Patient characteristics and specimens
A total of 107 enrolled patients were predominantly male (67, 62.6%). The mean age of 59 yrs. (range = 0 to 85 yrs.) and there was no significant age difference according to gender. Comorbidities in five patients were unknown due to missing data. Most patients (74/107, 69.2%) had at least one underlying comorbidity that predisposed them to IFD including diabetes mellitus (18/74, 24.3%), solid-organ malignancy/tumour (18/74, 24.3%), immunosuppressive therapy for non-malignant conditions (13/74, 17.6%), hematologic malignancy (12/74, 16.2%), hematopoietic stem cell transplant (HSCT) (5/74, 6.7%), HIV/AIDs (4/74, 5.4%), and end-stage renal disease (2/74, 2.7%). A total of 19 patients (17.8%) had confirmed IFD, another 12 (11.2%) had probable IFD, and the rest had no evidence of infection. Approximately one-third (15/54, 27.8%) of the true positive molecular tests for any yeast/fungi were found in patients with confirmed or probable IFD.
A total of 114 clinical specimens were tested from these patients including 39 (34.2%) BALs and 75 (65.8%) other types of sterile fluids and tissues; 7 patients had ≥2 specimens tested (Table 1). BALs and other pulmonary specimens (lung/bronchial/pleural aspirates or fluids) (n=51, 44.7%) were the most tested sterile fluids. A wide range of different tissue types were tested representing the disseminated nature of IFD. A total of 55 (48.2%) specimens had yeast/fungi recovered from fungal cultures. Twenty (17.5%) specimens only had bacterial cultures done because yeast/fungal culture was not ordered - most of these specimens (n=16, 80%) had negative Gram and CW stains and bacterial cultures, but 1 BAL and 2 abdominal fluid specimens grew Candida albicans (despite negative CW), 1 abdominal fluid showed yeast in the Gram stain and grew C. albicans, and 1 sinus aspirate grew Aspergillus fumigatus.
Fungi identified from contrived and clinical specimens
The FBR-PCR/S assay accurately identified 32/33 (97%) of the yeast/fungi inoculated into the contrived BAL specimens except for one specimen containing A. terreus (See Methods). Another sixty-six fungal isolates were recovered from fifty-five clinical specimens. Candida spp. (n=36, 54.5%) [C. albicans (n=18), C. dublinensis (n=4), C. glabrata (n=5), C. kefyr (n=3), C. krusei (n=2), C. parapsilosis (n=1) and C. tropicalis (n=2)] and Aspergillus spp. (n=14, 16.7%) [A. flavus (n=2), A, fumigatus (n=7), A. terreus (n=1), A. niger (n=1), and 3 other Aspergillus spp.] were the most identified species either fungal culture or FBR-PCR/S. Other fungal species identified included Alternaria spp. (n=1), Coccidioides immitis (n=2), Cryptococcus neoformans (n=1), Exophilia dermatiditis (n=1), Fonsecaea spp. (n=1), Fusarium merismoides (n=1), Histoplasma capsulatum (n=1), Penicillium spp. (n=2), Pseudallescheria boydii complex (n=2), Trichophyton rubrum (n=1) and Rhizopus oryzae (n=3). One BAL sample was also PCR positive for Pneumocystis jirovecii using specific PCR primers.
Resolution of discrepant results
Discordant results were initially observed in 30 clinical specimens including 16 BALs and 14 other types of clinical specimens. Table 2 details the resolution of discrepant results. Of the 30 discordant results, 19 (63.3%) specimens [BALs (n=10) and other clinical specimens (n=9)] were resolved in favour of the molecular assay results (Table 2A), while 11(36.7%) specimens [BALs (n=6) and other clinical specimens (n=5)] were resolved in favour of fungal culture results (Table 2B). BALs were prone to contamination from patient’s airway colonization with Candida spp. and/or Aspergillus spp., which gave initial discrepant results, but most were resolved in favour of the FBR-PCR/S result after repeat testing and clinical review (Table 2A).
FBR-PCR/S analysis made a critical difference to patient management and clinical outcome in 4 unusual cases where fungal cultures were negative (Table 2A). Brain, cheek, and parotid tissue (Specimens 11 to 13) were harvested in the operating room from a critically ill 25 yo diabetic female with rapidly progressive severe necrotizing left facial and sinus infection where sequential specimens remained culture negative but broad-based aseptate hyphae were seen on Grocott’s stains in histopathology sections(30). Rapid PCR diagnosis of rhino cerebral Mucormycosis dure to Rhizopus oryzae allowed optimal treatment and management. For Specimen 15, FBR-PCR/S results allowed appropriate management of this patient’s intra-abdominal abscesses and institution of anti-fungal therapy with cessation of broad-spectrum antibacterial agents. Specimen 18 was harvested under ultrasound guidance in a 49 yo female with a new onset acute myelogenous leukemia and large hepatosplenic lesions thought to be due to candidiasis that were fungal/bacterial culture negative. Diagnosis of hepatosplenic Mucormycosis due to Rhizomucor pusillus enabled immediate appropriate anti-fungal management and drainage, and the patient proceeded to allogenic stem cell bone marrow transplant. Although specimen 19 fungal cultures eventually grew scant amounts of Histoplasma capsulatum after almost 6 weeks of incubation, FBR-PCR/S testing allowed for rapid confirmation of Histoplasmosis, which was also consistent with histopathology sections showing yeast with broad-based budding on Grocott’s and PAS stains.
Molecular Assay Performance
The performance of the molecular assay compared to fungal culture is shown in Table 3A for BALs, and Table 3B for other clinical non-BAL specimens. For BALS, the FBR-PCR/S assay had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 88.5%, 100%, 100% and 61.1% compared to culture. Fungal culture had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 61.1%, 88.5% and 100% compared to the molecular assay for BALs. Overall, fungal culture and the molecular assay had similar diagnostic efficiency (90.2%) for BAL specimens (Table 3A).
For other clinical specimens, the FBR-PCR/S assay and fungal culture had similar performance with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 66.7%, 87.0%, 66.7% and 87.0% compared to culture. Overall, fungal culture and the molecular assay had similar diagnostic efficiency (81.3%) for other types of clinical specimens (Table 3B).
Table 3C shows that fungal culture and the molecular detection had similar performance compared to whether the clinical specimen showed fungal elements on CW stain and microscopic examination. Although a negative CW stain and microscopic examination has excellent specificity and NPV, it has poor sensitivity and PPV for fungal infection. Both BALs and other clinical specimens with negative culture and PCR results were microscopy negative. Although specimens positive by microscopy (n=10, 8.8%) demonstrated variable culture and/or PCR positivity; 6 specimens were positive by both methods, 2 were only positive by culture, and 2 were only positive by PCR. Another fifty-six (49.1%) specimens were microscopy negative but grew a variety of yeast/fungi and demonstrated variable culture and/or PCR positivity; 37 were positive by both methods, 14 were only positive by culture and 5 were only positive by PCR.
Implementation of the Molecular Assay
Figure 1 shows an overview of the FBR-PCR/S procedure workflow and the time required for each assay step. The FBR-PCR/S assay workflow uses fast-PCR protocols and automated commercial sequence alignment and interpretation so that results can be reported in ~8 h or within the technologist’s dayshift; divided between specimen processing/extraction and fast PCR amplification/gel interpretation (~4.5 h) and fast cycle sequencing and interpretation (~3.5 h) (Figure 1). Due to the longer sequence length provided by the LSU primers (>550 bp) this would be the preferred single target for initial detection followed by ITS (~350 bp). To ensure an optimal pre-test probability and the quality and quantity of specimen available FBR-PCR/S tests should be ordered by the Infectious Diseases service in consultation with a clinical microbiologist.
Previous reports of FBR-PCR/S evaluations in non-selected clinical cases have been limited, and primarily reported from large laboratories in Europe of the United States(18, 21-23, 31). Lass-Florl and colleagues(2013/Austria)(18) evaluated an ITS fungal PCR in 206 tissues and sterile fluid samples (n=190 patients) with negative microscopy and found a sensitivity, specificity, PPV and NPV of 57.1%, 97%, 80% and 91.7%. Valero and colleagues(2016/Spain)(23) developed a fungal PCR using two ITS primers and 4 probes specific for specific fungal pathogen groups and testing of 60 tissue and sterile fluid samples showed comparable sensitivity (83.3%) and specificity (100%) to our assay. Zeller and colleagues (2017/Austria) (20) evaluated an ITS fungal PCR in 105 tissues and sterile fluids (n=98 patients) and found a sensitivity, specificity, PPV and NPV of 87.7%, 90.3%, 76% and 95.5% respectively. Gomez and colleagues (2017/USA) (22) used a dual target (i.e., ITS 2 and D2 region of 28S) to evaluate 117 tissues and sterile fluids from 117 patients with confirmed IFD compared to 116 clinical samples from 108 patients with suspected IFD. Performance of their fungal PCR assay was better in the targeted IFD group [sensitivity (96.6%) and specificity (98.25%)] than in patients suspected of IFD [sensitivity (62.8%) and specificity (71.3%)](22). Ala-Houhala and colleagues (2017/Finland)(21) used a dual target ITS fungal PCR to test 37 tissue and sterile fluid specimens from 279 patients and found a sensitivity, specificity, PPV and NPV of 60.5%, 91.7%, 54.2% and 93.4% respectively. Stempak and colleagues (2019/USA) (31) also showed that fungal PCR testing had equivalent performance on analyses of 65 sterile fluid and tissue samples selected based on having all reference methods done (i.e., stains, DNA probes, culture, histopathology). This group did not recommend the routine use of fungal PCR however, because no IFD cases were found that were not diagnoses by the reference methods, and the referred out molecular assay had a prohibitive cost.
Fungal PCR had an excellent performance compared to culture in microscopy positive specimens, and an equivalent performance in microscopy negative specimens. In fact, most patients with IFD were diagnosed by fungal PCR analysis of microscopy negative specimens so testing should be done in non-selected patients without overt immunosuppression. Rampini and colleagues (2016/Switzerland)(19) have also demonstrated similar efficacy of their fungal ITS PCR compared to conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients. They evaluated 251 clinical specimens using both the fungal ITS PCR compared to fungal culture and demonstrated a high concordance of 89.6% and equivalent analytical performance with a sensitivity, specificity, PPV and NPV of 87.7%, 90.3%, 76% and 95.5% respectively(19).