiff (PAS) staining. T. whipplei strains can be cultured in an axenic culture medium supplemented with amino acids, but the slow growth rate means that culture is not an option for routine diagnosis of T. whipplei infection.
Symptoms of Whipple’s disease
In classical Whipple’s disease patients suffer from chronic diarrhoea, weight loss and fever. Molecular methods have detected isolated or systemic T. whipplei infection in almost every organ [joints, central nervous system (CNS), heart valves, skin, eye, lymph node, bone and lung], even in the absence of intestinal involvement. Depending on the location of infection, the symptoms may vary substantially. Often, the diagnosis of Whipple’s disease is delayed as the result of misdiagnosis as sero-negative rheumatoid arthritis, culture-negative endocarditis or neurological disorders. The involvement of the CNS is especially dramatic, as damage caused by the bacteria is often irreversible and antibiotic treatment may no longer be effective enough to cure the infection [3].
Transmission and asymptomatic carriage
To complicate the picture even more T. whipplei has been found in healthy carriers at an estimated prevalence in the population of <1–4% [4, 5]. This means that the detection of T. whipplei in stool or saliva may not be indicative of Whipple’s disease and in this case does not necessarily require antibiotic treatment. A higher prevalence has been found in high risk populations for direct or indirect faecal–oral transmission, such as sewage workers [5], homeless people and family members of Whipple´s disease patients. As T. whipplei is common in the environment, it is assumed that Whipple’s disease patients must have an immunological predisposition for developing a chronic infection instead of being only transiently colonized [6].
The current transmission model assumes that T. whipplei is taken up orally, probably in early childhood, leading to temporary asymptomatic carriage, self-limiting gastroenteritis, fever, or cough [1, 7, 8]. In most cases a protective humoral and cellular immune response prevents T. whipplei infection. However, in predisposed persons T. whipplei may spread systemically over the years resulting in Whipple´s disease.
Diagnosis of Whipple’s disease
Currently, Whipple’s disease is most often detected through PAS staining of biopsies from the lower duodenum or jejunum showing PAS-positive macrophages in the lamina propria. However, PAS staining can give false-positive results because of other infections, for example with nontuberculous mycobacteria, and also false-negative results because of low bacterial load [9]. Therefore, every positive PAS result should be confirmed by an independent method. Here, molecular techniques such as PCR are, so far, irreplaceable for providing a direct, valid species diagnosis. Several in-house PCR protocols are now successfully used to detect T. whipplei DNA [10, 11]. In patients without gastrointestinal manifestation of classical Whipple’s disease, sample specimens from the clinically affected organs, e.g. heart valves, lymph nodes, synovial tissue, cerebrospinal fluid (CSF) or brain biopsies, may be PAS-positive, whereas duodenal biopsies remain negative [1]. PCR was suggested for screening stool and saliva samples as the prevalence and load of T. whipplei is far higher in Whipple´s disease patients than in healthy controls [4]. Here, however, positive PCR results are no proof of infection compared to the direct detection of T. whipplei DNA in affected organs. Analysis of peripheral blood is also possible, but a negative PCR result will not rule out infection [12]. As with all PCR assays, results need to be carefully interpreted as the assay is prone to laboratory contamination (especially nested PCR protocols) or false-positive results because of nonspecific reaction conditions or primer design. Importantly, some positive PCR results in the past have been shown to be due to cross-reactivity, e.g. with Actinomyces odontolyticus [13].
Improved diagnostics of T. whipplei
A break-through for the diagnosis of Whipple’s disease that is specific and less prone to contamination is modern real-time PCR [5, 14]. We evaluated a real-time PCR assay targeting T. whipplei-specific segments within the rpoB gene on test strains and over 1000 clinical specimens in a national reference laboratory [14]. This assay proved to be specific, sensitive and substantially faster than a conventional in-house assay. The protocol includes two specific hybridization probes and, to our knowledge for the first time in T. whipplei diagnostics, a melting curve analysis. Both are crucial for the robustness and reliability of the assay. This applies especially to polymicrobial samples, such as saliva or stool, which contain numerous uncultured bacterial species with unknown DNA sequences. Here, the problem of unexpected probe binding with false-positive results remains and, therefore, PCR results should always be interpreted in the context of clinical and histopathological findings. An initial diagnosis of Whipple´s disease should not rely on only one isolated PCR result, and a confirmatory PCR (using a different target sequence, sequence analysis of ribosomal RNA sequence or genotyping PCR) is mandatory. In inconclusive cases a second PCR with an independent sample specimen is recommended.
Emerging techniques for the detection of T. whipplei
Besides PCR and PAS staining, additional methods such as immunohistochemistry or fluorescence in situ hybridization (FISH) are offered by specialized laboratories. These techniques are, as yet, not part of the routine work-up but provide promising insights. FISH uses fluorescently labelled probes that hybridize specifically with their target sequence in the intact bacterial cells (usually the 16S rRNA). Thus, FISH not only provides direct identification of T. whipplei but also visualizes the pathogen directly in the tissue context. Surprisingly, we found T. whipplei to be by far the most abundant cause of culture-negative endocarditis among the rare pathogens [15]. FISH revealed impressive infected areas in heart valves densely scattered with T. whipplei. In gut biopsies FISH reveals the amount and localization of single microorganisms in the tissue (Fig. 1). As with all microscopic techniques, however, FISH is less sensitive than PCR and will only give information on post-operatively obtained tissue and exclusively on the section investigated. Thus, a low bacterial load in the tissue might be missed. However, FISH is so far the only method bridging the gap between specific molecular biology and histopathology and, thus, might find broader application in the future.
Sampling for T. whipplei
Tissue specimens, such as small bowel biopsies in classical Whipple´s disease or samples of the affected organ in isolated T. whipplei infection, should be examined by PAS staining and PCR (Fig. 2). In the event of positive results, CSF should be tested by PCR to check for CNS involvement. For isolated T. whipplei infections gastrointestinal involvement should be controlled as well. Fluid samples, such as CSF, etc., should be examined by PCR.
For histological examination, PAS staining and FISH the samples should be fixed in 10% formalin and transported at room temperature. For PCR the samples need to be native (no formalin pre-treatment!) and can be transported at room temperature within one day. Samples can be stored for a few days at 4°C and should be kept at –80°C for long-term storage.
Conclusions
The recent development of real-time PCR protocols with hybridization probes for the specific detection of T. whipplei provides accurate and fast results in the challenging clinical situation of Whipple´s disease. However, due to the variety of clinical symptoms, asymptomatic carriage, isolated and systemic infection, as well as false positive and negative results all examinations need careful interpretation in specialized centres. Clinical and histopathological facts always have to be taken into account. Emerging techniques such as FISH might in the future close the gap between molecular biology and histopathology. Together clinical and microbiological expertise are the key to the fast and successful treatment of Whipple´s disease. Similarly, after initial diagnosis and initiation of treatment, it is highly recommended to follow each patient in specialized centres during and after antibiosis to keep relapses at bay.
Acknowledgements
We thank the Robert Koch Institute for its continuous support.
Funding Sources
This study was supported by the Robert Koch Institute (RKI). The epifluorescence microscope was a gift from the Sonnenfeld-Stiftung.
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The authors
Alexandra Wießner1, Annette Moter1* MD, Judith Kikhney1,2 PhD
1 Center for Biofilms and Infection, German Heart Institute Berlin, Berlin, Germany
2 Institut für Mikrobiologie und Hygiene, Charité University medicine Berlin, Berlin, Germany
*Corresponding author
E-mail: moter@dhzb.de