Background
Materials and methods
Patients and clinical specimens
Microbiological analyses
Molecular methods
Data analysis
Results
Patient characteristics and specimens
Fungal culturea | Bronchoalveolar lavages (BALs) | Lung/Bronchial/Pleural | Cerebrospinal fluids (CSFs) | Other sterile fluidsb | Other sterile tissuesc | Total |
---|---|---|---|---|---|---|
Positive | 34 | 3 | 2 | 9 | 7 | 55 |
Negative | 5 | 6 | 3 | 9 | 16 | 39 |
Not Ordered | 0 | 3 | 9 | 7 | 1 | 20 |
Total | 39 (34.2%) | 12 (10.5%) | 14 (12.3%) | 25 (22%) | 24 (21%) | 114 |
Fungi identified from contrived and clinical specimens
Resolution of discrepant results
Specimen No. | Specimen typea | Stain results | Standard methodsb | Initial molecular results | Sequence results | Results of resolution (repeat PCR and clinical review) |
---|---|---|---|---|---|---|
1 | BAL LUL | CW = fungal elements | C. albicans, A. fumigatus, A. flavus | ITS/LSU targets POS, β-globin POS | ITS = C. albicans LSU = C. albicans | True positive PCR Fungal culture contaminated |
2 | BAL LLL | Gram stain = Hvy WBCs + mixed bacteria; CW = NEG | Aspergillus spp. | ITS/LSU targets NEG, β-globin POS | N/A | True negative PCR No pulmonary disease Fungal culture contaminated |
3 | BAL RLL | Gram stain – Hvy WBCs; CW = NEG | Aspergillus spp., Penicillium spp. | ITS/LSU targets NEG, β-globin POS | N/A | True negative PCR No pulmonary disease Fungal culture contaminated |
4 | BAL RUL | Gram stain − Hvy WBCs; CW = NEG | A. fumigatus | ITS/LSU targets NEG, β-globin POS | N/A | True negative PCR No pulmonary disease Fungal culture contaminated |
5 | BAL LLL | Gram stain = Hvy WBCs; CW = Hvy yeast seen | A. fumigatus, dermatiaceous fungus | ITS/LSU targets POS, β-globin POS | ITS = Exophiala dermatitidis LSU = Exophiala dermatitidis | True positive PCR Other BAL samples grew A. fumigatus, E. jeanselmi Interstitial pneumonia |
6 | BAL RLL | Gram stain = Hvy WBCs + mixed bacteria; CW = NEG | A. fumigatus | ITS/LSU targets POS (weak), β-globin POS | ITS = A. fumigatus LSU = A. fumigatus | True positive PCR Clinical diagnosis of pulmonary Aspergillosis |
7 | BAL LUL | Gram stain = Sct. WBCs + mixed bacteria; CW = NEG | C. albicans + oropharyngeal flora | ITS/LSU targets NEG, β-globin POS | N/A | True negative PCR Pneumocystis jirovecii PCR POS |
8 | BAL RLL | Gram stain = Hvy WBCs; CW = Hvy yeast seen | C. albicans, yeast not C. albicans | ITS/LSU targets POS, β-globin POS | Mixed sequence: ITS = C. albicans and C. glabrata LSU = C. albicans and C. glabrata | True positive PCR Consistent with oropharyngeal colonization and overgrowth of Candida spp. |
9 | BAL RLL | Gram stain = Hvy WBCs + mixed bacteria; CW = NEG | A. niger, A. fumigatus | ITS/LSU targets POS, β-globin POS | ITS = A. niger LSU = A. niger | True positive PCR Other BAL samples grew both Aspergillus spp. Clinical diagnosis of airway colonization |
10 | BAL RLL | Gram stain = Hvy WBCs + mixed bacteria; CW = NEG | C. albicans, A. fumigatus | ITS/LSU targets POS, β-globin POS | ITS = C. albicans LSU = C. albicans | True positive PCR Clinical diagnosis of metapneumovirus/enterovirus infection C. albicans consistent with airway colonization; A. fumigatus contaminant |
11c | Brain tissue | Gram stain − Hvy WBCs; CW = NEG | No growth after 4 weeks | ITS POS LSU NEG β-globin POS | ITS: Rhizopus oryzae LSU: No data | True positive PCR Pathology sections positive for broad aseptate hyphae Clinical diagnosis of rhinocerebral mucormycosis |
12c | Cheek tissue | CW = fungal elements | No growth after 6 weeks | ITS POS LSU POS β-globin POS | ITS: Rhizopus oryzae LSU: Rhizopus oryzae | True positive PCR Pathology PAS and GMS section stains showed broad aseptate hyphae Clinical diagnosis of rhinocerebral mucormycosis |
13c | Parotid gland tissue | CW = no fungal elements | C. albicans | ITS POS LSU POS β-globin POS | ITS: Rhizopus oryzae LSU: Rhizopus oryzae | True positive PCR Clinical diagnosis of rhinocerebral mucormycosis |
14 | Sinus tract fluid | Gram stain = Hvy WBCs with mixed bacteria including yeast | C. albicans | ITS POS LSU POS β-globin POS | ITS: C. glabrata LSU: C. glabrata | True positive PCR MALDI-TOF MS confirmed C. glabrata and isolate had elevated fluconazole MIC. Fungal culture initially mis-identified |
15d | Abdominal abscess tissue/fluid | Gram stain = Hvy WBCs + mixed bacteria; CW = NEG | No growth after 6 weeks | ITS POS LSU POS β-globin POS | ITS: C. albicans LSU: C. albicans | True positive PCR Clinical diagnosis of intra-abdominal abscess |
16 | Shoulder tissue (intermedullary) | Gram stain − = Hvy WBCs; CW = NEG | Alternaria spp. | ITS NEG LSU NEG β-globin POS | N/A | True negative PCR Clinical diagnosis of Cutibacterium acnes joint infection Fungal culture contaminated |
17 | R hip tissue | Gram stain = No WBCs; CW = NEG | Environmental fungus isolated (not further identified at reference laboratory) | ITS weak band LSU weak band β-globin POS | ITS: poor sequence LSU: poor sequence | True negative PCR No evidence of IFD Fungal culture contaminated |
18e | Liver aspirate | Gram stain = no WBCS; CW = NEG | No growth after 4 weeks | ITS POS LSU POS β-globin POS | ITS: Rhizomucor pusillus LSU: Rhizomucor pusillus | True positive PCR Pathology sections positive for broad aseptate hyphae Clinical diagnosis of hepatosplenic mucormycosis |
19f | Lung tissue/fluid | Gram stain = Few WBCs; CW = NEG | No growth after 6 weeks | ITS POS LSU POS β-globin POS | ITS: Histoplasma capsulatum LSU: No data | True positive PCR Pathology of lung tissue showed necrotizing granulomas with yeast morphologically consistent Clinical diagnosis of Histoplasmosis |
Specimen No. | Specimen typea | Stain results | Standard methodsb | Initial molecular results | Sequence results | Results of clinical review | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | BAL LUL | CW = fungal elements | Negative | ITS/LSU targets POS, β-globin POS | ITS = Oxyporus corticola LSU = Oxyporus corticola Repeat testing using conventional ITS primers showed A. terreus | False positive PCR No pulmonary disease | ||||||
2 | BAL RLL | CW = no fungal elements | C. albicans, A. flavus | ITS/LSU targets NEG, β-globin POS (weak) | N/A | False negative PCR No pulmonary disease. Repeat testing gave same results. Likely sample deficiency | ||||||
3 | BAL LLL | Gram stain − Hvy WBCs; CW = NEG | C. dublinensis, A fumigatus | ITS/LSU targets POS; β-globin POS | Sequencing indeterminate as mixed sequences could not be resolved for accurate identification | Indeterminate PCR Mixed sequences Clinical diagnosis of Aspergillosis | ||||||
4 | BAL LUL | Gram stain − Hvy WBCs; CW = NEG | C. glabrata, A. terreus | ITS/LSU targets POS; β-globin POS | ITS = C. glabrata LSU = C. glabrata | False negative PCR Clinical diagnosis of invasive Aspergillosis with cavitary lung lesion | ||||||
5 | BAL | Gram stain and CW = NEG | C. glabrata | ITS POS (weak)/LSU target NEG, β-globin POS | ITS (short sequence) = Fusarium merismoides, a plant pathogen | False positive PCR Clinical diagnosis of primary lung adenocarcinoma | ||||||
6 | BAL RML | Gram stain = Hvy WBCs; CW = NEG | C. albicans | ITS/LSU targets NEG, β-globin POS | N/A | False negative PCR Clinical diagnosis of aspiration pneumonia. C. albicans consistent with airway colonization | ||||||
7 | CSF | Gram stain = few yeast | Cryptococcus neoformans | ITS NEG LSU NEG β-globin POS (weak) | N/A | False-negative PCR Repeat PCR/Sequencing Negative Likely sample deficiency given weak β-globin band | ||||||
8 | Bone(mandible) | Gram stain = few bacteria; CW = NEG | C. albicans | ITS NEG LSU NEG β-globin POS (weak) | N/A | False negative PCR Repeat PCR/Sequencing Negative Likely sample deficiency given weak β-globin band | ||||||
9 | Peritoneal fluid | Gram stain − Hvy WBCs; CW = NEG | A. flavus | ITS NEG LSU NEG β-globin POS | N/A | False negative PCR Repeat PCR/Sequencing ITS POS/LSU POS with Aspergillus spp. split identification | ||||||
10 | Dialysate fluid | Gram stain = Hvy WBCs; CW not done | C. tropicalis | ITS NEG LSU NEG β-globin POS | N/A | False negative PCR Repeat PCR/Sequencing Negative Fungal culture of other samples grew same organism | ||||||
11 | Lung tissue | Gram stain = Hvy WBCs; CW = NEG | Coccidiodes immitis | ITS NEG LSU NEG β-globin POS | N/A | False negative PCR Clinical diagnosis of pulmonary Coccidioidomycosis |
Molecular assay performance
Standard methodsb | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
FBR-PCR/S Assayc | Positive | 54 | 0 | 54 |
Negative | 7 | 11 | 18 | |
Total | 61 | 11 | 72 | |
cSensitivity (88.5%, 54/61), specificity (100%, 11/11), PPV (100%, 54/54), NPV (61.1%, 11/18) and efficiency 90.2% (65/72) |
FBR-PCR/S Assay | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
Standard methodsd | Positive | 54 | 7 | 61 |
Negative | 0 | 11 | 11 | |
Total | 54 | 18 | 72 | |
dSensitivity (100%, 54/54), specificity (61.1%, 11/18), PPV (88.5%, 54/61), NPV (100%, 11/11) and efficiency 90.2% (65/72) |
Standard Methodsb | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
FBR-PCR/S Assayc | Positive | 14 | 7 | 21 |
Negative | 7 | 47 | 54 | |
TOTAL | 21 | 54 | 75 |
FBR-PCR/S Assay | ||||
---|---|---|---|---|
Positive | Negative | TOTAL | ||
Fungal culturec | Positive | 14 | 7 | 21 |
Negative | 7a | 47 | 54 | |
Total | 21 | 54 | 75 | |
cBoth methods had sensitivity (66.7%, 14/21), specificity (87.0%, 47/54), PPV (66.7%, 14/21), NPV (87.0%, 47/54) and efficiency 81.3% (61/75) |
CW Stain/Microscopy | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
FBR-PCR/S Assayb | Positive | 8 | 42 | 50 |
Negative | 2 | 62 | 64 | |
Total | 10 | 104 | 114 | |
bSensitivity (80%, 8/10), specificity (59.6%, 62/104), PPV(16%, 8/50), NPV (96.9%, 62/64) and efficiency 61.4% (70/114) |
CW Stain/Microscopy | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
Standard methodsc | Positive | 8 | 51 | 59 |
Negative | 2 | 53 | 55 | |
Total | 10 | 104 | 114 | |
cSensitivity (80%, 8/10), specificity (59.6%, 62/104), PPV (13.6%, 8/59), NPV (93.4%, 53/55) and efficiency 53.5% (61/114) |