Abstract
               We report the case of a kidney transplantation patient on chronic immunosuppressive therapy presenting with subacute meningitis. The final diagnosis of cryptococcal meningitis was delayed due to 2 false-negative cryptococcal results on a molecular diagnostic panel. Caution with such platforms in suspected cryptococcal meningitis is needed.</jats:p

Burnham, Carey-Ann

Dubberke, Erik

Franklin, Alexander

Lainhart, William

O\u27Halloran, Jane A

Powderly, William

Open Forum Infectious Diseases

English

Jane A O’Halloran

Alexander Franklin

William Lainhart

Carey-Ann Burnham

William Powderly

Erik Dubberke

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Pitfalls Associated With the Use of Molecular Diagnostic Panels in the Diagnosis of Cryptococcal Meningitis

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Washington University School of MedicineDigital Commons@BeckerOpen Access Publications2017Pitfalls associated with the use of moleculardiagnostic panels in the diagnosis of cryptococcalmeningitisJane A. O'HalloranWashington University School of Medicine in St. LouisAlexander FranklinWashington University School of Medicine in St. LouisWilliam LainhartWashington University School of Medicine in St. LouisCarey-Ann BurnhamWashington University School of Medicine in St. LouisWilliam PowderlyWashington University School of Medicine in St. LouisSee next page for additional authorsFollow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubsThis Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in OpenAccess Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact engeszer@wustl.edu.Recommended CitationO'Halloran, Jane A.; Franklin, Alexander; Lainhart, William; Burnham, Carey-Ann; Powderly, William; and Dubberke, Erik, ,"Pitfallsassociated with the use of molecular diagnostic panels in the diagnosis of cryptococcal meningitis." Open Forum InfectiousDiseases.4,4. 1-3. (2017).https://digitalcommons.wustl.edu/open_access_pubs/6438AuthorsJane A. O'Halloran, Alexander Franklin, William Lainhart, Carey-Ann Burnham, William Powderly, and ErikDubberkeThis open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/6438BRIEF REPORT • OFID • 1Open Forum Infectious DiseasesB R I E F  R E P O R TPitfalls Associated With the Use of Molecular Diagnostic Panels in the Diagnosis of Cryptococcal MeningitisJane A. O’Halloran,1 Alexander Franklin,2 William Lainhart,3 Carey-Ann Burnham,3  William Powderly,1 and Erik Dubberke11Division of Infectious Diseases,  2Department of Medicine, and 3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USAWe report the case of a kidney transplantation patient on chronic immunosuppressive therapy presenting with subacute meningitis. The final diagnosis of cryptococcal meningitis was delayed due to 2 false-negative cryptococcal results on a molec-ular diagnostic panel. Caution with such platforms in suspected cryptococcal meningitis is needed.Keywords. cryptococcal meningitis; encephalitis; men-ingitis; molecular diagnostic techniques; polymerase chain reaction. Worldwide, Cryptococcus is the most common cause of fungal meningitis [1]. Delayed diagnosis of cryptococcal meningitis is associated with increased morbidity and mortality [2, 3]. Culture remains the gold standard for diagnosis of cryptococcal disease [4]. The limitations of culture are that it can be time consuming and labor intensive. Cryptococcal antigen (CrAg) lateral flow assays are rapid, specific, and may be more sensitive than culture when the burden of organism is low [4]. However, in patients presenting with symptoms of meningo-encephali-tis, the causative organism is frequently not clinically apparent, there can be a large potential differential diagnosis requiring consideration, and there may be limited available cerebral spinal fluid (CSF) for testing. Efforts have been made in recent years to develop assays that would allow testing for a wide variety of pathogens associated with meningo-encephalitis in a short time frame and using small volumes of CSF. The FilmArray Meningitis/Encephalitis (ME) Panel (BioFire Diagnostics, Salt Lake City, UT) uses multiplex polymerase chain reaction (PCR) to test for 14 targets using just 200 µL of CSF with a reported hands-on time of 2 minutes and a turnaround time of approxi-mately 1 hour [5]. As with all diagnostic tools, it is paramount that clinicians interpret results from PCR panels such as these in the context of the patient’s clinical presentation, and that they are aware of any pitfalls associated with such tests. Herein we report a case where the clinical outcome for the patient was neg-atively impacted by the use of a PCR panel.CASE REPORTA 54-year-old woman sought medical attention for a head-ache of 4  days’ duration, associated with nausea but without focal neurological signs. She had undergone deceased donor kidney transplantation for lupus nephritis–induced end-stage kidney disease 5 years previously and was receiving tacrolimus, mycophenolate mofetil (MMF), and prednisolone as immuno-suppressive therapy. She was diagnosed with migraine and pre-scribed a short course of hydrocodone. She did not have a past history of migraines. Over the proceeding month, she presented with persistent headache twice more to her primary care pro-vider, and on 3 occasions to the emergency department where she underwent computed tomography and magnetic resonance imaging (MRI) of the brain, both of which were within normal limits.Two months after the headache onset, the patient presented to her local hospital with headaches, nausea and vomiting, and fever. She was commenced on broad-spectrum antimicrobial therapy. Her CSF revealed a white cell count (WCC) of 145 cells/cm3 (27% neutrophils, 63% lymphocytes, 10% mono-cytes), glucose of 36 mg/dl, and protein of 72 mg/dl. Gram stain and routine bacterial cultures were negative. Fungal and myco-bacterial cultures were not performed. A FilmArray Meningitis/Encephalitis (ME) Panel detected human herpes virus 6 (HHV-6) but was negative for the remaining 13 components tested for by the panel (Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, cytomegalovirus, enterovirus, her-pes simplex virus 1 and 2, human parechovirus, varicella zoster virus, and Cryptococcus neoformans/gattii). Antimicrobial ther-apy was discontinued, and the patient was discharged on anal-gesia with symptoms attributed to HHV6 infection.In the weeks that followed, the patient continued to clinic-ally deteriorate, developing difficulty with her gait and fecal incontinence. New onset of abdominal pain, now 3  months after the headache onset, prompted her to once again seek med-ical assessment. The patient was readmitted, and her work-up revealed acute pancreatitis of unknown etiology, with a lipase of 18 800 units/L. At this time, MMF was held and she continued on tacrolimus and prednisolone immunosuppression. On day Open Forum Infectious Diseases®© The Author(s) 2017. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.DOI: 10.1093/ofid/ofx242Received 28 August 2017; editorial decision 30 October 2017; accepted 30 October 2017.Correspondence: J. A. O’Halloran, MB BcH BAO, PhD, Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, 4523 Clayton Ave, Campus Box 8051, St. Louis, MO 63110 (janeaohalloran@wustl.edu).2 • OFID • BRIEF REPORT11 of her admission, the patient became febrile to 38.6°C. Blood cultures were obtained, and she was initiated on empiric mer-openem; subsequently, linezolid and micafungin were added. On day 19, after a fall, she was noted to be encephalopathic with psychomotor slowing, which triggered a repeat neurolog-ical workup. A brain MRI did not reveal any acute intracranial processes. Lumbar puncture (LP) revealed a WCC count of 320 cells/cm3 (58% neutrophils, 37% lymphocytes), a glucose of 8  mg/dl, and protein of 121  mg/dl. No opening pressure was recorded. Again, a FilmArray ME Panel was performed, but it was negative for all pathogens included in the panel. CSF bac-terial culture was negative.On hospital day 20, the patient was transferred to a tertiary care academic center. On arrival, she was afebrile but remained encephalopathic, with altered speech. She intermittently fol-lowed simple commands and had evidence of meningismus. A  high-volume (10  mL of CSF) LP was performed, and the IMMY cryptococcal antigen lateral flow assay was positive at a titer of 1:20. Her serum CrAg was also positive at 1:10. The patient was initiated on liposomal amphotericin and flucytosine for management of cryptococcal meningitis. CSF from the LP performed prior to transfer was obtained and was also found to be positive at a titer of 1:40. All cultures, including blood and CSF, were negative. The patient’s symptoms improved on liposomal amphotericin and flucytosine, and she was ultimately discharged 3 weeks later on fluconazole.DISCUSSIONThis case highlights some important issues associated with the use of PCR panels such as the FilmArray ME Panel in clin-ical practice. The clinical assay performance of the FilmArray ME Panel was assessed in a multicenter study that examined 1560 residual CSF samples from patients undergoing lumbar puncture at 11 sites across the United States [6]. In this study, the results from the FilmArray ME Panel were compared with what was considered by the study team to be an appropriate comparator method. The comparator was CSF culture for the bacteria and bidirectional PCR sequencing for the viruses and fungi included in the panel. The sensitivity for Cryptococcal neoformans/gattii was reported at 100% (1/1) and was included in the overall assay sensitivity despite being calculated from a single positive specimen. Specificity for Cryptococcus spp. was reported as 99.7% (1555/1559). Of note, the comparator method for Cryptococcus in this study was PCR and not culture or CrAg testing. The investigators reviewed the records of the 4 patients who had positive results by the FilmArray ME Panel but negative results by the comparator PCR and determined that 2 were falsely negative by the comparator, and thus true positives detected by the FilmArray ME Panel. Not included in the published results but included in the FilmArray ME Panel package insert were comparisons with the test results of the clinical labs. The FilmArray ME Panel was positive for 1 of the 8 (12.5%) specimens positive for CrAg, and positive for 2 of 3 (66.7%) that were culture positive. Of the 7 that were positive for CrAg but negative by the FilmArray ME Panel, all patients were on antifungals at the time of specimen collection or had a history of Cryptococcal meningitis.Interestingly, a smaller preclinical study of a 16-target FilmArray ME Panel highlighted the importance of an appro-priate comparator test [7]. When the 16-target assay was performed on 342 stored CSF specimens, 8 of 14 (57%) CrAg-positive specimens were positive by the FilmArray ME panel, with 1 specimen that was CrAg negative/FilmArray positive also testing positive by sequencing. The false-negative results came from specimens with relatively low CrAg titers and/or high PCR crossing thresholds, and therefore were likely related to low burden of disease. This may also explain why the FilmArray ME Panel performed well in subjects with first pres-entations of cryptococcal meningitis in a study performed in sub-Saharan Africa where the median quantitative culture was 8950 CFU/mL (IQR, 118–113  500 CFU/mL) [8]. Decreased sensitivity was observed in this study on follow-up CSF spec-imens obtained from therapeutic lumbar puncture in patients receiving appropriate antifungal therapy and presumably hav-ing lower fungal burdens. In those with positive cryptococcal cultures and quantitative colony count <100 CFU/mL, the sen-sitivity was 50% (6/12).In the case reported herein, the patient was treated with the calcineurin inhibitor tacrolimus, a class of drugs known to have in vitro antifungal activity. In solid organ transplant recipients, tacrolimus has previously been associated with lower mortality rates in the setting of cryptococcal infection [9]. It is therefore possible that tacrolimus therapy may have contributed to the lower CrAg titers in this case, and potentially to the false-neg-ative cryptococcal result obtained on the FilmArray ME Panel, although there are no specific data available in relation to the latter. It is also noteworthy that while echinocandins generally have little activity against Cryptococcus neoformans alone, in vitro synergy between caspofungin and tacrolimus has been reported [10].To our knowledge, this is the first reported case of a patient whose clinical outcome was negatively impacted by a false-neg-ative cryptococcal PCR on the FilmArray ME panel. The patient had known risk factors for cryptococcal meningitis with a typ-ical clinical presentation and CSF profile, which ought to have prompted a more thorough investigation. This case serves to highlight to clinicians the importance of understanding the diagnostic accuracy of tests performed in order to ensure that they are appropriately interpreted, particularly in the setting where a sensitive, cheap, and easily performed alternative diag-nostic test exists. Compared with culture and CrAg, the sensi-tivity of the FilmArray ME Panel for Cryptococcus in the United States appears to be in the mid-60% range at best. Patients with a low burden of disease or who are already on antifungals are BRIEF REPORT • OFID • 3more likely to have a false-negative FilmArray ME Panel for Cryptococcus. This case also highlights the potential downside of testing for a multitude of pathogens, several of which may not be clinically relevant. In this case, the initial FilmArray ME panel performed was positive for HHV6, and the patient symp-toms and abnormal CSF profile were initially inappropriately attributed to this, and possibly contributed to the delay in the diagnosis of this patient’s cryptococcal meningitis.In conclusion, clinicians should be aware that a negative cryptococcal result on this platform does not rule out crypto-coccal meningitis, and standard assessment with CrAg and culture should be performed when the clinical scenario is con-sistent with cryptococcal infection.AcknowledgmentsDisclosures. Dr. Burnham reports grants from Cepheid, grants and personal fees from Accelerate Diagnostics, grants from bioMerieux, grants from Aperture Bio, grants from Theravance, personal fees from Thermo Fisher Scientific, and personal fees from BD outside the submitted work. Dr. Dubberke reports personal fees from Alere and personal fees from BioFire outside the submitted work. Dr. Powderly reports grants and personal fees from Merck Labs, grants from Astellas, and personal fees from Gilead out-side the submitted work. All other authors have no reported conflicts of interest.Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.References1. Rajasingham R, Smith RM, Park BJ, et  al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis 2017; 17:873–81.2. Brizendine KD, Baddley JW, Pappas PG. Predictors of mortality and differences in clinical features among patients with Cryptococcosis according to immune status. PLoS One 2013; 8:e60431.3. Aye C, Henderson A, Yu H, Norton R. Cryptococcosis—the impact of delay to diagnosis. Clin Microbiol Infect 2016; 22:632–5.4. Nalintya E, Kiggundu R, Meya D. Evolution of cryptococcal antigen testing: what is new? Curr Fungal Infect Rep 2016; 2016:1–6.5. Wootton SH, Aguilera E, Salazar L, et al. Enhancing pathogen identification in patients with meningitis and a negative Gram stain using the BioFire FilmArray(®) Meningitis/Encephalitis panel. Ann Clin Microbiol Antimicrob 2016; 15:26.6. Leber AL, Everhart K, Balada-Llasat JM, et al. Multicenter evaluation of BioFire FilmArray Meningitis/Encephalitis panel for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens. J Clin Microbiol 2016; 54:2251–61.7. Hanson KE, Slechta ES, Killpack JA, et al. Preclinical assessment of a fully auto-mated multiplex PCR panel for detection of central nervous system pathogens. J Clin Microbiol 2016; 54:785–7.8. Rhein J, Bahr NC, Hemmert AC, et  al; ASTRO-CM Team. Diagnostic perfor-mance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda. Diagn Microbiol Infect Dis 2016; 84:268–73.9. Singh N, Alexander BD, Lortholary O, et  al; Cryptococcal Collaborative Transplant Study Group. Cryptococcus neoformans in organ transplant recipients: impact of calcineurin-inhibitor agents on mortality. J Infect Dis 2007; 195:756–64.10. Del Poeta M, Cruz MC, Cardenas ME, et al. Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743 873) with calcineurin inhibitors FK506 and L-685 818 against Cryptococcus neoformans. Antimicrob Agents Chemother 2000; 44:739–46.

Pitfalls associated with the use of molecular diagnostic panels in the diagnosis of cryptococcal meningitis

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Pitfalls associated with the use of molecular diagnostic panels in the diagnosis of cryptococcal meningitis

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