The cornerstone to improving outcomes in invasive fungal infections in children is early, accurate diagnosis. With the sensitivity of blood culture for candidemia estimated to be approximately 50%, and the likelihood of Aspergillus growing in culture from bronchoalveolar lavage even worse, newer molecular fungal biomarkers are paramount to achieving that overall clinical goal. Often the patients at risk for invasive fungal infections are fragile enough that a blood-based strategy is all that can be safely employed. This brief review will highlight newer developments and thoughts focused only on blood-based fungal biomarkers for immunocompromised children (Table 1).
The oldest of the major molecular fungal biomarkers, a noninvasive serum test for the early diagnosis of invasive aspergillosis, is the Platelia Aspergillus galactomannan (GM) enzyme immunoassay (Bio-Rad, Marnes-la-Coquette, France) that has been available in Europe since 1997 and was approved by the US Food and Drug Administration (FDA) in 2003 for use in adult patients, whereas US pediatric approval came in 2006. The commercially approved assay uses a sandwich enzyme-linked immunosorbent assay technique using a rat anti-GM monoclonal antibody (EB-A2) which recognizes galactofuran epitopes of the GM molecule as both a capture and detector antibody. The antigen is released from the fungus as pure polysaccharide, but the number and nature of epitopes may vary between strains, species and over time. The enzyme-linked immunosorbent assay detects only antigens with 4 or more 1→5-β-D-galactofuranosyl residues, so technically the antigens released and detected are better called galactofuranose (galf) antigens.1 The GM assay is positioned to detect only Aspergillus, but over the years there are reports of fungal cross-reactivity, more commonly with Fusarium, Paecilomyces, Blastomyces, Histoplasma, Cryptococcus, Trichosporon, Penicillium and others. Several reports have even suggested the possible utility of the Aspergillus GM assay in diagnosing fusariosis to exploit this cross-reactivity.
Since its approval, there have been numerous publications in the last decade examining the diagnostic utility of the GM assay in individual subpopulations and under different clinical scenarios. The cutoff value in serum adopted by the current international guidelines is an optical density index of 0.5, and a meta-analysis found that the GM assay possesses an overall sensitivity of 71% and a specificity of 89%, highest in hematology malignancy patients and hematopoietic stem cell recipients, but that the sensitivity drops to only 22% in solid organ transplant recipients.2 The GM assay is consistently most useful in hematologic malignancy patients undergoing chemotherapy and those following hematopoietic stem cell transplantation, but has high false-negative results in patients with chronic granulomatous disease.3 In addition, sensitivity is greater in neutropenic patients,4 likely because of a more angioinvasive fungal pathogenesis, and is unfortunately lowered in the setting of mold-active antifungal therapy.5 Previously, piperacillin– tazobactam was found to yield false-positive reactions with the GM assay; however, in recent years, because of manufacturing changes, this antibacterial drug no longer cross-reacts with the GM assay.6
There have been limited reports with the GM assay in children, with the major studies recently reviewed.7 Although early pediatric data suggested an unacceptably high false-positive rate, that misconception has been disproven by multiple subsequent studies that support the utility of the GM assay in pediatric patients, including similar sensitivity and specificity profile in children as seen in adult patients.8–12 It appears that age and weight are not factors in GM assay performance in children. The current pediatric-specific European guidelines for the use of GM suggest similar test operating characteristics and cutoff values compared with adult patients, and that the use of GM for monitoring in children is reasonable.13
1,3-β-D-Glucan is an integral cell wall glucose polysaccharide found in the majority of fungi. Factor G, a coagulation factor of the horseshoe crab, is a highly sensitive natural detector of 1,3-β-D-glucan via a modified limulus endotoxin assay. Factor G is prepared from the amebocytes of either the North American horseshoe crab or the Japanese horseshoe crab, resulting in different chromogenic reactivities and different cutoff values for the various assays. These 2 different species of horseshoe crab are the basis for 4 different commercially available assays, each relying on lysate from the respective specific horseshoe crab. Fungitell (Associates of Cape Cod, East Falmouth, MA) is approved and available in the US and Europe and is based on the North American horseshoe crab, whereas the 3 other assays, Fungitec G (Seikagaku, Tokyo, Japan), Wako (Wako, Hyogo, Japan) and Maruha (Maruha-Nichiro, Tokyo, Japan) are only available in Japan and use the Japanese horseshoe crab as their analytical foundation.
Unlike the GM assay, this assay is more nonspecific and detects several different fungi, including Aspergillus spp., Candida spp., Fusarium spp., Trichosporon spp., Saccharomyces cerevisiae, Acremonium, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii and Pneumocystis jiroveci. The 1,3-β-D-glucan assay does not detect Cryptococcus and the yeast form of Blastomyces dermatitidis (which produce low levels of 1,3-β-D-glucan) or Absidia, Mucor and Rhizopus (which produce no 1,3-β-D-glucan). Importantly, this assay does not identify the genus of the fungi detected, but only the presence of the fungal cell wall component. A meta-analysis of adult hematology/oncology patients determined that the sensitivity and specificity of 2 consecutive tests as 49.6% and 98.9%, respectively.14
The 1,3-β-D-glucan assay was FDA-approved in 2004. False positives with the assay include blood products, hemodialysis, surgical gauze, piperacillin–tazobactam, ampicillin–clavulanate, mucositis and others (reviewed by Clancy and Nguyen15). The initial pediatric study examined normal values in uninfected children and found that 1,3-β-D-glucan baseline values, and therefore possibly cutoff values, were approximately one-third higher in children than in adults.16 This concern was validated in a study of 61 neonates where the optimal cutoff for distinguishing candidiasis was 125 pg/mL (instead of the standard 80 pg/mL used in adult patients).17 Therefore, the pediatric cutoff value is currently unclear and requires dedicated prospective study and the assay should be interpreted with caution in children. Over the last decade, there have been very few reports in children, all only small case series, except 1 Chinese study of 130 pediatric patients using a different assay kit that showed a sensitivity of 81.8%; however, it is unclear if those values are reproducible with more readily available commercial assays.18
FUNGAL POLYMERASE CHAIN REACTION
Nucleic acid–base detection of fungal DNA through polymerase chain reaction (PCR) has been investigated for years. There are reports of fungal genus-specific PCR assays (largely Aspergillus and Candida), and more pan-fungal approaches, with most studies focusing on invasive aspergillosis. Aspergillus PCR is somewhat mechanistically challenging, as it is unclear in what form Aspergillus circulates in the bloodstream, validated clinically by the very low sensitivity of blood cultures for invasive aspergillosis. A meta-analysis of blood-based Aspergillus PCR in high-risk hematology patients found a sensitivity of 84% and a specificity of 76%, but that specificity increased to 94% with at least 2 positive PCR results.19
PCR technology has several attractive attributes, such as theoretically higher sensitivity, because of the nature of the molecular platform used. However, PCR has several known technical drawbacks, including a lack of methodologic standardization, including sample preparation, specific primers and gene targets, and reaction format. To address these concerns, a European Aspergillus PCR Initiative was established to optimize protocols and accelerate routine clinical use.20 Data in children are limited and results have been mixed, with sensitivities ranging from approximately 30% to 80% depending on the study, specific patient population and selection of PCR assay. This wide range mirrors, in some part, the similar challenges found from PCR studies of adult patients.
Candida MANNAN ANTIGEN AND ANTI-MANNAN ANTIBODY
Cell wall mannan comprises approximately 7% of Candida cell dry weight and is a major circulating antigen during infection. The Candida mannan antigen and anti- mannan antibody assays are available in Europe (Bio-Rad Platelia Candida antigen and Platelia Candida antibody) and are felt to be best used in combination, resulting in a sensitivity of 83% (increasing from approximately 60% for the individual assays) and a specificity of 86%. Sensitivity appears to be best for Candida albicans, followed by Candida glabrata and Candida tropicalis.21 These assays have unfortunately only been reported in small case series in children so pediatric-specific studies will need to be completed to develop recommendations.
FUTURE OF PEDIATRIC FUNGAL BIOMARKERS
Although GM can be readily used in children with likely the same limitations as in adult patients, the 1,3-β-D-glucan assay requires more pediatric research for fundamental kinetics, such as cutoff values and studies in larger pediatric series. GM and the 1,3-β-D-glucan assay are the only approved fungal biomarkers listed above that are incorporated as indirect testing in the international consensus diagnostic guidelines for invasive fungal disease.22 Fungal PCR in children is still largely unclear, but likely includes the same technical limitations seen in adult patients. Similarly, the recently FDA-approved T2 detection methodology,23 using magnetic resonance to detect Candida colony-forming units, is promising for the detection of Candida in adults, but has never been examined in children to date.
The future optimal diagnostic strategy for invasive fungal infections will likely not involve a single assay, with all its inherent limitations, but a strategy that utilizes multiple tools to increase performance. Therefore, we need to fully establish the operating characteristics of each fungal biomarker, individually and in combination, and specifically in children to design the optimal approach, The International Pediatric Fungal Network (www.ipfn.org) is a multinational consortium that investigates the epidemiology, diagnosis, treatment and outcomes of pediatric invasive fungal infections.24 A recently begun clinical study (NCT02220790) called “BIOmarkers in Pediatric Invasive Candidiasis” (BIOPIC) will prospectively enroll 500 children at high risk for developing invasive candidiasis and test 4 currently approved molecular assays for detection of Candida: 1,3-β-D-glucan, mannan antigen, anti-mannan antibody and the T2Candida platform. In addition, extra sera, DNA and RNA will be captured to discover novel biomarkers. This 4-year study will be the largest study of any fungal biomarker in any age group, and for the first time will generate pediatric-specific data from which we hope to develop guidelines for an optimal strategy to use blood-based fungal biomarkers in children.
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