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Diagn Microbiol Infect Dis. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Diagn Microbiol Infect Dis. 2016 March ; 84(3): 268–273. doi:10.1016/j.diagmicrobio.2015.11.017.
Diagnostic Performance of a Multiplex PCR assay for meningitis in an HIV-infected population in Uganda Joshua Rhein1,2, Nathan C Bahr1, Andrew C Hemmert3, Joann L Cloud3, Satya Bellamkonda3, Cody Oswald3, Eric Lo3, Henry Nabeta2, Reuben Kiggundu2, Andrew Akampurira2, Abdu Musubire1,2, Darlisha Williams1,2, David B Meya1,2, and David R Boulware1 on behalf of the ASTRO-CM Team
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1Division
of Infectious Disease and International Health, Department of Medicine, University of Minnesota, Minneapolis, MN, USA 2Infectious Disease Institute, Makerere University, Kampala, Uganda 3BioFire Diagnostics, LLC, Salt Lake City, UT, USA
Abstract
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Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first-episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity, and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.
Keywords
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meningitis; PCR; immunocompromised; HIV; diagnostics; cryptococcal meningitis
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Corresponding author: Joshua Rhein, Infectious Disease Institute, P.O. Box 22418, Mulago Hospital Complex, Kampala, Uganda. [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflicts of Interest: Drs. Hemmert and Cloud, as well as Ms. Bellamkonda, Mr. Oswald, and Mr. Lo are employees of BioFire Diagnostics. All other authors have no conflict of interest.
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1. Introduction Meningitis is a common clinical condition in Sub-Saharan Africa. In the African meningitis belt, bacterial pathogens such as Neisseria meningitidis and Streptococcus pneumoniae have historically been among the most common etiologies, resulting in an estimated 800,000 cases between 1996 and 2010 (1). The higher prevalence of HIV infection in Eastern and Southern Africa has had a dramatic impact on the etiology of meningitis, with an emergence of Cryptococcus neoformans (2–8) at an estimated 1 million cases of cryptococcal meningitis occurring in 2008 (9). In immunocompromised hosts, the broad spectrum of potential diagnoses creates the need for multiplex assays to streamline diagnosis. Rapid diagnosis of meningitis is essential to optimize survival and minimize unnecessary healthcare costs related to isolation procedures and empiric treatment.
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Additionally, as cryptococcal meningitis is now the most common cause of meningitis in Africa, (2–8) recurrent second episodes create a diagnostic dilemma of differentiating culture-positive relapse from paradoxical immune reconstitution inflammatory syndrome (IRIS) after initiating antiretroviral therapy. The gold standard to distinguish relapse from IRIS remains CSF culture, which can often take 5–14 days in the setting of culture-positive relapse. India ink is unhelpful in this setting, as unviable Cryptococcus are often still present in CSF with IRIS (10). This delay in diagnosis creates a clinical problem. Persons with IRIS likely benefit from anti-inflammatory therapy (11, 12), yet the use of corticosteroids in persons with relapse may lead to detrimental outcomes (13). Similarly, the treatment for relapse with amphotericin is associated with potentially severe toxicities and may not be of benefit for persons with paradoxical IRIS (14).
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This study aimed to comprehensively evaluate the utility of using a new multiplex PCR panel (FilmArrray Meningitis/Encephalitis panel, BioFire Diagnostics, LLC, Salt Lake City, Utah) in determining the microbiologic etiologies of meningitis in an HIV-infected adult population with suspected meningitis in Kampala, Uganda. With a single PCR test, the FilmArray Meningitis/Encephalitis panel allows detection of several common pathogens (bacteria, viruses, and fungi) that cause meningitis, including Cryptococcus.
2. Materials and Methods 2.1 Setting and Patients
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We conducted a prospective cohort study of 69 HIV-infected adult inpatients presenting with suspected meningitis between January 2014 and May 2014 at Mulago National Hospital, a tertiary referral hospital in Kampala, Uganda. This was conducted as part of a diagnostic sub-study for the Adjunctive Sertraline for the Treatment of HIV-Associated Cryptococcal Meningitis (ASTRO-CM) pilot trial (ClinicalTrials.gov: NCT01802385). We only enrolled patients who presented with suspected meningitis and provided written informed consent for lumbar puncture (LP). If HIV-serological status was unknown, rapid HIV testing was performed. Institutional review board (IRB) approval occurred at all relevant organizations.
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2.2 Routine CSF Examination
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A total of 119 CSF specimens were prospectively collected by lumbar puncture at time of meningitis diagnosis (n=51) or 2–28 days after cryptococcal meningitis diagnosis (n=68) in the case of therapeutic lumbar punctures. The Makerere University Microbiology Laboratory performed CSF testing on the day of collection to determine white cell count and protein level. Cryptococcal meningitis was diagnosed immediately at the bedside using cryptococcal antigen (CRAG) lateral flow assay (IMMY, Norman, Oklahoma). Quantitative CSF fungal culture was performed on fresh CSF in the Makerere Microbiology Laboratory with five 1:10 serial dilutions of 100µL of CSF, as previously described (15). Among persons with cryptococcal meningitis, therapeutic LPs were routinely performed using manometers on day 3, 7, 10, and 14 of amphotericin therapy and additionally as needed for intracranial pressure control. Quantitative CSF fungal cultures were performed with every CSF collection, with the exception of 1 specimen that had a positive culture without quantification. Among CSF specimens negative by CRAG, Gram stain, bacterial culture, and tuberculosis testing were performed. 2.3 FilmArray Multiplex PCR System
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With diagnostic or therapeutic LPs (specific for cryptococcal meningitis participants), a 1mL aliquot of CSF was cryopreserved at −80°C, as volume allowed. Cryopreserved CSF specimens were then shipped in dry ice to BioFire Diagnostics, where 200 mcL of CSF was analyzed using the FilmArray Meningitis/Encephalitis system (Figure 1). The system employs a reagent freeze-dried pouch that stores components necessary for sample preparation, reverse transcription, PCR and detection (16–18). The user injects hydration solution and sample combined with sample buffer into the pouch. Sample extraction, purification, and multiplexed-nested PCR are performed in the enclosed pouch with the FilmArray instrument. Using endpoint melting curve analysis, a result is generated for each of 16 targeted pathogens: 6 bacterial, 8 viral, and Cryptococcus neoformans/gattii. FilmArray operators were blinded to prior microbiology results.
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CSF from study subjects with known tuberculosis, by culture or GeneXpert (Cepheid, Sunnyvale, CA), which is part of the work-up for the main ASTRO-CM trial, were not included in this sub-study due to biosafety concerns in the transport and analysis by FilmArray. An Investigation Use Only (IUO) version of the FilmArray Meningitis/ Encephalitis panel was used to test these samples. Performance characteristics of this FilmArray panel had not been evaluated by the FDA at the time of this study. Confirmatory PCR testing was carried out using Argene® (Biomerieux, Marcy l'Etoile, France) for viral pathogens and genesig® Streptococcus pneumoniae PCR (Primerdesign, Southamptom, United Kingdom). Epstein-Barr virus (EBV) was not validated by additional confirmatory testing due to its ubiquitous nature and unclear significance in this immunocompromised population, as described previously (19). 2.4 Statistical Analysis The diagnostic performance of multiplex PCR for cryptococcal meningitis was compared to a reference standard of positive CSF cryptococcal culture in patients with cryptococcal meningitis. Diagnostic performance was determined only from subjects who had CSF Diagn Microbiol Infect Dis. Author manuscript; available in PMC 2017 March 01.
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available at diagnosis. Given the diagnostic difficulties associated with second episodes of cryptococcal meningitis, individuals presenting to the study with a previous history of treated cryptococcal meningitis were excluded from the analysis of diagnostic performance, with multiplex PCR results instead assessed by the ability to determine the presence (fungal relapse) or absence (paradoxical IRIS with sterile cultures) of cryptococcal growth on CSF cultures. To assess persistence of infection while receiving antifungal therapy, the negative predictive value for detecting ongoing culture positive cryptococcal meningitis was determined among follow up CSF specimens obtained from therapeutic LPs. Finally, the presence of other CNS pathogens was determined by FilmArray multiplex PCR in every CSF specimen collected.
3. Results Author Manuscript
We enrolled a cohort of 69 HIV-infected persons with suspected meningitis at Mulago Hospital in Kampala, Uganda. The cohort consisted of 49 men and 20 women with a median age of 32 (IQR, 25–44) years and median CD4 count of 33 (IQR, 11–69) cells/µL. Among participants, 52% (36/69) presented with a first episode of cryptococcal meningitis (median quantitative culture 8,950 CFU/mL, IQR 118–113,500 CFU/mL), 12% (8/69) had a previous history of cryptococcal meningitis (three of whom had microbiological relapse, range: 90–9,700 CFU/mL) and 36% (25/69) did not have active or prior cryptococcal meningitis.
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A total of 84 CSF specimens were obtained from individuals with first-episode cryptococcal meningitis, of which 18 were obtained at time of diagnosis and 66 were obtained from therapeutic lumbar punctures conducted 2–28 days after diagnosis (Figure 2). Ten CSF specimens were obtained from 8 individuals presenting with second-episode cryptococcosis, with 2 CSF specimens being obtained from therapeutic lumbar punctures. All 25 CSF specimens obtained from individuals without Cryptococcus were obtained at time of diagnosis. In total of the 119 CSF specimens tested on the FilmArray Meningitis/ Encephalitis system, 117 specimens had both quantitative CSF fungal cultures performed and generated a FilmArray result on first testing. One specimen had a positive culture reported for Cryptococcus without quantification (and was positive by FilmArray), and one specimen from a patient without cryptococcal meningitis had a failure of the internal PCR control with a test invalid reported. 3.1 Diagnostic Performance for Cryptococcal Meningitis
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The assay provided 100% sensitivity and specificity compared to cryptococcal culture in diagnostic CSF specimens collected at hospital admission (n=18 positive; n=24 negative). Overall, FilmArray performance was related to the burden of Cryptococcus present in the CSF specimen (Figure 3). In comparing the CSF Cryptococcus CFU/mL versus FilmArray positivity among all sample time points, there was 96% (49/51) sensitivity for detecting ≥100 Cryptococcus CFU/mL CSF (95%CI: 87–99%). Overall, a negative FilmArray result had 95% (39/41) predictive value for a CSF quantitative culture being ≤100 CFU/mL or sterile in CRAG positive CSF (95%: 83%–99%). This included CSF from individuals receiving therapy for firstepisode cryptococcal meningitis (n=66, from 31 individuals) as
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well as those with second episodes of cryptococcal meningitis (n=10, from 8 individuals). In CSF specimens obtained from individuals receiving antifungal therapy (n=76), FilmArray results for Cryptococcus predicted conversion to culture sterility with 71% (20/28) negative predictive value (i.e. when FilmArray results were negative for Cryptococcus, culture was sterile).
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A novel application was in assessing the CSF of persons with a history of cryptococcal meningitis presenting with a second symptomatic episode, where CSF was cryptococcal antigen positive. In 8 individuals with second episodes of symptomatic recurrence of cryptococcal meningitis, FilmArray test results were positive for Cryptococcus in all cases of CSF culture-positive fungal relapse (n=3, with quantitative cultures of 90 CFU/mL, 90 CFU/mL, and 9,700 CFU/mL) and negative for Cryptococcus in all cases having sterile cultures (n=5) with presumed paradoxical immune reconstitution inflammatory syndrome or with persistently elevated intracranial pressure (and a normal CSF white cell count and normal CSF protein). 3.2 Pathogen Detection in CSF
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In this population of persons living with HIV/AIDS with suspected meningitis in Uganda, the FilmArray Meningitis/Encephalitis panel detected six distinct pathogens in CSF. Cryptococcus was very common (n=39/69; including both first and second episodes, where Cryptococcus was detected by both CSF cultures and the FilmArray panel), as expected. Cases of cryptococcal meningitis were frequently associated with viral co-infection with EBV and other viruses. Of those that did not have cryptococcal meningitis, 1% (1/69) had bacterial meningitis, 7% (5/69) had non-EBV viral pathogens identified by FilmArray, and 10% (7/69) had EBV identified only. No pathogen was identified in 25% (17/69) of participants (Figure 4). EBV was frequently detected (n=45) in this immunocompromised population. Other viruses detected include cytomegalovirus (CMV; n=2), varicella zoster virus (VZV; n=2) and human herpes virus 6 (HHV-6; n=1). Streptococcus pneumoniae was detected in one subject by FilmArray PCR; this specimen had gram positive cocci detected on Gram’s stain although CSF bacterial culture was negative. The five specimens containing non-EBV viral pathogens were confirmed by Argene PCR and S. pneumoniae by genesig PCR.
4. Discussion
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In our population of HIV-infected Ugandans with suspected meningitis, we observed excellent detection of first-episodes of cryptococcal meningitis, the most common etiology in our setting, using the BioFire FilmArray Meningitis/Encephalitis panel. In addition, this PCR-based platform demonstrated a novel diagnostic utility among patients with a history of cryptococcal meningitis and recurrent symptoms, specifically the ability to differentiate between culture-positive relapse and paradoxical IRIS with sterile cultures. Furthermore, the assay detected several additional pathogens in CSF, in particular EBV at high rates. Similar to a prior investigation in Uganda, herpes simplex virus (HSV) was not detected as a cause of viral meningitis (19).
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PCR testing appears to be useful in cryptococcal disease. While CRAG testing performs extremely well in detecting initial infection, and the low cost of the CRAG lateral flow assay underscores its central role in the initial diagnosis of cryptococcal meningitis, CRAG can remain positive for months to years (10). Therefore, while the multiplex PCR test cannot replace CRAG as the preferred assay for first episodes of cryptococcosis (8), our findings suggest that PCR may have a role in the differentiation of culture-positive cryptococcal meningitis relapse from paradoxical IRIS or other non-cryptococcal related etiologies in cases of symptom recurrence in patients with a previous history of cryptococcosis. In this scenario, a positive test could avoid inappropriate use of steroids in patients with culturepositive relapse, upon detection of Cryptococcus species by PCR. Given the small number of second episodes of recurrent cryptococcosis in this study, however, further validation studies are required before PCR can be fully endorsed as a reliable and cost-effective test for differentiating relapse from paradoxical IRIS. Furthermore, PCR appears to allow for rapid detection of CSF culture sterility and thus a potential role in better tailoring the duration of amphotericin B therapy and hospitalization (20). The potential of shortening hospital durations could have a substantial cost savings in middle and high income countries. In addition to the benefits of PCR as a stand-alone test in known cryptococcosis, the inclusion of Cryptococcus in a multiplex panel allows detection when cryptococcal disease might not suspected, for example in unknown immune deficiency. In the United States, for example, 3600 cases of cryptococcal meningitis occur annually, with one-third in HIVseronegative individuals (21). Cryptococcal diagnosis in areas of low prevalence is often delayed and/or accidental (22, 23), and inclusion on a multiplex assay could lead to more rapid recognition.
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An operational limitation of our study is that multiplex PCR testing was not performed in on-site (in the case of FilmArray, results would have been available in about an hour), and we are therefore unable to make firm conclusions regarding the impact of introducing a multiplex PCR system on outcomes, such as timing of initiating appropriate therapy, hospital duration, avoidance of unnecessary diagnostic testing or antimicrobials, and costeffectiveness. It seems apparent, however, that the introduction of on-site multiplex PCR testing would result in more rapid and accurate diagnosis with minimization of unnecessary testing, a shortened time to initiation of targeted therapy, and in the case of cryptococcosis at least, potentially shorter hospital durations.
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The detection of EBV and other herpesviruses in this highly immunocompromised population is of unclear significance (19). Of note, EBV was left out of the recently FDAapproved panel which is now commercially available, and the high prevalence of EBV noted in this study should be interpreted with caution, since confirmatory PCR testing was not performed. The increasing use of multiplex PCR testing for simultaneous detection of several herpesviruses should shed more light on the frequency and clinical significance among more diverse populations. Multiplex PCR detected one case of bacterial meningitis which was positive by Gram’s stain and who had received antecedent ceftriaxone. Thus, in partially treated bacterial meningitis, PCR assays have the potential to provide a confirmed diagnosis.
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While our results are encouraging, it must be noted that the FilmArray Meningitis/ Encephalitis system had not yet been approved by the U.S. FDA, and additional studies to validate the role and cost-effectiveness of multiplex PCR in the diagnosis and monitoring of CNS infections are needed across a broad range of settings. The addition of Toxoplasma gondii, Mycobacterium tuberculosis, and John Cunningham (JC) virus PCR testing would be useful for multiplex panels used in immunocompromised populations such as those with advanced HIV, transplant patients, or in the case of JC virus, in persons with multiple sclerosis (24). The multiplex PCR panel tested in this study did not differentiate between C. neoformans and C. gattii. Given the predominance of C. neoformans in our study population, further evaluation of CSF of individuals with C. gattii infection is required. Our study is also limited by the relatively small sample size and the high burden of cryptococcal disease present, consistent with the ongoing reality in sub-Saharan Africa (8). Lastly, as with any new technology, cost may be a barrier for rapid adoption in a resource-limited setting such as Uganda, and further cost-effectiveness analyses are required.
5. Conclusion In summary, the FilmArray multiplex PCR panel offers a promising platform for the rapid diagnosis of meningitis. PCR testing appears to be particularly useful in detection of Cryptococcus species, predicting culture sterility, and differentiating IRIS from culturepositive relapse among persons with recurrent symptoms. Further additions to the panel focusing on immunosuppressed populations and minimizing the real world costs of the technology would make the panel more useful and the overall effectiveness of the assay requires further evaluation.
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Acknowledgements Research support for aspects of this project was received from the National Institutes of Allergy and Infectious Disease, the National Institute of Neurologic Disorders and Stroke and the Fogarty International Center of the National Institutes of Health (R01NS086312, U01AI089244, T32AI055433, R25TW009345).
ASTRO-CM Team members Jane Francis Ndyetukira, Cynthia Ahimbisibwe, Florence Kugonza, Sruti S. Velamakanni, Alice Namudde, Alisat Sadiq, Tadeo Kiiza Kandole, Julian Kaboggoza, Eva Laker, Tony Luggya, Liliane Tugume, Elissa Butler, Jonathan Dyal, Julie Neborak, Lexie King, Nathan Yueh, Kathy Huppler Hullsiek, Ryan Halupnick, Paul R Bohjanen, Andrew Kambugu.
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22. Katchanov J, Von Kleist M, Arasteh K, Stocker H. Time-to-amphotericin B' in cryptococcal meningitis in a european low-prevalence setting: Analysis of diagnostic delays. QJM. 2014; 107:799–803. [PubMed: 24722846] 23. Liao CH, Chi CY, Wang YJ, Tseng SW, Chou CH, Ho CM, et al. Different presentations and outcomes between HIV-infected and HIV-uninfected patients with cryptococcal meningitis. J Microbiol Immunol Infect. 2012; 45:296–304. [PubMed: 22608531] 24. Evangelopoulos ME, Koutoulidis V, Kilidireas K, Evangelopoulos DS, Nakas G, Andreadou E, et al. Immune reconstitution inflammatory syndrome mimicking progressive multifocal leucoencephalopathy in a multiple sclerosis patient treated with natalizumab: A case report and review of the literature. J Clin Med Res. 2015; 7:65–68. [PubMed: 25368707]
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Highlights We evaluated the utility of multiplex PCR in HIV-associated meningitis in Uganda. Multiplex PCR identified 6 distinct CNS pathogens from 69 patients with meningitis. At diagnosis, PCR detected Cryptococcus with excellent sensitivity and specificity. PCR differentiated between fungal relapse and paradoxical IRIS with sterile cultures. Automated multiplex PCR offers promising platform for rapid diagnosis of meningitis.
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Author Manuscript Author Manuscript Figure 1. The FilmArray multiplex PCR system
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The FilmArray system employs a reagent freeze-dried pouch that stores components necessary for sample preparation, reverse transcription, PCR and detection. The user injects hydration solution and sample combined with sample buffer into the pouch. A nested multiplex PCR is performed in a two-step process and, using endpoint melting curve analysis, a result is generated for each of 16 common pathogens.
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Figure 2. Cohort Composition
CSF specimens in the cohort were collected at diagnosis and in cases of cryptococcal meningitis, as part of follow-up therapeutic LP for control of intracranial pressure.
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Figure 3. FilmArray PCR positivity in comparison to Cryptococcus quantitative CSF culture at all time points of antifungal therapy
Performance characteristics of FilmArray Meningitis/Encephalitis panel compared to CSF cryptococcal quantitative culture by colony forming units (CFU)/mL of CSF. Among initial diagnostic specimens 100% (18/18) were FilmArray positive compared to culture growth, and 100% (24/24) were FilmArray negative when the CSF was sterile. Numbers indicate n=117 specimens with a quantitative culture result per group. One participant had a positive culture without quantification (and positive FilmArray), and one cryptococcal-negative subject had a failure of the internal PCR control with invalid FilmArray test reported and were not included in the analysis.
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Author Manuscript Author Manuscript Figure 4. Distribution of Pathogens Detected in CSF and the Relationship to Other Potential Coinfections
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Cryptococcus was the most common meningitis etiology in this cohort, followed by EBV only (n=7), viral pathogens (n=5), and bacterial meningitis (n=1). No pathogen was identified in 17 persons. Cases of cryptococcal meningitis were frequently associated with viral co-infection with EBV and other viruses (right inset).
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Diagn Microbiol Infect Dis. Author manuscript; available in PMC 2017 March 01. Published in final edited form as: Diagn Microbiol Infect Dis. 2016 March ; 84(3): 268–273. doi:10.1016/j.diagmicrobio.2015.11.017.
Diagnostic Performance of a Multiplex PCR assay for meningitis in an HIV-infected population in Uganda Joshua Rhein1,2, Nathan C Bahr1, Andrew C Hemmert3, Joann L Cloud3, Satya Bellamkonda3, Cody Oswald3, Eric Lo3, Henry Nabeta2, Reuben Kiggundu2, Andrew Akampurira2, Abdu Musubire1,2, Darlisha Williams1,2, David B Meya1,2, and David R Boulware1 on behalf of the ASTRO-CM Team
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1Division
of Infectious Disease and International Health, Department of Medicine, University of Minnesota, Minneapolis, MN, USA 2Infectious Disease Institute, Makerere University, Kampala, Uganda 3BioFire Diagnostics, LLC, Salt Lake City, UT, USA
Abstract
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Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first-episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity, and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.
Keywords
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meningitis; PCR; immunocompromised; HIV; diagnostics; cryptococcal meningitis
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Corresponding author: Joshua Rhein, Infectious Disease Institute, P.O. Box 22418, Mulago Hospital Complex, Kampala, Uganda. [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflicts of Interest: Drs. Hemmert and Cloud, as well as Ms. Bellamkonda, Mr. Oswald, and Mr. Lo are employees of BioFire Diagnostics. All other authors have no conflict of interest.
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1. Introduction Meningitis is a common clinical condition in Sub-Saharan Africa. In the African meningitis belt, bacterial pathogens such as Neisseria meningitidis and Streptococcus pneumoniae have historically been among the most common etiologies, resulting in an estimated 800,000 cases between 1996 and 2010 (1). The higher prevalence of HIV infection in Eastern and Southern Africa has had a dramatic impact on the etiology of meningitis, with an emergence of Cryptococcus neoformans (2–8) at an estimated 1 million cases of cryptococcal meningitis occurring in 2008 (9). In immunocompromised hosts, the broad spectrum of potential diagnoses creates the need for multiplex assays to streamline diagnosis. Rapid diagnosis of meningitis is essential to optimize survival and minimize unnecessary healthcare costs related to isolation procedures and empiric treatment.
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Additionally, as cryptococcal meningitis is now the most common cause of meningitis in Africa, (2–8) recurrent second episodes create a diagnostic dilemma of differentiating culture-positive relapse from paradoxical immune reconstitution inflammatory syndrome (IRIS) after initiating antiretroviral therapy. The gold standard to distinguish relapse from IRIS remains CSF culture, which can often take 5–14 days in the setting of culture-positive relapse. India ink is unhelpful in this setting, as unviable Cryptococcus are often still present in CSF with IRIS (10). This delay in diagnosis creates a clinical problem. Persons with IRIS likely benefit from anti-inflammatory therapy (11, 12), yet the use of corticosteroids in persons with relapse may lead to detrimental outcomes (13). Similarly, the treatment for relapse with amphotericin is associated with potentially severe toxicities and may not be of benefit for persons with paradoxical IRIS (14).
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This study aimed to comprehensively evaluate the utility of using a new multiplex PCR panel (FilmArrray Meningitis/Encephalitis panel, BioFire Diagnostics, LLC, Salt Lake City, Utah) in determining the microbiologic etiologies of meningitis in an HIV-infected adult population with suspected meningitis in Kampala, Uganda. With a single PCR test, the FilmArray Meningitis/Encephalitis panel allows detection of several common pathogens (bacteria, viruses, and fungi) that cause meningitis, including Cryptococcus.
2. Materials and Methods 2.1 Setting and Patients
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We conducted a prospective cohort study of 69 HIV-infected adult inpatients presenting with suspected meningitis between January 2014 and May 2014 at Mulago National Hospital, a tertiary referral hospital in Kampala, Uganda. This was conducted as part of a diagnostic sub-study for the Adjunctive Sertraline for the Treatment of HIV-Associated Cryptococcal Meningitis (ASTRO-CM) pilot trial (ClinicalTrials.gov: NCT01802385). We only enrolled patients who presented with suspected meningitis and provided written informed consent for lumbar puncture (LP). If HIV-serological status was unknown, rapid HIV testing was performed. Institutional review board (IRB) approval occurred at all relevant organizations.
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2.2 Routine CSF Examination
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A total of 119 CSF specimens were prospectively collected by lumbar puncture at time of meningitis diagnosis (n=51) or 2–28 days after cryptococcal meningitis diagnosis (n=68) in the case of therapeutic lumbar punctures. The Makerere University Microbiology Laboratory performed CSF testing on the day of collection to determine white cell count and protein level. Cryptococcal meningitis was diagnosed immediately at the bedside using cryptococcal antigen (CRAG) lateral flow assay (IMMY, Norman, Oklahoma). Quantitative CSF fungal culture was performed on fresh CSF in the Makerere Microbiology Laboratory with five 1:10 serial dilutions of 100µL of CSF, as previously described (15). Among persons with cryptococcal meningitis, therapeutic LPs were routinely performed using manometers on day 3, 7, 10, and 14 of amphotericin therapy and additionally as needed for intracranial pressure control. Quantitative CSF fungal cultures were performed with every CSF collection, with the exception of 1 specimen that had a positive culture without quantification. Among CSF specimens negative by CRAG, Gram stain, bacterial culture, and tuberculosis testing were performed. 2.3 FilmArray Multiplex PCR System
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With diagnostic or therapeutic LPs (specific for cryptococcal meningitis participants), a 1mL aliquot of CSF was cryopreserved at −80°C, as volume allowed. Cryopreserved CSF specimens were then shipped in dry ice to BioFire Diagnostics, where 200 mcL of CSF was analyzed using the FilmArray Meningitis/Encephalitis system (Figure 1). The system employs a reagent freeze-dried pouch that stores components necessary for sample preparation, reverse transcription, PCR and detection (16–18). The user injects hydration solution and sample combined with sample buffer into the pouch. Sample extraction, purification, and multiplexed-nested PCR are performed in the enclosed pouch with the FilmArray instrument. Using endpoint melting curve analysis, a result is generated for each of 16 targeted pathogens: 6 bacterial, 8 viral, and Cryptococcus neoformans/gattii. FilmArray operators were blinded to prior microbiology results.
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CSF from study subjects with known tuberculosis, by culture or GeneXpert (Cepheid, Sunnyvale, CA), which is part of the work-up for the main ASTRO-CM trial, were not included in this sub-study due to biosafety concerns in the transport and analysis by FilmArray. An Investigation Use Only (IUO) version of the FilmArray Meningitis/ Encephalitis panel was used to test these samples. Performance characteristics of this FilmArray panel had not been evaluated by the FDA at the time of this study. Confirmatory PCR testing was carried out using Argene® (Biomerieux, Marcy l'Etoile, France) for viral pathogens and genesig® Streptococcus pneumoniae PCR (Primerdesign, Southamptom, United Kingdom). Epstein-Barr virus (EBV) was not validated by additional confirmatory testing due to its ubiquitous nature and unclear significance in this immunocompromised population, as described previously (19). 2.4 Statistical Analysis The diagnostic performance of multiplex PCR for cryptococcal meningitis was compared to a reference standard of positive CSF cryptococcal culture in patients with cryptococcal meningitis. Diagnostic performance was determined only from subjects who had CSF Diagn Microbiol Infect Dis. Author manuscript; available in PMC 2017 March 01.
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available at diagnosis. Given the diagnostic difficulties associated with second episodes of cryptococcal meningitis, individuals presenting to the study with a previous history of treated cryptococcal meningitis were excluded from the analysis of diagnostic performance, with multiplex PCR results instead assessed by the ability to determine the presence (fungal relapse) or absence (paradoxical IRIS with sterile cultures) of cryptococcal growth on CSF cultures. To assess persistence of infection while receiving antifungal therapy, the negative predictive value for detecting ongoing culture positive cryptococcal meningitis was determined among follow up CSF specimens obtained from therapeutic LPs. Finally, the presence of other CNS pathogens was determined by FilmArray multiplex PCR in every CSF specimen collected.
3. Results Author Manuscript
We enrolled a cohort of 69 HIV-infected persons with suspected meningitis at Mulago Hospital in Kampala, Uganda. The cohort consisted of 49 men and 20 women with a median age of 32 (IQR, 25–44) years and median CD4 count of 33 (IQR, 11–69) cells/µL. Among participants, 52% (36/69) presented with a first episode of cryptococcal meningitis (median quantitative culture 8,950 CFU/mL, IQR 118–113,500 CFU/mL), 12% (8/69) had a previous history of cryptococcal meningitis (three of whom had microbiological relapse, range: 90–9,700 CFU/mL) and 36% (25/69) did not have active or prior cryptococcal meningitis.
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A total of 84 CSF specimens were obtained from individuals with first-episode cryptococcal meningitis, of which 18 were obtained at time of diagnosis and 66 were obtained from therapeutic lumbar punctures conducted 2–28 days after diagnosis (Figure 2). Ten CSF specimens were obtained from 8 individuals presenting with second-episode cryptococcosis, with 2 CSF specimens being obtained from therapeutic lumbar punctures. All 25 CSF specimens obtained from individuals without Cryptococcus were obtained at time of diagnosis. In total of the 119 CSF specimens tested on the FilmArray Meningitis/ Encephalitis system, 117 specimens had both quantitative CSF fungal cultures performed and generated a FilmArray result on first testing. One specimen had a positive culture reported for Cryptococcus without quantification (and was positive by FilmArray), and one specimen from a patient without cryptococcal meningitis had a failure of the internal PCR control with a test invalid reported. 3.1 Diagnostic Performance for Cryptococcal Meningitis
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The assay provided 100% sensitivity and specificity compared to cryptococcal culture in diagnostic CSF specimens collected at hospital admission (n=18 positive; n=24 negative). Overall, FilmArray performance was related to the burden of Cryptococcus present in the CSF specimen (Figure 3). In comparing the CSF Cryptococcus CFU/mL versus FilmArray positivity among all sample time points, there was 96% (49/51) sensitivity for detecting ≥100 Cryptococcus CFU/mL CSF (95%CI: 87–99%). Overall, a negative FilmArray result had 95% (39/41) predictive value for a CSF quantitative culture being ≤100 CFU/mL or sterile in CRAG positive CSF (95%: 83%–99%). This included CSF from individuals receiving therapy for firstepisode cryptococcal meningitis (n=66, from 31 individuals) as
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well as those with second episodes of cryptococcal meningitis (n=10, from 8 individuals). In CSF specimens obtained from individuals receiving antifungal therapy (n=76), FilmArray results for Cryptococcus predicted conversion to culture sterility with 71% (20/28) negative predictive value (i.e. when FilmArray results were negative for Cryptococcus, culture was sterile).
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A novel application was in assessing the CSF of persons with a history of cryptococcal meningitis presenting with a second symptomatic episode, where CSF was cryptococcal antigen positive. In 8 individuals with second episodes of symptomatic recurrence of cryptococcal meningitis, FilmArray test results were positive for Cryptococcus in all cases of CSF culture-positive fungal relapse (n=3, with quantitative cultures of 90 CFU/mL, 90 CFU/mL, and 9,700 CFU/mL) and negative for Cryptococcus in all cases having sterile cultures (n=5) with presumed paradoxical immune reconstitution inflammatory syndrome or with persistently elevated intracranial pressure (and a normal CSF white cell count and normal CSF protein). 3.2 Pathogen Detection in CSF
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In this population of persons living with HIV/AIDS with suspected meningitis in Uganda, the FilmArray Meningitis/Encephalitis panel detected six distinct pathogens in CSF. Cryptococcus was very common (n=39/69; including both first and second episodes, where Cryptococcus was detected by both CSF cultures and the FilmArray panel), as expected. Cases of cryptococcal meningitis were frequently associated with viral co-infection with EBV and other viruses. Of those that did not have cryptococcal meningitis, 1% (1/69) had bacterial meningitis, 7% (5/69) had non-EBV viral pathogens identified by FilmArray, and 10% (7/69) had EBV identified only. No pathogen was identified in 25% (17/69) of participants (Figure 4). EBV was frequently detected (n=45) in this immunocompromised population. Other viruses detected include cytomegalovirus (CMV; n=2), varicella zoster virus (VZV; n=2) and human herpes virus 6 (HHV-6; n=1). Streptococcus pneumoniae was detected in one subject by FilmArray PCR; this specimen had gram positive cocci detected on Gram’s stain although CSF bacterial culture was negative. The five specimens containing non-EBV viral pathogens were confirmed by Argene PCR and S. pneumoniae by genesig PCR.
4. Discussion
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In our population of HIV-infected Ugandans with suspected meningitis, we observed excellent detection of first-episodes of cryptococcal meningitis, the most common etiology in our setting, using the BioFire FilmArray Meningitis/Encephalitis panel. In addition, this PCR-based platform demonstrated a novel diagnostic utility among patients with a history of cryptococcal meningitis and recurrent symptoms, specifically the ability to differentiate between culture-positive relapse and paradoxical IRIS with sterile cultures. Furthermore, the assay detected several additional pathogens in CSF, in particular EBV at high rates. Similar to a prior investigation in Uganda, herpes simplex virus (HSV) was not detected as a cause of viral meningitis (19).
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PCR testing appears to be useful in cryptococcal disease. While CRAG testing performs extremely well in detecting initial infection, and the low cost of the CRAG lateral flow assay underscores its central role in the initial diagnosis of cryptococcal meningitis, CRAG can remain positive for months to years (10). Therefore, while the multiplex PCR test cannot replace CRAG as the preferred assay for first episodes of cryptococcosis (8), our findings suggest that PCR may have a role in the differentiation of culture-positive cryptococcal meningitis relapse from paradoxical IRIS or other non-cryptococcal related etiologies in cases of symptom recurrence in patients with a previous history of cryptococcosis. In this scenario, a positive test could avoid inappropriate use of steroids in patients with culturepositive relapse, upon detection of Cryptococcus species by PCR. Given the small number of second episodes of recurrent cryptococcosis in this study, however, further validation studies are required before PCR can be fully endorsed as a reliable and cost-effective test for differentiating relapse from paradoxical IRIS. Furthermore, PCR appears to allow for rapid detection of CSF culture sterility and thus a potential role in better tailoring the duration of amphotericin B therapy and hospitalization (20). The potential of shortening hospital durations could have a substantial cost savings in middle and high income countries. In addition to the benefits of PCR as a stand-alone test in known cryptococcosis, the inclusion of Cryptococcus in a multiplex panel allows detection when cryptococcal disease might not suspected, for example in unknown immune deficiency. In the United States, for example, 3600 cases of cryptococcal meningitis occur annually, with one-third in HIVseronegative individuals (21). Cryptococcal diagnosis in areas of low prevalence is often delayed and/or accidental (22, 23), and inclusion on a multiplex assay could lead to more rapid recognition.
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An operational limitation of our study is that multiplex PCR testing was not performed in on-site (in the case of FilmArray, results would have been available in about an hour), and we are therefore unable to make firm conclusions regarding the impact of introducing a multiplex PCR system on outcomes, such as timing of initiating appropriate therapy, hospital duration, avoidance of unnecessary diagnostic testing or antimicrobials, and costeffectiveness. It seems apparent, however, that the introduction of on-site multiplex PCR testing would result in more rapid and accurate diagnosis with minimization of unnecessary testing, a shortened time to initiation of targeted therapy, and in the case of cryptococcosis at least, potentially shorter hospital durations.
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The detection of EBV and other herpesviruses in this highly immunocompromised population is of unclear significance (19). Of note, EBV was left out of the recently FDAapproved panel which is now commercially available, and the high prevalence of EBV noted in this study should be interpreted with caution, since confirmatory PCR testing was not performed. The increasing use of multiplex PCR testing for simultaneous detection of several herpesviruses should shed more light on the frequency and clinical significance among more diverse populations. Multiplex PCR detected one case of bacterial meningitis which was positive by Gram’s stain and who had received antecedent ceftriaxone. Thus, in partially treated bacterial meningitis, PCR assays have the potential to provide a confirmed diagnosis.
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While our results are encouraging, it must be noted that the FilmArray Meningitis/ Encephalitis system had not yet been approved by the U.S. FDA, and additional studies to validate the role and cost-effectiveness of multiplex PCR in the diagnosis and monitoring of CNS infections are needed across a broad range of settings. The addition of Toxoplasma gondii, Mycobacterium tuberculosis, and John Cunningham (JC) virus PCR testing would be useful for multiplex panels used in immunocompromised populations such as those with advanced HIV, transplant patients, or in the case of JC virus, in persons with multiple sclerosis (24). The multiplex PCR panel tested in this study did not differentiate between C. neoformans and C. gattii. Given the predominance of C. neoformans in our study population, further evaluation of CSF of individuals with C. gattii infection is required. Our study is also limited by the relatively small sample size and the high burden of cryptococcal disease present, consistent with the ongoing reality in sub-Saharan Africa (8). Lastly, as with any new technology, cost may be a barrier for rapid adoption in a resource-limited setting such as Uganda, and further cost-effectiveness analyses are required.
5. Conclusion In summary, the FilmArray multiplex PCR panel offers a promising platform for the rapid diagnosis of meningitis. PCR testing appears to be particularly useful in detection of Cryptococcus species, predicting culture sterility, and differentiating IRIS from culturepositive relapse among persons with recurrent symptoms. Further additions to the panel focusing on immunosuppressed populations and minimizing the real world costs of the technology would make the panel more useful and the overall effectiveness of the assay requires further evaluation.
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Acknowledgements Research support for aspects of this project was received from the National Institutes of Allergy and Infectious Disease, the National Institute of Neurologic Disorders and Stroke and the Fogarty International Center of the National Institutes of Health (R01NS086312, U01AI089244, T32AI055433, R25TW009345).
ASTRO-CM Team members Jane Francis Ndyetukira, Cynthia Ahimbisibwe, Florence Kugonza, Sruti S. Velamakanni, Alice Namudde, Alisat Sadiq, Tadeo Kiiza Kandole, Julian Kaboggoza, Eva Laker, Tony Luggya, Liliane Tugume, Elissa Butler, Jonathan Dyal, Julie Neborak, Lexie King, Nathan Yueh, Kathy Huppler Hullsiek, Ryan Halupnick, Paul R Bohjanen, Andrew Kambugu.
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Highlights We evaluated the utility of multiplex PCR in HIV-associated meningitis in Uganda. Multiplex PCR identified 6 distinct CNS pathogens from 69 patients with meningitis. At diagnosis, PCR detected Cryptococcus with excellent sensitivity and specificity. PCR differentiated between fungal relapse and paradoxical IRIS with sterile cultures. Automated multiplex PCR offers promising platform for rapid diagnosis of meningitis.
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Author Manuscript Author Manuscript Figure 1. The FilmArray multiplex PCR system
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The FilmArray system employs a reagent freeze-dried pouch that stores components necessary for sample preparation, reverse transcription, PCR and detection. The user injects hydration solution and sample combined with sample buffer into the pouch. A nested multiplex PCR is performed in a two-step process and, using endpoint melting curve analysis, a result is generated for each of 16 common pathogens.
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Figure 2. Cohort Composition
CSF specimens in the cohort were collected at diagnosis and in cases of cryptococcal meningitis, as part of follow-up therapeutic LP for control of intracranial pressure.
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Figure 3. FilmArray PCR positivity in comparison to Cryptococcus quantitative CSF culture at all time points of antifungal therapy
Performance characteristics of FilmArray Meningitis/Encephalitis panel compared to CSF cryptococcal quantitative culture by colony forming units (CFU)/mL of CSF. Among initial diagnostic specimens 100% (18/18) were FilmArray positive compared to culture growth, and 100% (24/24) were FilmArray negative when the CSF was sterile. Numbers indicate n=117 specimens with a quantitative culture result per group. One participant had a positive culture without quantification (and positive FilmArray), and one cryptococcal-negative subject had a failure of the internal PCR control with invalid FilmArray test reported and were not included in the analysis.
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Author Manuscript Author Manuscript Figure 4. Distribution of Pathogens Detected in CSF and the Relationship to Other Potential Coinfections
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Cryptococcus was the most common meningitis etiology in this cohort, followed by EBV only (n=7), viral pathogens (n=5), and bacterial meningitis (n=1). No pathogen was identified in 17 persons. Cases of cryptococcal meningitis were frequently associated with viral co-infection with EBV and other viruses (right inset).
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