The diagnosis of infectious diseases in transplant populations is not bound by laws of parsimony. Due to impaired innate and adaptive immune responses following induction and maintenance immunosuppression, solid organ transplant (SOT) and hematopoietic transplant (HSCT) recipients are at increased risk for community-acquired and nosocomial infections, opportunistic and uncommon pathogens, as well as multiple pathogens simultaneously. Their immunosuppressed state also renders transplant recipients more susceptible to progressive and disseminated infections while extensive healthcare contact and antimicrobial exposure increase the likelihood of infections with multidrug resistant (MDR) organisms, together conferring a high degree of morbidity and mortality. In light of this increased susceptibility to infection, accurate and rapid diagnostics applicable to this population are essential. Though gold standard methods including culture and histopathologic examination continue to occupy a central role in the diagnosis of infectious diseases, the last decade has seen a significant shift toward culture-independent techniques. These have included single-target and multiplexed molecular testing, mass-spectrometry, and magnetic resonance-based methods which have together greatly expanded the array of pathogens identified, increased processing speed and throughput, allowed for detection of resistance, and ultimately improved the outcomes of infected transplant recipients. More recently, a new generation of diagnostics with immense potential has emerged including multiplexed molecular panels directly applicable to blood and blood culture specimens, next-generation metagenomics, and gas chromatography mass spectrometry (Figure 1). Here, we review emerging diagnostic methods applicable to the practice of transplant infectious diseases.
Molecular Methods for Bloodstream Infections
Due to a combination of impaired host defenses and increased nosocomial contact, transplant recipients are at increased risk for bloodstream infections. In the last decade, MDR Gram-negative organisms including extended-spectrum beta-lactamase (ESBL), carbapenem-resistant Enterobacteriaceae (CRE), and drug-resistant Pseudomonas have developed into serious yet increasingly common hazards to hospitalized patients, compounding the threat posed by already established Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus. Transplant patients are at increased risk for MDR bloodstream infections due to a multitude of factors ranging from chronic organ insufficiency, prolonged hospitalization, surgical site infections, immunosuppressive agents, intravascular catheters, prophylactic and therapeutic antimicrobials, and the potential for donor-derived infection.1-4 Infections with MDR Gram-negative organisms including ESBL and carbapenemase-producing Enterobacteriacae, Pseudomonas, Stenotrophomonas, Burkholderia, and Acinetobacter and resistant Gram-positive organisms like MRSA and vancomycin-resistant Enterococcus are associated with increased mortality for recipients of kidney, liver, lung and heart transplants, and HSCT.3-7 An equally worrisome trend in the last decade has been the rise of drug-resistant Candida species, now the fifth most common hospital-acquired pathogen and an important cause of morbidity and mortality for transplant patients, especially in liver SOT.8 In addition to increasing azole and echinochandin resistance among established pathogens like C. albicans and C. glabrata, newly emerging species pose a significant threat to transplant recipients.9 Chief among these is Candida auris, an emerging species that has triggered global health concerns and has the potential for multidrug resistance, nosocomial outbreaks, and donor-derived transmission.10-12
Drug resistance poses challenges to the selection of appropriate prophylactic, empiric, and targeted antibiotics for suspected or confirmed bloodstream infections in any circumstance; these difficulties are magnified in the context of transplantation due to poor organ function and drug-drug interactions that further constrain antibiotic choices. The negative impact of increasing multidrug resistance is particularly concerning in the setting of transplant since inadequate empiric antimicrobial therapy in this population is strongly linked to increased mortality.13 Due to delays in identification and antimicrobial susceptibility testing (AST) of bloodstream pathogens using conventional methods, transplant clinicians must often select empiric antimicrobial therapy based on clinical suspicion and local resistance patterns. Unfortunately, empiric therapy based on “best-guess” estimation of pathogen and resistance patterns is often incorrect, including in up to 50% of bacteremic and 70% of fungemic patients who receive inadequate antimicrobial coverage before obtaining culture results.14,14a
To obviate intrinsic delays associated with culture-based testing for bloodstream infections, molecular techniques that rapidly identify organisms from positive blood culture bottles and directly from blood samples have been developed. In addition to organism identification, several of these methods can detect genetic determinants of resistance including the mecA gene that confers methicillin resistance in S. aureus, and genes associated with ESBL (CTX-M) or carbapenemase production (KPC, OXA-48, NDM etc). Such methods are generally of moderate complexity, requiring trained operators but benefit from minimal sample processing and automated test interpretation. Several Food and Drug Administration (FDA)-approved and rapid molecular tests for direct detection from blood culture broth are currently commercially available in the United States (Table 1).
Molecular assays that directly detect organisms directly from blood specimens are not currently FDA-approved for use in the United States, but several are available in Europe and Asia. Circumventing the blood culture step offers several advantages including a requirement for lower blood volumes (1 versus 10 mL for blood culture) and greater sensitivity for fastidious or unculturable organisms.15 Eliminating the growth phase does, however, result in lower sensitivity for most pathogens due to a combination of low organism burden in peripheral blood, low specimen volumes, and circulating human nucleic acids and other blood components that interfere with polymerase chain reaction (PCR)-based amplification, leading to false negative results. Nucleic acid amplification of DNA from dead or contaminating organisms may lead to false positive results, impacting test specificity.15 False negatives may occur in the context of polymicrobial blood cultures with all aforementioned methods.
Molecular Detection From Positive Blood Culture Bottles
Several FDA-approved assays for detection of organisms from positive blood cultures are available in the United States (Table 1). These employ various molecular methods including (1) fluorescence in-situ hybridization (QuickFISH and PNA FISH BC assays [OpGen, MD, previously AdvanDx] and Accelerate PhenoTest BC kit [Accelerate Diagnostics, AZ]),16 (2) DNA-microarray (Verigene GP and GN assays [Luminex Corporation, US]), (3) nucleic-acid-amplification (Cepheid Xpert MRSA/SA blood culture assay [Cepheid, CA) and FilmArray (Biofire Diagnostics, US]), or (4) combined methods (ePlex [GenMarkDx., CA] blood culture identification [BCID] panels).
The QuickFISH BC assays benefit from the shortest turn around times (TAT) of 20–30 minutes among all the aforementioned assays (compared with 90 min to 6.5 h for others), potentially leading to earlier institution of targeted and appropriate antimicrobial therapy. However, the assay detects a limited panel of organisms, provides no resistance information and only processes once patient sample at a time. Importantly, the assay does not distinguish MRSA from MSSA, or between clinically important Candida species such as C. glabrata and C. krusei, potentially leading to inappropriate antimicrobial coverage and limiting its overall utility in real world settings.
The PNA FISH BC assays offers no real advantages over the QuickFISH iteration, with longer TAT of 90 minutes and the additional drawbacks of potentially misidentifying 2 clinically important organisms (Micrococcus as Staphylococcus and S. anginosus as Enterococcus), which may lead to inadequate antibiotic use and other unnecessary interventions.
The Accelerate PhenoTest BC kit is the only platform to provide minimal inhibitory concentration (MIC)-based resistance information (also known as phenotypic resistance information), allowing clinicians to directly determine whether an infecting organism is susceptible or resistant to a test antibiotic, rather than inferring susceptibility from the presence of absence of resistance genes as is done with genotypic assays. Phenotypic assays have the potential for improved performance since genetic determinants of resistance may not always correlate with susceptibility (decreasing the specificity of genotypic assays) and novel or emerging mechanisms may be missed by genotypic assays (decreasing the sensitivity of genotypic assays). Conversely, genotypic information can be useful, especially in cases of MDR organisms where the choice of therapy may be guided by specific underlying resistance mutations (eg, using ceftazidime-avibactam for a KPC-producing CRE but not for an NDM-producing CRE for which the drug is not active). Additionally, the assays’ organism panel is relatively limited, the throughput is low, and provision of AST information requires 6.5 hours (versus genotypic assays that require <2.5 h). Notably, S. pneumoniae the most common bacterial cause of community-acquired pneumonia is not differentiated from other Streptococcus species.
The Verigene GP and GN assays offer a slightly broader range of bacterial targets when compared with the QuickFISH and Accelerate PhenoTest but these assays do not detect fungi including Candida species, which are important causes of BSI in transplant patients. E. coli, an important pathogen cannot be distinguished from Shigella species. Verigene assays provide genotypic resistance information including the 5 canonical genes for carbapenemase production (KPC, IMP, NDM, OXA, VIM) but the GP assay does not detect a novel variant of methicillin resistance among MRSA islolates (mecC). The TAT (2–2.5 h) is longer than competing assays and a single 1 sample can run at a time per processor. However, adding multiple processors per reader can enhance throughput.
Unlike the other listed assays, the Cepheid Xpert MRSA/SA is a monoplex test targeting S. aureus only. Up to 16 samples can be processed at once if a larger instrument is purchased and the limit of detection for S. aureus is 1000-fold lower than other assays. As with the Verigene, this assay cannot detect mecC. Notably, in coinfection with MSSA and MRSA may lead to false negative results. In FDA trials, the assay sensitivity was 100% but in high volume centers, real world incidence of false negatives (~0.5–1%) can be significant and lead to inappropriate deescalation of empiric anti-Staphylococcal antibiotics. Therefore, individual centers should perform internal verification of the assay and monitor for false negatives by comparing with culture results.17
The FilmArray Blood Culture Identification Panel benefits from an expanded organism panel including Candida and important organisms not covered by aforementioned assays, such as Acetinobacter, Listeria, and Hemophilus. Genotypic resistance information is provided but the range of carbapenemase resistance genes detected is limited to KPC. Additionally, mecC is not detected. Other drawbacks include a lack of specificity for important pathogens such as Klebsiella and Enterococcus. The platform offers a scalable configuration that allows up to 16 specimens to be processed at once.
Perhaps the most promising assay, the ePlex by GenMark Diagnostics combines a very expanded organism and resistance detection panel with the potential for high throughput and a short TAT of 90 minutes. The assay provides a much more expansive array of organisms than other methods, including clinically important Gram-positive rods (Bacillus, Lactobacillus, Cutibacterium acnes), anaerobes (Fusobacterium, Bacteroides), and oxidase negative Gram-negative rods (Cronobacter and Stenotrophomonas maltophilia). Beyond the 5 most common Candida species (C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis), the fungal pathogens panel (BCID-FP) identifies several less common but clinically important species, such as C. kefyr, C. dubliniensis, and C. guillermondii and other important yeasts and molds (Cryptococcus, Rhodotorula, and Fusarium) that are known causes of disease in transplant patients. Importantly, C. auris is included in the panel and can be differentiated from C. famata, a weakness of other platforms like the Vitek 2.18 The assay also detects important genetic determinants including mecA/C, vanA/B, and common genetic determinants of ESBL and carbapenemase production.
Molecular Detection From Blood Specimens
Assays for direct molecular detection of organisms directly from blood specimens without the need for a blood culture step are not yet FDA approved in the United States at the time of this writing. However, several such assays are available in parts of Asia and Europe. T2MR system assays identify organisms directly from blood and are available in the United States but include magnetic resonance-based detection in addition to PCR so are classified separately in this review. Some of the most common are the SepsiTest (Molzym, Germany), The LightCycler SeptiFast (Roche Molecular System, Switzerland), and the Magicplex Sepsis Real-time Test (Seegene, Korea). The SepsiTest, which is based on broad range PCR of ribosomal DNA followed by nucleic acid sequencing, detects numerous clinically important bacteria, yeasts and molds as well as the parasite Plasmodium on blood samples down to 1 mL within 8 to 12 hours of collection. In a multicenter study, the SeptiTest was moderately sensitive (87%) and specific (86%) in comparison to blood cultures.19 The Magiplex assay employs multiplex real-time PCR to detect a wide array of organisms and a limited set of resistance genes (vanA, vanB, and mecA) is applied directly to 1 mL of whole blood. The sensitivity was low (29%) in comparison to blood culture in a large study.20 The SeptiFast is a real-time multiplex PCR assay that employs fluorescence resonance energy transfer probes targeting internal transcribed spacer sequences within ribosomal DNA. This method detects common bacterial and fungal pathogens directly from a whole blood sample of 1.5 mL within 6 hours. When combined with blood culture results, the Septifast may improve the negative predictive value for complicated bloodstream infections for Gram-positive organisms and Candida species.21 The assay detected a higher proportion of bloodstream infections in immunocompromised patients including organ transplant recipients than in immunocompetent patients with endocarditis.22
Molecular Detection of Candida Species From Blood Specimens
Infection with Candida spp. frequently complicates solid organ and hematopoietic stem cell transplants. Though still considered the current standard for detection of candidemia, blood cultures are only 50% sensitive for invasive candidiasis.23 Direct detection of Candida spp. DNA in blood is an alternative to culture and can be accomplished with PCR assays, most of which are laboratory developed and internally validated. These vary in sensitivity and specificity, with overall estimated sensitivity of 95% (range 88%–98%) and specificity of 92% (88%–95%) per a 2011 meta-analysis.24 No commercial kits are FDA approved for use in the United States but Fungiplex Candida (Bruker, Germany) is CE-marked for use in Europe (Table 2). Of note, the T2MR system also utilizes PCR to detect candidemia and is both CE marked and FDA approved (see below for discussion of this platform).
Despite a battery of conventional microbiologic testing, the diagnosis remains unclear in up to 60% of cases of community-acquired pneumonia, gastrointestinal infections, and meningoencephalitis.25,26 Multiplex PCR assay (Table 3) allows for the simultaneous detection of several target sequences using multiple primer sets and have the potential to detect genes associated with antimicrobial resistance, 2 salient factors in the selection of targeted therapy.
Respiratory multiplex PCR employs an automated system that incorporates sample preparation, nucleic acid extraction and PCR based detection of separate targets directly from an unprocessed clinical sample within 1–5 hours. Nasopharyngeal swab samples, nasal washes/aspirates and bronchoalveolar lavage (BAL) can be used for sample collection depending on the assay. At present, there are several FDA-approved respiratory multiplex assays, namely: NxTAG Respiratory Pathogen Panel (RPP; Luminex, TX), Verigene Respiratory (RP) Flex (Nanosphere, IL), FilmArray Respiratory Panel (RP; BioFire Diagnostics, UT), eSensor Respiratory Viral Panel (RVP; GenMark, CA), and the Unyvero Lower Respiratory (Curetis, CA). These respiratory panels (Table 3) detect predominantly viral pathogens whereas Unyvero targets bacterial pathogens and genes associated with antimicrobial resistance.
Respiratory multiplex PCR assays allow for early diagnosis and subsequent initiation of appropriate therapy, detection of copathogens not identified by conventional methods, and institution of early isolation practices. All aforementioned strengths are critical in the care of immunocompromised hosts but few studies have assessed these assays in this population. The FilmArray RP was evaluated on BAL and/or nasopharyngeal samples from 87 immunocompromised patients (48% HSCT, 37% SOT, and 18% hematologic malignancy).64 Only viral pathogens were detected in this patient cohort. The positive predictive value was 86% while the negative predictive value was 100%. Initial iterations of the BioFire RP (v 1.6) were poorly sensitive for adenovirus (67%) but an updated version (v1.7) reported improved performance (91%) due to inclusion of more adenovirus types. However, in a large pediatric study, the BioFire RP v1.7 had low sensitivity for Adenovirus species A, C, D, and F and missed several cases in immunocompromised, including transplant patients.65,66 Therefore, this assay should be avoided as a standalone test for Adenovirus infection in a transplant population.
A similar study evaluated Unyvero on BAL samples of 522 immunocompromised patients suspected to have pneumonia.67 Assay results were available within 5 hours and the reported sensitivity and specificity were 68.4% and 86%, respectively. Surprisingly, the assay was not able to identify Streptococcus pneumoniae in the collected samples so users should be cautious when using this assay to rule out pneumococcal pneumonia. In contrast, the detection of Gram-negative pathogens had acceptable sensitivity and a good positive likelihood ratio.
Diarrheal disease is associated with significant morbidity, poor graft survival, and increased risk of death in SOT recipients.68 Early recognition of infection and distinguishing infectious from noninfectious etiologies (including drug induced injury and graft versus host disease) allows for early intervention. Multiplex PCR assays on stool samples benefit from shorter TAT compared with conventional testing methods (stool culture, microscopy, and antigen testing) and offer the convenience of simultaneous testing of a wide array of potential enteric pathogens using a single stool specimen.
FDA-approved multiplex tests for gastrointestinal pathogens include the FilmArray Gastrointestinal Panel (Biofire Diagnostics, UT), xTAG Gastrointestinal Pathogens Panel (GPP; Luminex, TX), and Verigene Enteric Pathogens (Nanosphere, IL) (Table 3). There are limited published data for these assays among immunocompromised patients. The xTAG GPP was used to work-up diarrhea in 102 adult SOT recipients.69 Of 147 collected samples, at least one pathogen was detected in 25 (17%). Toxigenic C. difficile, Norovirus, and Salmonella spp. were detected in 7.5%, 4.1%, and 2.7%, respectively. xTAG GPP was evaluated in 49 adult kidney transplant recipients who experienced 54 severe diarrhea events.70 Of the 39 positive samples, the assay identified 15 instances of coinfection (38%) compared to none with standard testing. Bacterial, viral, and parasitic pathogens were identified in 32 of 39 (82%), 16 of 39 (41%), and 2 of 39 (5%), respectively. Enteropathogenic E coli (15 of 39, 38%), Campylobacter spp. (15 of 39, 38%), and Norovirus (14 of 39, 36%) were commonly detected in this cohort. FilmArray, xTAG, and Verigene detect Adenovirus serotypes F40 and F41, the most common causes of gastroenteritis in children. However, they do not detect other important serotypes including Adenovirus species C, which are more common causes of infection in transplant patients and should therefore not be used to rule out Adenovirus gastrointestinal disease in this population.71
Despite the advantages of multiplex PCR, there are limitations to consider. These assays detect the nucleic acid of viable and nonviable pathogens or free DNA/RNA72; hence, it is difficult to distinguish between disease, asymptomatic carriage, or viral shedding. C. difficile is frequently detected in these multiplex GI platforms,73-75 possibly representing colonization rather than true infection. Given the ability to detect >1 pathogen,76 interpretation of results’ clinical significance can be challenging. In contrast to culture techniques, no antimicrobial susceptibility information is provided with current assays.
The FilmArray meningitis/encephalitis (ME) panel (Biofire Diagnostics, UT) was FDA-approved for detection of CNS pathogens, including 6 bacterial, 7 viral, and 2 fungal pathogens (Table 3), in cerebrospinal fluid (CSF) samples with a quick TAT of 1 hour. In a multicenter study evaluating the FilmArray ME panel versus conventional techniques (ie, culture for bacteria and monoplex PCR for other pathogens) in normal hosts,77 the assay was 100% sensitive for the detection of 9 of 14 pathogens including E. coli K1, H. influenzae, S. pneumoniae, CMV, HSV-1, HSV-2, Human paraechovirus, VZV, and Cryptococcus neoformans/gattii. Specificity was uniformly high (≥99.2%).
In another study of hospitalized patients, which included 8 immunocompromised patients (HIV/AIDS, steroid use, active chemotherapy, transplant), the FilmArray ME detected HHV-6 in a HSCT recipient with encephalitis who had an undetectable HHV-6 by PCR.78 The FilmArray ME may be suboptimal for the diagnosis of CNS cryptococcosis. In a published report, the assay failed to detect Cryptococcus species in CSF twice in a kidney transplant recipient causing delays in diagnosis. Cryptococcus was eventually identified using a conventional cryptococcal antigen lateral flow assay.79 In another study of 14 patients (12 HIV positive and 2 HIV negative patients) with positive CSF cryptococcal antigen testing, only 5 had concordant positive culture and Film Array ME results.80 Among the 9 negative FilmArray ME negative cases, 2 had positive culture while 7 had negative cultures. The sensitivity of the FilmArray ME for cryptococcal meningitis was found to be 71.4% with a specificity of 100%. Based on these data, a negative FilmArray ME result for Cryptococcus does not rule out cryptococcal meningitis, especially for at-risk transplant patients.
Aspergillus PCR for Diagnosis and Resistance Testing
Invasive pulmonary aspergillosis (IPA) is the most common cause of fungal pneumonia in recipients of lung transplantation and HSCT and carries significant morbidity and mortality for all transplant populations. Detection of Aspergillus species in transplant recipients poses significant diagnostic and clinical challenges. American and European guidelines recommend diagnosing syndromes of IPA based on clinical risk factors, syndromic presentation, radiographic findings, detection of fungal antigens (galactomannan and 1,3-beta-d-glucan), histopathology, and culture.81-83 While PCR-based detection of Aspergillus species on respiratory specimens or blood was not formally recommended as a diagnostic tool in most recent IDSA guidelines82 and such assays are not currently FDA approved in the United States, European guidelines have suggested their use in conjunction with conventional diagnostics. Recently, American Thoracic Society Clinical Practice Guidelines for microbiologic testing of fungal infections have endorsed the inclusion of Aspergillus PCR testing on blood or BAL as part of the initial evaluation of IPA in both SOT and HSCT patients84 and the revised Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium now include Aspergillus PCR as mycological evidence for probable invasive aspergillosis.85 As a whole, PCR is emerging as an increasingly useful approach for diagnosis, particularly as assays become more standardized. The European Aspergillus PCR Initiative (EAPCRI) is working toward improving assay standardization with specific recommendations for sample processing including DNA extraction.86
PCR-based methods for Aspergillus fall into 3 general categories: (1) laboratory-developed tests independently developed by individual clinical microbiology laboratories which specifically detect Aspergillus DNA (ie, “home grown / home brewed” assays), (2) commercial kits made available by various biotechnology companies, and (3) commercially available multiplex PCR platforms detecting multiple potentially pathogenic organisms including Aspergillus species. Genetic sequences within the 18s rRNA, 18s rDNA, 5.8S rDNA, 28s rRNA, mitochondrial DNA, and internal transcribed spacer regions have been used as PCR targets.
To date, 3 meta-analyses have investigated the performance characteristics of commercially available PCR assays for whole blood or serum in published studies and have reported pooled sensitivities and specificities of 79.2% (95% CI, 71.0-85.5) and 79.6% (95% CI, 69.9-86.6), 84% (95% CI, 75-91) and 76% (95% CI, 65-84),87 and 88% (95% CI, 75-94) and 75% (95% CI, 63-84),88 respectively. In these meta-analyses, the sensitivity and specificity of PCR for 2 consecutive positive samples were 59.6% (95% CI, 40.7-76.0) and 95.1% (95% CI, 87.0-98.2), 64% (95% CI, 38-84) and 95% (95% CI, 88-98), and 75% (95% CI, 54-88) and 87% (95% CI, 78-93), respectively, an approach that was associated with reduced sensitivity but increased specificity for IPA.
Meta-analyses evaluating the performance of PCR on BAL have also been published, showing overall higher but also more variable sensitivities compared with blood PCR testing. The mean sensitivity and specificities of 90.2% (95% CI, 77.2-96.1) and 96.4% (95% CI, 93.3-98.1),89 91% (95% CI, 79-96) and 92% (95% CI, 87-96),90 and 79% (95% CI, 36-100) and 94% (95% CI, 75-100),91 respectively. Importantly, the specificity of PCR assays on BAL is lower for lung transplant recipients (sensitivity 88%, specificity 100%), reducing the ability of these assays to differentiate IPA from colonization.92 Therefore at this time, in the lung transplant population, Aspergillus PCR testing on BAL should only be used in conjunction with clinical, radiographic and additional microbiologic evidence to make the diagnosis of IPA.
Commercially produced PCR kits detecting Aspergillus spp. DNA are available from multiple developers and include AsperGenius (PathoNostics, The Netherlands), MycoGENIE (Ademtech, France), MycAssay (Microgen Bioproducts/Lab21, formerly Myconostica, United Kingdom), Bio-Evolution Aspergillus (Bio-Evolution, France), VYOO (Analytik Jena, Germany), SepsiTest (Molzym, Germany), and Magicplex (Seegene, South Korea) (see Table 2 for references and comparative evaluation of performance).
In a recent review, the performance characteristics of these Aspergillus PCR assays were overall similar with improved sensitivity on BAL than serum, in hematological patients and in patients not on antifungals.93 The AsperGenius kit additionally reports the detection of Aspergillus terreus DNA, a species intrinsically resistant to polyenes (including amphotericin-B). The MycAssay kit is notable for a broad range of detection, reporting the detection of 15 separate Aspergillus spp., including species intrinsically resistant to azoles or polyenes.30 In addition to organism detection, the AsperGenius and MycoGENIE kits detect mutations in the CYP51A gene, which is a primary mechanism for resistance to azole antifungals and an increasing reality worldwide.27,38 In fact, the prevalence of azole resistance among A. fumigatus isolates is up to 15% in certain parts of Europe and though still low (<1%) in the United States, is expected to rise in the coming decade.94
Platforms differ from kits in that they utilize PCR-based technology as a component of a standalone devoted device in a clinical microbiology laboratory. Such platforms include IRIDICA (Abbott Laboratories, US; recently discontinued),48 RenDx Fungiplex (Renishaw, United Kingdom), and the Roche SeptiFast (Roche, Switzerland); see Table 2 for references and comparative evaluation of performance. These platforms report detection of DNA of either Aspergillus fumigatus alone or multiple Aspergillus spp., as well as DNA of other fungal and bacterial pathogens. Few studies have been conducted assessing the sensitivity and specificity of these platforms. The future diagnostic utility of some of these platforms is questionable, particularly given the discontinuation of the PLEX-ID system likely as a result of financial, logistical, and regulatory considerations.48
PCR-based diagnostics for IPA benefit from increased sensitivity when compared with culture (30%–60%) and antigen-based methods including serum galactomannan (sensitivity 71%, specificity 89%),95 BAL galactomannan (sensitivity 85%, specificity 92%),96 and serum 1,3-beta-d-glucan (sensitivity 64%, specificity 92%).97 Moreover, PCR assays often have significantly shorter time to result reporting. Limitations include the inability to differentiate colonizing from infectious organisms (especially in the setting of lung SOT) as well as the inability of PCR to differentiate living or dead organisms.
Metagenomic next-generation sequencing (mNGS) is an unbiased, hypothesis-independent diagnostic method that identifies pathogens directly from clinical samples. In contrast to PCR-based and 16S ribosomal RNA-based genetic sequencing that target specific primers, mNGS aims to sequence the entirety of the nucleic acid present in a clinical sample. Most mNGS platforms sequence DNA, excluding microbes with RNA-based genomes (RNA viruses in particular). To address this limitation, cDNA can be synthesized from total RNA. mNGS assays also carry the potential for detection of antimicrobial resistance by identifying genetic determinants of resistance.98-100 Current mNGS platforms may vary in sequencing technologies and data analyses methods. The diagnostic capabilities of various mNGS assays are dependent on their associated organism databases. These databases can be continually updated based on newly available organism genome sequences.101 Multiple studies have demonstrated the clinical utility of mNGS performed on various biological samples including respiratory specimen, cerebrospinal fluid, and blood.
Blood cultures have long been considered the gold standard for the identification of bloodstream infections, due to high specificity and the ability to subsequently perform AST. However, blood cultures are insensitive for fastidious bacteria, Candida, and other fungi. In the setting of febrile neutropenia, the yield of blood cultures is <30%.102,103 Metagenomic testing on blood specimens has the potential to greatly expand the array of organisms tested and the sensitivity for fastidious organisms. A commercially available mNGS assay on plasma (Karius, Karius Inc, US) was evaluated in a cohort of HSCT recipients. Samples were collected before conditioning, at the time of stem cell infusion, at weekly intervals posttransplant and at the onset of symptoms.104 Karius testing was useful in 3 of 20 patients, including a case of Chlamydia trachomatis and MRSA infection before development of bacteremia. The Karius assay has also been employed for the diagnosis of non-BSI, including pneumonia, brain abscesses, sinusitis, and intestinal. In a study of 9 patients with underlying SOT, HSCT or active leukemia with proven fungal infections by histopathology or culture of clinical specimens (2 lung, 1 small bowel, 1 endotracheal, 1 brain, 1 sternal tissue, 1 pancreatic, 1 aortic, and 1 sinus), the Karius assay identified 7 of 9 fungi down to species level. In an eighth case, fungal DNA was identified but below level of detection of the assay.105
Noncommercial laboratory-developed mNGS assays have also been evaluated in immunocompromised populations. In a multicenter prospective study of 101 immunocompromised patients (including 22 SOT and 18 HSCT recipients), an mNGS assay on blood identified more clinically important organisms than conventional testing (36 versus 11) and was associated with a higher negative predictive value (98.4%)106 suggesting utility as a tool for antimicrobial stewardship.
Encephalitis has been associated with a multitude of infectious pathogens, many of which are difficult to diagnose. Additionally, infectious encephalitis is often clinically indistinguishable from noninfectious cases, such as autoimmune encephalitis. The current diagnostic approach is hypothesis-driven and includes culture, serology, antigen-based assays, and PCR targeted to clinical, radiographic and epidemiologic factors. In the setting of SOT and HSCT, mNGS has the potential to improve diagnostic yield, as demonstrated by multiple published reports (Table 4).
In 8 of 17 reported cases, novel pathogens including Arenavirus (3 of 17), Astrovirus (4 of 17), and human beta-Coronavirus (1 of 17) were involved. In 2 cases, mNGS revealed unexpected etiologies of encephalitis (Candida species and Hepatitis E). mNGS may have a role in better defining the etiology of meningoencephalitis of unclear etiology in potential organ donors. Donor-derived encephalitis secondary to tick-borne virus encephalitis119 and Arenavirus107 were confirmed using mNGS. In a multicenter study of 58 patients with confirmed meningoencephalitis (including HSCT or SOT patients), 19 (33%) were diagnosed by conventional testing and mNGS, 26 (45%) by conventional testing alone, and 13 (22%) by mNGS alone. Based on these data, mNGS can serve as an adjunctive test but cannot yet replace conventional testing for encephalitis as a substantial proportion of pathogens was missed when using mNGS alone.120
As with encephalitis, the causative agents of pneumonia are often undiagnosed. Several studies have investigated the utility of mNGS on respiratory specimens or blood for the diagnosis of respiratory infection of immunocompromised hosts. In a cohort of 29 lung transplant recipients, a laboratory developed mNGS assay identified only 5 cases,121 all of which were viral. In a study of 22 HSCT recipients with a respiratory illness, BAL samples were evaluated by both standard of care microbiologic testing and mNGS.122 mNGS identified 12 potential pathogens, 6 missed by conventional testing, and another 6 confirmed by conventional testing. mNGS may also have a role in the diagnosis of fungal infections in the setting of HSCT. In a study that included 5 HSCT patients with confirmed fungal pneumonia on lung biopsy (by histopathology and/or culture), mNGS identified fungal organisms in all 5 (Rhizopus microsporus , Mucor racemosus , and Rhizopus oryzae ).123 Histopathology demonstrated fungal elements in all whereas fungal culture was positive in only 2 (Table 5).
Explify Respiratory (IDbyDNA, US) is a commercially available mNGS assay that is performed on BAL. The assay detects hundreds of bacterial, fungal, and viral respiratory pathogens within 3–6 days. In a study of immunosuppressed pediatric patients, results of mNGS were concordant with standard microbiologic testing for bacteria (90.2%) and viruses (94.1%) but not fungi (66.7%). In 44% of children, mNGS identified possible pathogens missed by standard testing.124 More clinical data are needed to demonstrate utility of this assay among adult transplant patients.
Magnetic Resonance-based Methods
The T2MR platform (T2 Biosystems, US) is a standalone device that employs PCR technology combined with magnetic resonance to detect superparamagnetic nanoparticles bound to amplicon-specific probes.125 The T2MR system is validated for detection of pathogens directly from blood. Akin to other molecular diagnostic approaches, it does not require a culture-based step; whole blood samples can be directly evaluated for the presence of DNA from pathogenic organisms. Two panels manufactured by T2 Biosystems are currently approved by the FDA: T2Bacteria and T2Candida (Table 2).44,45,126 A third panel, T2Candida auris, is currently research-use-only but is being investigated for detection of the intrinsically multiantifungal resistant C. auris.46 These assays do not provide genotypic resistance information.
The sensitivity of T2Candida in a heterogeneous group of patients (that included transplant recipients) with suspected candidemia was 91.1% in a multicenter trial, along with a specificity of 99.4%,44 when compared with conventional blood cultures, which are around 50% sensitive for candidiasis.23 In another study, T2Candida was 89% sensitive and performed better than blood cultures for patients on antifungals, suggesting that the assay can identify lower burdens of organisms, nonviable and/or growth-stunted Candida organisms.47 Despite significantly improved sensitivity over cultures, the T2Candida assay does not always translate into real-world utility. The true sensitivity of the assay is likely lower than the 91% reported in the initial validation study as a large number of samples included in the study were contrived specimens with yeast concentrations around 10–100 times higher than the mean concentration in blood cultures of candidemic patients (11–100 Candida CFU/mL versus 1 CFU/mL, respectively).44 The limit of detection of the assay (1–3 CFU/mL) is in fact equal to or slightly higher than the concentration of yeast in patient blood cultures, leading to a potential for false negatives. Though the sensitivity of the assay is significantly higher than blood culture, clinicians may be unwilling to discontinue empiric antifungals in at-risk transplant patients unless candidemia is more definitively ruled out. The positive predictive value (PPV) of the assay varies greatly with the prevalence of candidemia in the population studied. In patients post-HSCT with an assumed candidemia prevalence of 20%, the PPV is 98% when compared with a PPV of 15% in immunocompetent patients without risk factors.127 Other limitations include an inability to detect other clinically important Candida species (C. guilliermondii, C. lusitanea, C. kefyr and C. auris), which account for 1–10% of Candida infections in transplant patients128 and a high incidence of indeterminate or invalid results which require repeat testing and may impede workflow.
In a single-center validation study, the T2Bacteria had a sensitivity of 83.3% and specificity of 97.6% compared with blood cultures for T2Bacteria-targeted organisms. The performance varied according to species tested with a sensitivity of 100% for P. aeruginosa and S. aureus versus 50% for K. pneumonia.126 In a more recent multicenter study of >1400 hospitalized patients with suspected bloodstream infections comparing T2Bacteria to blood cultures, the assay missed 10% (90% sensitivity) of T2Bacteria-targeted organisms and 57% (43% sensitivity) of all infecting organisms. Though the array of pathogens detected is limited and the sensitivity is less than ideal, the T2Bacteria panel performed better than culture for patients receiving antibiotics so may have particular value in this setting.129 Another advantage over blood cultures is the much shorter TAT of 3–5 hours, which may help with targeting antibiotics in transplant recipients with sepsis.
Gas Chromatography Mass Spectrometry
A novel method for use in the diagnosis of pulmonary invasive fungal infections is detecting exhaled volatile organic compounds produced during fungal metabolism using gas chromatography mass spectrometry. Initially studied in IPA, the detection of exhaled metabolites specific to Aspergillus species including monoterpenes and sesquiterpenes using gas-chromatography mass spectrometry yielded 94.0% sensitivity and 93.0% specificity for IPA when compared with diagnosis by clinical criteria.130 This assay has the potential to detect invasive infections caused by multiple fungal pathogens, including Aspergillus and Mucorales species (down to species level), which are challenging to isolate or identify using conventional microbiologic techniques alone.131 The technique is currently limited by difficulties with standardization of breath collection (affected by variations in respiratory rate and tidal volumes as well as age, race, sex, and other factors) but is being optimized for commercial use.
Conclusions and Future Vision
Emerging diagnostics in the field of microbiology, including multiplex PCR panels, metagenomics, magnetic-resonance based testing, and breath-based diagnostics, have the potential to revolutionize the diagnosis of infectious diseases among SOT and HSCT recipients. Although more data are needed to best establish the utility of these modalities for specific transplant patient subgroups and clinical syndromes, early reports suggest that these methods complement, enhance, and sometimes outperform conventional microbiology diagnostics. Within the next decade, another wave of diagnostics including Matrix Assisted Laser Desorption/Ionization Time Of Flight mass spectrometry directly applicable to clinical specimens, cell-free plasma DNA sequencing, pathogen-specific Immuno-Positron Emission Tomography scanning, and other modalities promise to further expand the diagnostic armamentarium at the disposal of transplant providers.
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