Molecular Diagnostics for Invasive Molds: From Lab to Bedside

BACKGROUND

Fungal infections are increasingly recognized as a cause of morbidity and mortality, particularly in immunocompromised patients. As the global population of individuals who are immunocompromised due to novel chemotherapies, immunomodulatory therapies, and improved transplantation strategies grows, it is imperative to develop and use improved methods to diagnose invasive fungal disease (IFD) in this high-risk population. A key innovation for the diagnosis of IFD in high-risk populations has been the development and clinical scale-up of molecular tests. While encompassing a wide range of technical variation, these tests fundamentally rely upon the detection of genetic material specific to a suspected (or unsuspected) fungal pathogen, which can in some cases also be used to deduce phenotypic characteristics of the pathogen, such as drug resistance. These tests include polymerase chain reaction (PCR) and metagenomic sequencing-based platforms (Table 1). In this review, we discuss the state of current molecular IFD diagnostics in high-risk, immunocompromised populations, with a focus on molds. We provide practical guidance on the utilization of commercially available assays and platforms for the identification of fungus.

Table 1.

Molecular testing modalities for invasive fungal disease

Testing Modality	Pathogen Specific or Agnostic	How it Works	Number of Pathogens Detected	Examples of Assays
Single-pathogen PCR	Very pathogen-directed	PCR amplification of family-specific, genus-specific, or species-specific fungal DNA	+	MycAssay Aspergillus
Multiplex PCR	Somewhat pathogen-directed	Parallel PCR amplification of family-specific, genus-specific, or species-specific fungal DNA, using a single specimen (ie, multiple individual PCR tests performed at once)	++	Fungal Plus PCR Profile
Broad range PCR	Pathogen-agnostic	PCR amplification of highly conserved fungal genetic regions (typically ribosomal subunit and internal transcribed spacer sequences), followed by Sanger sequencing of amplified DNA for fungal identification	+++	University of Washington Broad Range PCR assay; ARUP ITS rDNA Sequencing
Next-generation sequencing	Pathogen-agnostic	High throughput parallel sequencing of microbial DNA (“shotgun sequencing”), followed by matching with a known pathogen library	+++	Karius; NeXGen Fungal/AFB NGS assay; MicroGenDx

The development of molecular tests for IFD has occurred in parallel with changes in both complexity and capacity in the field of medical mycology. Recent years have seen declines in the number of clinical mycologists and skilled technicians who are able to identify mold phenotypically, and many hospital clinical microbiology laboratories have shifted fungal diagnostics to large commercial laboratories.¹ Taxonomic nomenclature continues to change and increase in complexity for a number of medically important fungi. Furthermore, advances in proteomics,² molecular diagnostics,³ and biomarker identification have allowed for rapid diagnosis of fungal infections either directly from cultured isolates or from clinical specimens.

The diagnosis of invasive fungal infections has historically relied on the identification of isolates grown in culture from clinical samples, as well as evidence of infection on microscopy and histopathology. From tissue pathology, special stains such as Grocott methenamine silver or Periodic acid-Schiff stains can help microbiologists visualize the presence of hyphal forms. On pathologic specimens, important consideration is given to whether there is evidence of tissue invasion. While species or genus level identification is not possible from tissue pathology alone, information such as hyphal morphology or presence of pigmentation can give clues as to the identification of the fungi. Fungal identification from culture typically involves phenotypic characterization of hyphae and conidia, allowing for genus and often species level identification. Growth of fungi in culture can take up to 4 weeks,⁴ and the sensitivity of this method is low.⁵ Once growth does occur, phenotypic identification requires considerable time and expertise.

The limited sensitivity of pathologic examination and culture, plus the concern that invasive sampling, is often not feasible or safe among many immunocompromised individuals, has motivated development and use of orthogonal testing in this population, such as biomarkers and molecular assays to more rapidly identify fungal infections. The technology required for these diagnostic modalities is rapidly improving and commercial availability is expanding.^6,7 However, substantial uncertainty remains about whether the current body of clinical evidence supports routine use of advanced, and often expensive, molecular diagnostics.

Molecular diagnostics have mostly been studied and used in immunocompromised adults and children. Molecular testing may be more useful in immunocompromised patients who have a higher pretest probability of fungal infection.^8-10 In particular, the extent of neutropenia has been shown to play a role in the reliability of some molecular testing.^11,12 Meanwhile, antifungal prophylaxis, which is relatively common among immunocompromised populations, may reduce the sensitivity of these tests, though more data are needed to evaluate the impact of antifungal prophylaxis or therapy on the reliability of molecular testing.^13-15 Understanding the scope of—and gaps in—clinical evidence supporting the use of these diagnostic modalities is critical for clinicians, microbiologists, and laboratorians who contribute to the care of immunocompromised individuals.

Singleplex and Multiplex Polymerase Chain Reaction Platforms

Fungal PCR assays that detect a single fungal family, genus, or species are becoming increasingly available for clinical practice. Assays designed to detect Aspergillus spp and Mucorales are commercially available for clinical use, though their acceptable specimen types and performance characteristics vary greatly and few have been evaluated in clinical practice independent of manufacturer-sponsored studies (Table 2). Standardization of these assays and clinical evaluation in different clinical centers and settings are needed to understand the role of these assays in routine practice.

Table 2.

Single-pathogen and multiplex fungal polymerase chain reaction panels in clinical use

Assay Name	Manufacturer (Location)	Organisms Detected/Gene Targets	Specimen Type/Population Studied	Sensitivity/Specificity
MycAssay Aspergillus	Myconostica (UK)	18 different Aspergillus species (including A fumigatus, Aspergillus flavus, A terreus, and Aspergillus niger) Targets 18S rRNA gene	BAL, serum HM	Respiratory specimens Sensitivity: 86%–94% Specificity: 46%–75% Serum specimens Specificity 97.6%54
AsperGenius	PathoNostics (Netherlands)	Multiplex real-time PCR (A fumigatus, A terreus, and pan-Aspergillus species) Can detect 4 mutations in CYP51A gene of A fumigatus that are typically associated with azole resistance (TR34, L98H, Y121 F, and T289 A)	BAL HM and other ICH	Respiratory specimens Sensitivity: 68%–84% Specificity: 80%–92%39,55
MycoGenie Aspergillus-Mucorales	Ademtech (France)	Real-time duplex PCR 28S rDNA of Aspergillus genus and the Mucorales order Does not allow identification to the species level	Serum CAPA, Other ICH	Aspergillus Sensitivity: 71% Specificity: 92% Mucorales Sensitivity: 80% Specificity: 99%56
MucorGenius	PathoNostics (Netherlands)	Rhizopus spp, Mucor spp, Lichtheimia spp, Cunninghamella spp, and Rhizomucor spp Targets 28S rRNA (exact sequences have not been disclosed by the manufacturer)	Serum, BAL HM, SOTR, and other ICH	Sensitivity: 85.2%–90% Specificity: 89.8%–100%57-59
Fungal Plus PCR Profile	Eurofins-Viracor (USA)	Multiplex PCR Pan-Aspergillus species, A fumigatus, A terreus, Mucorales, and Nocardia sp	BAL HM, SOTR, and other ICH	Aspergillus Sensitivity: 35% Specificity 97% Mucorales Sensitivity: 0% Specificity: 98.5%60

Abbreviations: BAL, bronchoalveolar lavage; CAPA, COVID-19-associated pulmonary aspergillosis; HM, patients with a hematologic malignancy; ICH, immunocompromised hosts; SOTR, solid organ transplant recipients.

In the United States, several reference laboratories offer fungal PCR either for a single pathogen or as part of multiplex panels. For example, Eurofins Viracor offers the Fungal Plus PCR Profile for pan-Aspergillus species, Aspergillus fumigatus, Aspergillus terreus, Mucorales, and Nocardia that can be run on bronchoalveolar lavage (BAL) samples. The inclusion of A terreus is notable because this species is usually amphotericin B-resistant. ARUP Laboratories offers a PCR assay that detects Aspergillus genus organisms and also specifically detects A fumigatus. ARUP Laboratories also has a pan-Mucorales PCR that can be run on a variety of specimen types. The University of Washington offers a commercial pathogen-specific PCR for Aspergillus species, Mucorales, and Coccidioides species for tissue samples. Several similar assays are only available outside of the United States, including MycAssay Aspergillus (Myconostica, UK), AsperGenius (PathoNostics, Netherlands), MucorGenius (PathoNostics, Netherlands), and MycoGenie Aspergillus-Mucorales (Ademtech, France).

The role of single pathogen or multiplex PCR testing for clinical use has yet to be fully defined in the United States. PCR for Aspergillus in serum and respiratory specimens has been recently included in the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium consensus guidelines as a mycological criterion to define probable invasive aspergillosis, owing to the increasing use and standardization of PCR testing.¹⁶

PATHOGEN-AGNOSTIC PLATFORMS

Single-pathogen and multiplex PCR testing requires clinicians to speculate on the pathogenic species of interest and choose an assay that detects that pathogen. In contrast, pathogen-agnostic platforms, including broad-range PCR and next-generation metagenomic sequencing (NGS), can detect a wide number of bacterial, viral, fungal, and mycobacterial organisms in clinical specimens.

Broad Range Polymerase Chain Reaction

Broad range PCR assays involve amplification of highly conserved, specific fungal genetic sequences—typically 18S or 28S fungal ribosomal genes or internal transcribed spacer sequences—in clinical specimens, followed by Sanger sequencing for species determination. An advantage of this method is that it can theoretically detect any of thousands of fungi. These assays can be performed on a number of clinical specimens, including fresh frozen and paraffin-embedded tissue, as well as nonbloody body fluid. Downsides of this method include low sensitivity in the setting of low fungal burden, as well as challenges with Sanger sequencing speciation for polymicrobial infections.¹⁷ A number of commercial laboratories offer broad range PCR testing, including the University of Washington and ARUP. The University of Washington’s assay uses amplification of fungal 28S and internal transcribed spacer sequence DNA to detect fungal pathogens from any tissue source, followed by sequencing. This test is most clinically useful when fungal elements are seen on microscopy but are difficult to grow in culture.¹⁸

Literature describing clinical use of these assays has reported heterogeneous performance and utility. Sensitivity is highly dependent upon organism burden, with studies showing low (<60%) sensitivity in culture-negative specimens¹⁹ to greater than 96% in patients with IFD established through other diagnostic methods.²⁰ The real-world clinical utility of these tests as measured by their impact on clinical management has been variable. Some studies suggest substantial added benefit, while others report minimal added utility of these tests beyond the information provided by traditional fungal diagnostics.^20-22 However, there is growing consensus that these tests are most likely to be useful to diagnose IFD when obtained from tissue-based specimens that have corresponding inflammation or organisms seen on pathology,²³ in the context of culture-negative workup.^22,24

Next-Generation Metagenomic Sequencing

NGS for pathogen diagnostics refers to high throughput sequencing of all DNA elements in a clinical specimen using “shotgun sequencing.” Sequencing is followed by bioinformatics processing to filter out signals from human and nonpathogen DNA, and signaling DNA from potential pathogens. A number of NGS-based tests have been developed, including tests on blood, cerebrospinal fluid (CSF), and synovial fluid specimens. Due to its rapid processing time, low specimen volume requirements, and ability to theoretically identify thousands of pathogens, there has been growing interest in using NGS to diagnose fungal infections.

One of the most appealing uses of NGS is to diagnose IFD using sequencing of cell-free DNA (cfDNA) in blood. CfDNA NGS analyzes small fragments of DNA that are freely circulating in the bloodstream. Pathogens causing local infections release cfDNA into the bloodstream, and this DNA is extracted from plasma and sequenced using NGS.²⁵ CfDNA is particularly useful because it enables pathogen detection without the need to recover pathogens directly from the infected organ and does not require organisms to grow, potentially sparing patients from high-risk diagnostic procedures and shortening time to diagnosis. Karius is the most widely used cfDNA NGS assay. This test requires 1.2 mL of plasma and has a turn-around-time of 1 to 3 days. Through automated DNA extraction and use of a microbe database of over 20,000 microbe assemblies, 267 fungi can be detected. As with all cfDNA testing, Karius does not rely on organism growth.²⁶

The performance of Karius has been characterized within specific populations, including hematopoietic stem cell transplantation (HCT) recipients,²⁷ patients with neutropenic fever,¹¹ and patients with suspected endocarditis.²⁸ In one study of patients who had undergone HCT and had proven or probable pulmonary IFD, cfDNA was found to have high specificity, negative predictive value, positive predictive value, and moderate sensitivity.¹³ Among patients diagnosed with Aspergillus, cfDNA was complementary to serum galactomannan and when used together, were positive in over 80% of cases of proven or probable pulmonary aspergillosis. In a study of patients with hematologic malignancies undergoing BAL for pneumonia evaluation, Karius identified the cause in 23% of cases when noninvasive testing was negative, and results led to optimized antimicrobial therapy in 81% of patients.²⁷ However, the added diagnostic benefit was much higher for bacterial rather than fungal etiologies. In another study, cfDNA was able to identify pathogenic mold up to 3 weeks prior to clinical IFD diagnosis, and often prior to use of invasive diagnostics.²⁹

NGS assays have also been developed to use nonblood clinical specimens. Viracor Eurofins developed the NeXGen Fungal/AFB NGS assay (Eurofins Viracor, Lenexa, KS 66219) specifically for IFD and acid-fast bacteria. This test can be run on blood, tissue or respiratory samples, with a turn-around-time of less than 72 hours. The test is reportedly 96% specific and is validated for 94 fungal species. MicroGenDx is an NGS method that has been validated for CSF, synovial fluid, hardware/heart valve, sputum and saliva, respiratory swabs, tissue drainage, and urine. This test can be reimbursed by Medicare.³⁰ Additional studies using in-house, noncommercial assays NGS on respiratory specimens demonstrated increased identification of pathogens for patients on antibiotics, identification of fastidious organisms, and identification of acquired resistance genes,^31,32 suggesting future directions of NGS development for clinical care.

Clinically, there is substantial debate about the appropriate patients, syndromes, and timing to use NGS diagnostic tests. The test is often used to identify a pathogen in patients with an infectious syndrome but negative microbiologic workup. Studies in heterogeneous patient populations have differed regarding the clinical utility of Karius testing,^10,33 with some studies finding limited added diagnostic benefit compared to conventional testing.^34,35 Additional limitations include the potential for overtreatment with the initiation of antifungal therapeutics when a pathogen is detected that is later deemed to be clinically insignificant. Although Karius reports quantitative units of pathogens, as of yet, there is not a threshold to distinguish colonization from infection. In some cases, even when care was tailored to the Karius result, there was clinical uncertainty as to whether the organism was the true underlying driver of pathology.³⁵ Finally, the optimal timing of testing and the role of quantitative NGS results remain to be determined. As Karius remains costly, there is a trade-off between sending the test upfront to avoid additional expenses later and waiting for less expensive conventional methods to be performed first.

ANTIFUNGAL RESISTANCE TESTING

Molecular testing has a promising but unproven role in antifungal resistance testing. Traditional methods of antifungal resistance testing pose many challenges, including uncertain or unestablished breakpoints, frequent discordance between resistance testing and clinical outcomes, long turn-around times, and need to send fungal isolates to reference laboratories to test susceptibilities to many antifungals. Leveraging genetic mutation-based resistance testing could overcome many of these challenges.

Among the most well-studied arenas of molecular resistance testing in mold-related IFD is the use of novel assays to detect azole resistance in Aspergillus species, and several commercial tests have been developed. These tests assess for resistance-conferring mutations in the Cyp51 A gene, which encodes a protein necessary for ergosterol synthesis that is inhibited by azole antifungals and is the most common source of acquired azole resistance among Aspergillus.³⁶ AsperGenius (Pathonostics, Netherlands) is a multiplex PCR that can detect the three most common mutations of Cyp51A that confer resistance to triazoles (TR₃₄/L98H, Y121F, and TR₄₆/Y121F/T289A).^37,38 The assay can also distinguish both wild-type and mutant Cyp51 A DNA seen in mixed infections. This assay was evaluated on banked BAL samples, in which 14 out of 14 A fumigatus isolates were positive for resistance targets.³⁹ MycoGENIE (Ademtech, France) and Fungiplex are PCR assays that detect Aspergillus DNA and assess for 2 Cyp51 A mutations (MycoGENIE: L98H and TR₃₄ mutations; Fungiplex: TR34/TR46 mutations).⁴⁰ Other noncommercial tests probe for additional mutations that may enable differentiation of resistance to different azoles (eg, itraconazole-posaconazole cross-resistance vs itraconazole resistance alone).³⁷

Despite its promise, current molecular resistance testing for Aspergillus has limitations. First, while the Cyp51 A gene mutation has been well described in European populations, it may be less relevant to countries or regions where Cyp51 A mutations are a less-common driver of Aspergillus triazole resistance.^41,42 Second, not all azole resistance is mediated through Cyp51 A mutations, and current assays test for mutations implicated in only about half of Cyp51A-mediated Aspergillus resistance.⁴² Finally, validation studies to date have included only small numbers of clinical isolates, and the threat of false-negative and false-positive results could have important clinical consequences. Thus, the role of molecular resistance testing remains aspirational at this point.

TESTING IN PEDIATRICS

Clinical manifestations of IFD differ between children and adults.¹⁶ Several challenges routinely complicate diagnosis of IFD in children, including the frequent need to perform diagnostics on small amounts of fluid and tissue specimens, and the resulting loss of sensitivity for culture-based and nonculture based testing. The evidence supporting use of molecular IFD diagnostics in children lags behind the evidence base in adults,⁴³ and clinical decision-making for children often relies upon extrapolation from adult data.

The most robust clinical evidence for use of molecular fungal testing arises from studies of children with cancer and/or who received HCTs. A 2016 systematic review and metanalysis of molecular blood, BAL, or CSF testing as a means to screen for and diagnose IFD in pediatric cancer and HCT recipients identified studies that used multifungal or Aspergillus-specific PCR methods to screen for IFD (n = 3 studies) or diagnose IFD in febrile episodes (n = 8 studies).⁴⁴ Of note, most of these methods used in-house platforms rather than commercial tests. These studies collectively included 686 children, of whom 86 had proven or probable IFD. In studies using molecular testing for screening, specificity ranged from 43% to 85% and sensitivity ranged from 11% to 80%. In studies using molecular testing for diagnosis during febrile episodes, specificity ranged from 36% to 83% and sensitivity ranged from 0% to 100%. A total of 6 studies using PCR for diagnosis were included in a metanalysis, in which pooled specificity was 58% (95%CI 42%–72%) and sensitivity was 75% (95%CI 62%–86%). Notably, in the single study in this review that used universal antimold prophylaxis (voriconazole or amphotericin B) for children with acute leukemia, the sensitivity of a blood-based 28S Aspergillus/Candida PCR assay to distinguish proven/probable IFD from no IFD was 0%.⁴⁵ These data have been variably incorporated into clinical practice. The 2019 European Society of Clinical Microbiology and Infectious Diseases (ESCMID) - European Confederation of Medical Mycology (ECMM) pediatric aspergillosis diagnosis guideline reports an overall lack of pediatric data, including dearth of data from neonates, plus the wide variability in test performance, when concluding that no recommendation can be made regarding the use of PCR for the diagnosis of invasive aspergillosis in children.⁴⁶ Meanwhile, the 2020 European oncology and HCT guidelines endorse the use of PCR-based methods to diagnose IFD in these immunocompromised children,⁴⁷ citing inclusion of PCR in updated consensus definitions of IFD.¹⁶

There has been increasing interest in using broad range PCR and NGS to diagnose IFD in children. As in adults, these platforms could reduce the need for high-risk, invasive diagnostic sampling in children. Retrospective case series in clinically diverse pediatric populations have reported cases in which broad range PCR and NGS enabled clinical teams to tailor therapy or avoid invasive procedures in children with IFD.^48-50 Blood-based NGS may also yield faster diagnoses or direct subsequent tissue sampling for children with IFD.⁵¹ However, concerns about false-negative tests, overtesting, and interpretation of unexpected results are common themes in this literature.

IMMUNOCOMPETENT PATIENTS

There is a paucity of data on the performance of fungal diagnostic testing, particularly novel molecular diagnostics, in non-immunocompromised patients. In particular, important patient populations to consider are those in the intensive care unit or following viral infections including influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Guidelines specific to these populations have adopted similar diagnostic criteria to those used in immunocompromised populations for invasive aspergillosis, with the inclusion of serum and bronchoalveolar galactomannan and Aspergillus PCR as mycologic criteria for the diagnosis of probable disease.^16,52 The diagnostic criteria for other rare mold infections in this population are not well defined. Other molecular testing including NGS is not currently recommended for the diagnosis of IFD following SARS-CoV-2 infection, though one recent study of Karius testing for diagnosis of coronavirus disease 2019 (COVID-19)-associated aspergillosis (CAPA) showed promising results with a reported sensitivity of 87% and specificity of 97%.⁵³

SUMMARY

As more patients experience immunocompromising conditions and as environmental changes expand the range of common and emerging fungi, there is an urgent need to efficiently diagnose IFD to promptly initiate appropriate antifungal therapies. Compared to traditional diagnostic methods, molecular assays for IFD have several promising characteristics, including ease of use (less need for specialized microbiology technicians), noninvasive and small biologic sampling required, and faster turn-around-times. A growing literature supports the use of these tests in immunocompromised patients, though the low incidence and heterogeneous presentations and pathophysiology of IFD continues to cloud a robust understanding of sensitivity, specificity, and clinical performance of these tests. Additionally, the high cost of many of these assays (eg, NGS) requires targeted clinical implementation strategies to justify use. Several key questions remain: which patients are most likely to benefit from these tests? How do these tests perform in routine clinical care, for initial diagnostics, fungal disease surveillance, and treatment response? How can these tests be integrated with common biomarkers and other diagnostics? As these tools become widely available, users of these tests must grapple with these questions and be cognizant of specific limitations and evidence gaps for these tests.

KEY POINTS.

• Molecular diagnostic modalities for the identification of invasive fungal disease are rapidly being developed and utilized for high-risk individuals with immunocompromising conditions.

• Platforms range from pathogen-specific to pathogen-agnostic assays, and include single and multiplex-polymerase chain reaction (PCR), broad range PCR, and next-generation metagenomic sequencing.

• Benefits of fungal molecular diagnostic assays include ease of use, noninvasive and small biologic sampling, and faster turn-around-times as compared to traditional fungal culture methods.

CLINICS CARE POINTS.

• The population of individuals who are immunocompromised is expanding due to novel chemotherapies and immunomodulatory therapies, thus simultaneously increasing the population at high-risk of developing invasive fungal infections.

• Molecular diagnostics to identify invasive fungal diseases offer several advantages over traditional fungal culture methods, including faster turn-around-times, decreased reliance on specialized microbiology technicians, and decreased amount of specimen required.

• Since standardization across molecular fungal diagnostic tools does not yet exist, clinicians must interpret results with careful consideration of the host characteristics, timing of sample collection, type of biological specimen, and potential effects of anti-fungal therapies.