Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda

Abstract

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: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.

Keywords: Cryptococcal meningitis; Diagnostics; HIV; Immunocompromised; Meningitis; PCR.

Figure 1. The FilmArray multiplex PCR system

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.

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.

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.

Figure 4. Distribution of Pathogens Detected in CSF and the Relationship to Other Potential Co-infections

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).