Clinical metagenomics-assisted diagnosis of relapsed HIV-associated cryptococcal meningitis

Introduction

Cryptococcal meningitis is a common opportunistic infection among individuals living with AIDS associated with high mortality rates. It is estimated that there are 112,000 cryptococcal-related deaths annually among 4.3 million people living with HIV, accounting for 19% of all AIDS-related mortality. ¹ A proportion of the disease burden is due to relapses of previously treated infections and/or paradoxical immune reconstitution inflammatory syndrome (IRIS), which occurs among 8–49% of patients living with HIV with cryptococcal disease initiating antiretroviral therapy (ART).^{2, 3} To date, identification of cryptococcal relapse remains clinically challenging.

Clinical metagenomic next-generation sequencing (mNGS) is a rapidly growing approach to infectious disease diagnosis that involves sequencing the nucleic acid of clinical samples to identify microorganisms. Previous studies have demonstrated the potential utility of mNGS for detecting Cryptococcus in the central nervous system, the pulmonary system, and the liver.^4-6 Yet, few studies have focused on using the method in differentiating between an IRIS and a relapse of cryptococcal meningitis. This study reports the use of mNGS to aid in diagnosing recurrent cryptococcal meningitis in a person living with HIV experiencing recurring symptoms, despite negative culture results for Cryptococcus neoformans in the cerebrospinal fluid.

Case

A 30-year-old man living with HIV, with an initial CD4 count of 35 cells/mm³, presented to a hospital with intermittent fever, headache, and neck stiffness (Figure 1(a)). He was diagnosed with cryptococcal meningitis based on symptoms, detection of cryptococcal antigen (CrAg) with a titer of 1:512 in CSF, and a positive CSF culture of C. neoformans. We used third-generation sequencing (Pacific Biosciences) to sequence and assemble the isolate into a complete genome consisting of 14 chromosomes and one mitochondrion with a 3; 11 chromosomal translocation. We performed repeated lumbar punctures and external ventricular drain for intracranial pressure control and initiated induction and consolidation therapies with amphotericin B and flucytosine. When CSF culture was sterile, the patient received antiretroviral therapy with bictegravir, emtricitabine, and tenofovir alafenamide. He had resolution of symptoms and was discharged on day 42 with oral fluconazole for maintenance.OPEN IN VIEWER

The patient returned to the hospital with a recurrence of fever and headache on day 308. Brain MRI revealed progressive leptomeningeal enhancement. CSF from lumbar puncture showed positive CrAg with a titer of 1:4. He received re-induction therapy with amphotericin B and flucytosine. Brain biopsy performed on day 313 showed numerous round spore-like organisms with budding yeast (Figure 1(b)) despite no growth of pathogenic fungi in CSF. Although fungal culture was negative, when reads from metagenomic and metatranscriptomic sequencing performed on the CSF sample were mapped onto the TW1 genome and NCBI RefSeq, 589 TW1-specific reads were identified. NCBI BLAST search also revealed Cryptococcus-specific 18S/25S/28S ribosomal RNA and DNA (supplementary figure S1), indicating a relapse of the disease.

Discussion

Relapse of cryptococcal meningitis is generally defined as having recurrence of symptoms with recovery of viable organisms from previously sterile cerebrospinal fluid (CSF), whereas paradoxical cryptococcal IRIS describes patients with recognized pre-ART cryptococcal disease who subsequently deteriorate after initiating ART.^{7, 8} Differentiation between a relapse and CM-IRIS is difficult due to their similarities in clinical presentations, which can lead to a dilemma when making diagnosis and treatment decisions.

Current guidelines advice consideration of paradoxical cryptococcal IRIS after excluding other causes of recurrent symptoms among people who have started ART and use of short course oral steroid therapy if there is continued deterioration or the development of life-threating complications despite ART, multiple repeat lumbar punctures, and induction therapy. ² Therefore, in cases similar to the one reporting here, an IRIS might be considered. However, studies indicated management of IRIS with corticosteroids may increase the risk of relapse and further increase the risk of recurrent IRIS and resulting complications including death.^{9, 10} Thus, uncertainties in diagnosis may negatively impact the outcome.

Although diagnostics for cryptococcal meningitis have evolved from culturing to India ink and lateral flow assay over the past decade and have proven to be highly reliable with high sensitivities and specificities, culturing can take up to weeks and antigen tests can occasionally show false-negative results.^{11, 12} In this study, the application of mNGS allows for timely microorganism identification when isolates from positive cultures are not available.

One of limitations of mNGS is that microorganisms detected may be dead or dormant, and clinical importance should be assessed by combining with other clinical data. ¹³ In this case, the pathological finding of live-replicating budding yeasts was consistent with cryptococcosis and hence proved the diagnosis. However, from the point of view of molecular analysis, the existing reads of rRNA and DNA detected by mNGS considered as proof of relapse could be debated. Microbial DNA detection cannot distinguish between the living and dead portion of the microbiome due to persistence for longer periods of time compared to RNA. However, the degradation half-life of the rRNA was reported about 4 days under 20°C. ¹⁴ As we know, the 16S rRNA polymerase chain reaction (PCR) has been used in several recent studies to detect acute bacterial meningitis. ¹⁵ A similar approach in fungal infection warrants further investigation. Ribosomal RNA (rRNA) and transfer RNA (tRNA) comprise up to 98% of total cellular RNA in bacteria and several conditions related to degradation, such as starvation and slow- or no-growth conditions. ¹⁶ In our opinion, although no definite RNA load limit has been established, the detection of pathogen rRNA in human body fluids could play a role in differentiating infection processes and provide new culture-independent approaches for clinical practitioners to diagnose diseases.

In addition, when metagenomic sequencing results from culture-negative CSF were compared with the whole genome of C. neoformans isolated from the first cryptococcal meningitis event prior to ART initiation, an identical strain of C. neoformans was identified. To our knowledge, this is the first report of using a combination of whole genome sequencing and mNGS longitudinally to differentiate relapsed crypotococcal meningitis and IRIS clinically, demonstrating a timely decision to initiate re-induction therapy.

Conclusion

This report presents the case of person living with HIV with a relapse of cryptococcal meningitis and highlights the potential of metagenomics in aiding timely and precise diagnosis. While the current gold standard for diagnosis of relapse is a positive CSF culture after a previously sterile CSF, metagenomics has shown evidence of microorganisms in culture-negative samples, offering clinical practitioners insights previously unattainable. However, it is important to acknowledge the limitations of metagenomics in establishing causation and fulfilling Koch’s postulates. In this case, the detection of rRNA, which has a short degradation half-life, provided additional evidence for the presence of live, replicating yeasts. The integration of metagenomic findings with other clinical data, such as pathological examinations, can offer a more comprehensive understanding of the disease state.

Supplemental Material