Buenos Aires 01 de Mayo del 2022

False Negative Diagnostic Errors With Polymerase Chain Reaction For The Detection of Cryptococcal Meningoencephalitis

False Negative Diagnostic Errors With Polymerase Chain Reaction For The Detection of Cryptococcal Meningoencephalitis

       Paul Lewis, Cameron Lanier, Paras Patel, Whitney Krolikowski and Matthew Krolikowski

                                                                 Medical Mycology, 2020, 58, 408–410 - Brief Report



The accuracy of the BioFire FilmArray Meningitis/Encephalitis (ME) panel for the identification of Cryptococcus has recently been called into question.
The primary objective of this study was to assess the agreement between the BioFire ME polymerase chain reaction (PCR) and other markers of cryptococcal infection. This retrospective review identified five patients with cryptococcal meningoencephalitis, 4 of whom had a negative ME panel for Cryptococcus. All five cases had positive serum cryptococcal antigens, and three of five had a positive cerebrospinal fluid (CSF) culture for Cryptococcus. The BioFire ME panel does not appear to be reliable for ruling out Cryptococcus meningoencephalitis; multiple testing methods are recommended.


Cryptococcal meningoencephalitis is an infection of the meninges and brain parenchyma predominantly caused by Cryp[1]tococcus neoformans or Cryptococcus gattii.1 C. neoformans is often associated with immunosuppression, such as human immunodeficiency virus (HIV), glucocorticoid therapy, solid organ transplantation, cancer, or rheumatological disorders.2 Alternatively, C. gattii occurs in immunocompetent hosts.2 Diagnosis evolved away from direct microscopic and histopathologic ex[1]amination to rapid detection methods such as the cryptococcal antigen and polymerase chain reaction (PCR).3 The cryptococcal antigen, which detects glucuronoxylomannan within the polysaccharide capsule, has emerged as being inexpensive yet sensitive for detection.4
PCR is another alternative for rapid detection and diagnosis of viruses, bacteria, and fungi and is often described as highly sensitive and specific.5–8 The BioFire FilmArray Meningitis/Encephalitis (ME) Panel is a multiplex PCR that tests for 14 common pathogens, including Cryptococcus.5,9 Result are available in approximately 1 hour, compared to cultures that can take upward of 5 days.
In a multicenter evaluation on the sensitivity and specificity of the BioFire PCR of 1560 cerebrospinal fluid (CSF) specimens, only one sample resulted in a true positive, four were false positives, and no false negatives were reported.
Additionally, there has been a growing body of evidence that has shown the testing process may be flawed, including false negatives compared to other diagnostic techniques.7,10–12 Given this limitation, we retrospectively reviewed our cases of cryptococcal meningoencephalitis to determine if the BioFire ME panel resulted in false negatives based on positive cryptococcal antigen tests.


This retrospective review was conducted at Johnson City Medical Center, a 488-bed regional referral hospital. Patients were included if they were admitted inpatient status and were diagnosed with cryptococcosis by International Classification of Diseases, 10th revision (ICD-10) of B45, had symptoms of meningitis or meningoencephalitis, and had a BioFire ME panel performed.
The primary objective was to assess the discrepancy of BioFire ME PCR and other markers of cryptococcal infection including fungal culture, and IMMY cryptococcal antigen performed on serum and CSF. Background data collected included age, race, sex, immunosuppression status, and diagnosis.
CSF analysis included color, appearance, red blood cell count, white blood cell count and differentiation, glucose, and protein. Cryptococccal specific markers collected included BioFire ME panel cryptococcal PCR, cell culture, serum cryptococcal antigen, and CSF cryptococcal antigen.


There were five cases meeting criteria. All presented with central nervous system (CNS) symptoms: four reported headache, three presented with altered mental status or confusion, one complained of neck stiffness, and one had hydrocephalus. Fever was reported in three patients. The mean age was 49 years (range 29– 70), 4/5 were male, and all were white. There was one case with a positive result from the PCR and four cases with negative results for PCR. All five cases had a positive serum cryptococcal antigen. CSF culture was positive in three of the five cases. CSF analysis and cryptococcal assays are seen in Tables 1 and 2, respectively. It should be noted that results from the lumbar puncture on case 5 were not consistent with meningoencephalitis. However, magnetic resonance imaging of the brain demonstrated multiple lesions of the meninges and parenchyma, consistent with meningoencephalitis. The patient was subsequently started on amphotericin based on clinical diagnosis leading to improvement in the patient’s neurological symptoms. Repeat lumbar puncture was declined by the patient; however, the cryptococcal serum antigen was repeated 14 days later, which resulted at 1:1280


This retrospective review identified five patients with cryptococcal meningoencephalitis. All five patients had a detectable serum cryptococcal antigen, four of the five had a detectable CSF cryptococcal antigen, three of the five had cultures positive for Cryptococcus neoformans, and one of the five had a positive CSF PCR positive for Cryptococcus neoformans/gattii. Based on these results, CSF PCR for Cryptococcus is insufficient to rule out cryptococcal meningoencephalitis. For these cases, serum cryptococcal antigen appears to be the most robust; however, the numbers are insufficient to extrapolate these results externally.
These results are consistent with other recent reports. A case report of a post kidney transplant female experienced persistent headaches. BioFire ME Panel was only positive for human herpes virus 6(HHV-6), while negative for Cryptococcus. She returned with worsening symptoms; repeat ME panel remained negative. After transfer to another facility, serum and CSF cryptococcal antigens were positive.10 Other false negative reports have been reported recently, and research has been published to find an explanation as to why these errors in reporting have occurred.11,12 Two case reports described situations where both patients tested negative for PCR but later had positive culture results for C. neoformans. 12 The 60% sensitivity rate of the BioFire ME panel for Cryptococcus is worrisome for the overall utility of the test in patients. Authors have also pointed to the potential for patients with low disease burden or those receiving treatment are more likely to have a negative ME panel.10 Issues of misdiagnosis of Cryptococcus is not limited to false negatives. There were two false positives reported in the original sensitivity and specificity trial. The authors claim that positive cryptococcal result should be interpreted with clinical correlation, including antigen testing.
A high number of false positive results were seen with using BioFire ME across all organisms tested, and the study failed to be able to identify a conclusive answer as to why this occurred.7 Case 5 warrants further discussion, due to low serum anti[1]gen titers and a negative CSF antigen. Clinical correlation supported this case as early cryptococcal meningoencephalitis. This presentation has been previously described among HIV-positive individuals. Of 1201 patients presenting with suspected meningoencephalitis, 4% (54/1201) had negative CSF cryptococcal antigen, all of whom had their cryptococcal infection detected by a positive serum cryptococcal antigen.13 One hospital’s review of PCR reliability demonstrated a sensitivity rate calculating to 76%, with positivity rates only equaling 8.6%. Explanation for the low success rates were based on poor effective treatment in PCR-negative cases and contaminated PCR-positive cases. The net effect of change of PCR on patient treatment regimen was only seen in 3.2% of patient cases.14 Though published results of false positives seems to be small based on the published literature, there is enough concern where general recommendations have been made on laboratory procedures to attempt to reduce errors.15 Cost analysis research has examined standard of care using blood cultures versus the BioFire ME panel. While the costs of the ME Panel have is more than classical testing, the reduction in antibiotic use and subsequent total costs of treatment tended to have lower overall total costs. However, this differ[1]ence in cost was minimal.
Average costs per standard of care was $239.63 versus $239.14 when using the BioFire FA ME. While a reduction in costs is apparent the degree to how much that can truly be valued if false negatives continue to occur is questionable, especially when the cost-savings is low.9 PCR failed to identify four of five cases of cryptococcal meningoencephalitis. Serum cryptococcal antigen was detected in all five cases. Prescribers should exercise caution when utilizing the BioFire ME panel for ruling out cryptococcal meningoencephalitis. Suspicion of cryptococcosis should warrant multiple testing methods including serum and CSF antigens and cultures.



1. Perfect JR, Dismukes WE, Dromer F et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2010; 50: 291–322
2. Hagen F, Khayhan K, Theelen B et al. Recognition of seven species in the Crypttococcus gattii /Cryptococcus neoformans species complex. Fungal Genet Biol.2015; 78: 16.
3. Nalintya E, Kiggundu R, Meya D. Evolution of cryptococcal antigen testing:what is new? Curr Fungal Infect Rep. 2016; 10: 62–67.
4. Marcos JY, Pincus DH. Fungal diagnostics: review of commercially available methods. Methods Mol Biol. 2013; 968: 25–54.
5. Rhein J, Bahr NC, Hemmert AC et al. Diagnostic performance of a multiplex
PCR assay for meningitis in an HIV-infected population in Uganda. Diagn Microbiol Infect Dis. 2016; 84: 268.
6. Sidiq F, Hoostal M, Rogers S. Rapid identification of fungi in culture-negative clinical blood and respiratory samples by DNA sequence analyses. BMC Research Notes. 2016; 9: 1–8.
7. Leber A, Everhart K, Balada-Liasat J et al. Multi-center evaluation of BioFire FilmArray meningitis /encephalitis panel for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens. J Clin Microbiol. 2016; 54: 2251–2261.
8. Ahmad S, Khan Z, Mustafa A et al. Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol. 2002; 40: 2483–2489.
9. Soucek D, Dumkow L, VanLangen K et al. Cost justification of the BioFireFilm Array meningitis /encephalitis panel versus standard of care for diagnosing meningitis in a community hospital. Journ Pharm Pract. 2019; 32: 36–40.
10. O’Halloran J, Franklin A, Lainhart W et al. Pitfalls associated with the use of molecular diagnostic panels in the diagnosis of cryptococcal meningitis. Open Forum Infect Dis. 2017; 4: ofx242.
11. Lee C, Chui I, Yan C et al. False negative results caused by erroneous automated result interpretation algorithm on the FilmArray 2.0 Instrument. Clin Chem Lab Med. 2018; 56: e43–e45.
12. Chew K, Lee C, Cross G et al. Culture-confirmed cryptococcal meningitis not detected by Cryptococcus PCR on the BioFire Meningitis/Encephalitis Panel. Clin Microbiol Infect. 2018; 24: 791–792.
13. Ssebambulidde K, Bangdiwala AS, Kwizera R et al. Symptomatic cryptococical antigenemia presenting as early cryptococcal meningitis with negative CSF analysis. Clin Infect Dis. 2018 Sep 25. doi: 10.1093/cid/ciy817. 
14. Khoury Amit S, Geffen Y, Adler A. Clinical utility of pan-microbial PCR assays in the routine diagnosis of infectious disease. Diagn Microbiol Infect Dis. 2019; 93: 232–237.
15. Gomez CA, Pinsky BA, Liu A, Banaei A. Delayed diagnosis of tuberculosis meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray syndromic polymerase chain reaction panel. Open Forum Infect Dis. 2016; 4: ofw245