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Cryptococcal meningitis (CM)-related immune reconstitution inflammatory syndrome (IRIS) complicates antiretroviral therapy (ART) in 20–40% of ART-naïve persons with AIDS and prior CM. Pathogenesis is unknown.
We compared initial CSF cultures, inflammatory markers and cytokine profiles in ART-naïve AIDS patients who did or did not subsequently develop IRIS after starting ART. We also compared results obtained at IRIS events or CM-relapse.
Of 85 subjects with CM, 33 (39%) developed CM-IRIS and 5 (6%) developed culture-positive CM-relapse. At CM diagnosis, subjects subsequently developing IRIS had less inflammation, with decreased CSF leukocytes, protein, interferon-gamma (IFN-g), interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-a) compared with subjects not developing IRIS (P<.05). Initial CSF WBCs ≤25 cells/μL and protein ≤50 mg/dL were associated with development of IRIS (OR=7.2, 95%CI: 2.7 to 18.7, P<.001). Compared to baseline levels, we identified CSF elevations of IFN-g, TNF-a, G-CSF, VEGF, and eotaxin (CCL11) (P<.05) at IRIS but minimal inflammatory changes in those with CM relapse.
Patients who subsequently develop CM-IRIS exhibit less initial CSF inflammation at the time of CM diagnosis compared to those who do not develop IRIS. The inflammatory CSF cytokine profiles observed at time of IRIS can distinguish IRIS from CM-relapse.
Immune Reconstitution Inflammatory Syndrome (IRIS) has arisen as a troubling clinical problem in HIV/AIDS care. IRIS is characterized by a clinical “paradoxical reaction” most often without detectable or viable organisms. IRIS commonly occurs during the first six months of antiretroviral therapy (ART) in HIV-infected persons with CD4 counts <200 cells/μL and opportunistic infections (OIs), and IRIS associated with cryptococcal meningitis (CM) is emerging as a major problem, especially in resource poor regions [1–3]. Overall, CM is the most common fatal OI in AIDS patients world-wide  and is the initial AIDS-defining illness in 10–20% of patients with AIDS in Africa [5–7]. Clinical manifestations of cryptococcal IRIS (CM-IRIS) include fever, meningitis, seizures, lymphadenopathy, cutaneous lesions, or pneumonitis. Definitive diagnostic criteria are not clearly delineated, but evidence-based IRIS clinical case definitions are underway [8–12].
One limitation of providing appropriate medical care for CM-IRIS is the lack of understanding of IRIS pathophysiology. IRIS is proposed to occur from a dysregulated immune recovery and response to foreign antigen, producing symptoms that mimic the relapse of active infections [9, 13]. Current hypotheses regarding IRIS pathogenesis focus on possible imbalances of homeostatic mechanisms between effector and regulatory T-cells during immune recovery [13–15]. Unresolved is the nature of the inflammation occurring at the anatomical site of IRIS, since most studies have involved evaluation of inflammation in peripheral blood. Thus, we directly measured markers of inflammation in the target organ system, the cerebrospinal fluid (CSF), in persons with and without CM-IRIS.
A healthy immune response to cryptococcus (Figure 1) depends on coordinated interactions between antigen-presenting cells and effector T-cells thereby generating a type-1 helper T-cell (Th1) immune response. T-cells are involved directly in cryptococcal killing, and Th1 cytokines, particularly interferon-gamma (IFN-γ), enhance antibody and complement-dependent phagocytosis and killing by macrophages [16–26]. IFN-γ secreted in response to mannoprotein is thought to generate protective immunity to cryptococcus [27–29].
We hypothesized that an ineffective immune response at the time of initial CM may predispose to IRIS through ineffectual antigen clearance. Therefore, we prospectively characterized local inflammation in the CSF in ART-naïve patients during their initial CM as well as at time of CM-IRIS or CM relapse in order to better understand the immune pathways associated with IRIS so we can develop rational strategies to predict, prevent, and treat CM-IRIS.
A prospective cohort of 199 HIV-infected, ART-naïve Ugandans with a first episode of CM was recruited at Mulago Hospital, Kampala over a 26 month cumulative period from July 2006 to July 2009. Of these, 170 had prospective clinical data collected and 130 persons had CSF stored. CM treatment and outcomes were previously described in subjects recruited in 2006 . All subjects received CM induction therapy with amphotericin 0.7–1.0 mg/kg/day for 14 days followed by fluconazole at 400mg, per DHHS-IDSA guidelines . In 2009, fluconazole 800mg was prescribed until starting ART to achieve dose-dependent fungicidal activity [29, 32–34]. Eighty-five subjects survived to start ART, of whom 72 had CSF available for cytokine analysis. After ART initiation, all subjects were prospectively followed biweekly for the first 3 months and monthly thereafter for development of IRIS through 1 year. When IRIS was suspected, subjects underwent lumbar puncture (LP) to exclude culture-positive cryptococcal relapse or other etiologies. Steroids (prednisolone 60 mg/day for 28 days) were initiated if CSF results revealed an inflammatory profile consistent with IRIS. CM-IRIS was defined per the International Network for the Study of HIV-associated IRIS definition  www.inshi.umn.edu/definitions, with three complex cases decided by expert physician opinion. Written informed consent and IRB approval were obtained from the University of Minnesota and the Uganda National Council of Science and Technology.
CSF clinical laboratory parameters, including protein, cell count, glucose, were measured on site. All subjects had a negative bacterial CSF culture. All 199 persons had detectable CSF cryptococcal antigen (CRAG) measured at a College of American Pathologist (CAP)-accredited laboratory. Quantitative CSF cultures were performed using a calibrated loop with 10μL of CSF cultured on Sabouraud agar. Visible colonies were counted by two technicians with the average colony count recorded. CSF supernatant was frozen at −80°C, then shipped on dry ice (−20°C) to Minnesota. Multiplex cytokine profiling of 27 cytokines/chemokines were measured on 72 specimens using the Luminex platform (Human 27-Plex Panel, Bio-Rad, Hercules, CA).
To identify baseline predictors for developing future IRIS, we compared the clinical CSF parameters and cytokine profiles between groups using the non-parametric Mann Whitney U test (SPSS 17.0.1). Differentially expressed cytokines by univariate analysis with P<.2 were included in a multivariate logistic regression model. For the regression, cytokines were log2 transformed for normalization. Odds Ratio (OR) and 95% confidence interval (95%CI) presents risk. For a 25 person sample size with IRIS, we estimated >90% power to detect an effect size ≥1SD difference in means between groups.
Of 170 CM patients with baseline CSF results, 85 (50%) survived to initiate ART at a median of 5 weeks after CM diagnosis (Figure 2). Thereafter, 39% (n=33) developed paradoxical CM-IRIS with CNS manifestations at a median of 8 weeks on ART (IQR: 4 to 17 weeks). Another 10% (n=9) developed probable cryptococcal-related IRIS with non-CNS manifestations, such as lymphadenitis or pneumonitis, which newly developed on ART. Among patients who did or did not develop IRIS, clinical characteristics were similar. The median age was 36 years, and 58% were men. Clinical history included 33% with a history of TB and 16% receiving current TB treatment. Residual neurologic deficits were common, with 16% having visual impairment and 5% having gross hearing loss after hospital discharge.
Baseline demographic and clinical laboratory parameters were similar among those who did or did not develop CM-IRIS (Table 1). We identified no significant differences between groups in baseline CD4+ T-cell counts (P=.30), cryptococcal quantitative cultures (P=.8), or CRAG titers (P=.074). At the time of starting ART, the mean CD4 count was 30 cells/μL. The cohort’s median quantitative culture was 1.33×104 colony forming units (CFU)/mL of CSF (IQR: 1.7×103 to 3.65×104 CFU/mL) with a median CRAG titer of 1:1024 (IQR: 256 to 1024).
The clinical IRIS events presented with signs and symptoms of headache (85%), photophobia (32%), vomiting (28%), meningismus (25%), papilledema (25%) and seizures (14%). Lumbar puncture revealed: elevated intracranial pressure (median 305 mm, IQR: 178 to 450 mm CSF), elevated CSF white blood cells (WBCs) (median=31, IQR: 5 to 85 cells/μL), protein (median=80, IQR: 58 to 123 mg/dL), and sterile cultures, except for one subject who had a CSF quantitative culture of 70 CFU/mL at 5 weeks of ART (7 weeks of anti-fungal therapy). In this subject, CSF WBC counts increased from 5 to 75 cells/μL, and protein increased from 20 to 40 mg/dL. Overall, the median decline in CSF CRAG from baseline to IRIS was 8-fold (3 log2, IQR: 2 to 9 log2), but 25% of persons with IRIS had negative cultures accompanied by a <4-fold decrease in quantitative CRAG titer. Four subjects with CM-IRIS presented with seizures, which on CT and/or post-mortem exam were associated with cryptococcoma(s). The CD4 counts at IRIS events (median 80.5, IQR: 47 to 167 cells/μL) were similar to cohort control CD4 counts at 8–12 weeks (median 77, IQR: 41 to 121 cells/μL; P=.6).
72 CSF samples were evaluated (13 had insufficient volume collected for analysis), including 27 from subjects with CM-IRIS. Persons who subsequently developed CM-IRIS after starting ART had less inflammation present in their CSF at time of their initial CM. CSF WBC counts and protein levels were lower in persons who developed IRIS than those not developing IRIS after starting ART (P=.012; P=.004 respectively). CSF levels of interleukin (IL)-6, IL-8, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and eotaxin (CCL11) were also significantly lower in subjects who subsequently developed IRIS (Figure 3). The initial CSF CRAG titer, quantitative culture, and CSF glucose were comparable (P>.2).
For the initial, baseline pre-ART CSF specimen, each two-fold increase in IL-8 was associated with increasing protection from IRIS (OR=0.32; 95%CI: 0.17 to 0.62, P=.001) after initiating ART. Conversely, increasing levels of IL-1β were associated with increased risk of IRIS (OR=2.1; 95%CI: 1.2 to 3.6; P=.012) per 2-fold change, as were possibly CCL2 (monocyte chemotactic protein-1 (MCP-1)) (OR=1.56; 95%CI: 0.99 to 2.4; P=.053).
The combination of CSF WBC ≤25 cells/μL and CSF protein ≤50 mg/dL was more highly associated with increased risk of IRIS (OR=7.2; 95% CI: 2.7 to 18.7, P<.001) with a diagnostic sensitivity of 69% (27/39), specificity of 76% (35/46), positive likelihood ratio of 2.90, and negative likelihood ratio of 0.40. The overall proportion correctly classified by CSF WBC and protein was 73% (62/85). Thus, initial CSF parameters, including low levels of pro-inflammatory cytokines, low protein, and low cellular response were associated with the subsequent development of IRIS.
At time of CM-IRIS, CSF WBC, CSF protein, and CSF levels of multiple pro-inflammatory cytokines were increased compared with baseline CSF levels from the same subjects at the time of CM diagnosis (Table 1). We observed 3-fold elevations in median IFN-γ, TNF-α, G-CSF levels as well as 2-fold elevations in vascular-endothelial growth factor (VEGF) and eotaxin (CCL11) levels (P<.05) at the time of IRIS compared to the time of initial CM. In contrast, CSF levels of CCL2 (MCP-1) were decreased at the time of IRIS. Changes in IL-6 (P=.065), IL-8 (P=.39), or IL-17 (P=.099) levels at time of IRIS were not statistically significant.
In contrast to culture-negative CM-IRIS, CSF parameters in five persons with culture positive CM-relapse showed limited signs of inflammation. Compared with paradoxical CM-IRIS, CSF from patients with CM culture-positive relapse had lower levels of the pro-inflammatory cytokines: IFN-γ (P=.004), TNF-α (P=.022), IL-17(P=.018), and IL-12 (P=.006) as well as the cytokines IL-9 (P<.001) and IL-4 (P=.005). CSF WBC counts at CM relapse were similar to initial CM. The WBC counts in CM relapse were lower (mean 12, Range 5–30 cells/μL) than in CM-IRIS (mean 68 ±66 cells/μL) (P=.05).
Definite CM-relapse was diagnosed in four persons with positive CSF cultures after 12–25 weeks of ART. No clinical features differentiated relapse vs. IRIS at event presentation. In three subjects, CSF quantitative cultures grew 30,000 to 38,000 C. neoformans CFU/mL, with two-fold rises in CSF CRAG titer to 1:2048 and two of these subjects had virologic failure. A fourth subject had three separate episodes of recurrent meningitis at 12, 24, and 28 weeks of ART. The first episode was culture negative (i.e. CM-IRIS) and managed with therapeutic LPs to control pressure. The second episode occurred at 12 weeks, and they received corticosteroids for presumed refractory CM-IRIS; however the CSF quantitative culture was 900 CFU/mL (i.e. CM-relapse). This patient subsequently developed multiple intra-parenchymal, cystic brain cryptococcomas causing obstructive hydrocephalus, but CSF culture was negative at 28 weeks. The patient died, and post-mortem exam revealed intra-parenchymal cysts filled with intact, but dead cryptococcus. A fifth subject presented at 2 weeks of ART with a positive CSF culture which did not differentiate relapse from IRIS. This subject received two weeks of amphotericin with a complete clinical response followed by 4 weeks of fluconazole 400mg before presenting with recurrent meningitis. The CSF quantitative culture decreased from 25,000 to 5,000 CFU/mL. Based on the initial complete clinical response and known early fungicidal activity of amphotericin B [33, 36], the clinical picture favored CM-relapse over treatment failure or IRIS. Whether or not we included this fifth case as a relapse did not alter the overall statistical significance (P<.05) of the above CSF findings in CM-relapse.
A 6th case failed to improve with antifungal therapy and was classified a CM treatment failure. This subject received corticosteroids prior to their CM diagnosis. When starting ART, this subject had persistent headache, blindness, and subsequently developed fever, worsening headache, and new seizures. At 10 days of ART, the CSF opening pressure was 310 mm H2O, and culture grew 15,100 CFU/mL. At 14 days, a 6th cranial nerve palsy developed, but they refused further lumbar punctures and died at 4 weeks of ART.
In this large, prospective cohort of HIV-infected persons with AIDS and CM in Sub-Saharan Africa, a paucity of CSF inflammation at the time of initial CM diagnosis was associated with subsequent development of IRIS. This finding that persons who later develop IRIS had less initial CSF inflammation compared to those without IRIS suggests that persons at risk for IRIS had ineffectual protective immune responses, despite similar CD4+ T-cell counts and cryptococcal burdens.
Specifically, compared with non-IRIS patients, persons with future IRIS had lower CSF levels of the Th1-associated cytokine IFN-γ and of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α at the time of initial CM diagnosis. These attenuated cytokine responses were consistent with decreased local inflammation in the CSF and were associated with decreased levels of CSF protein and WBCs. Normally, an effective immune response to cryptococcus requires a Th1 T-cell response for cryptococcal clearance directly by T-cell cytotoxicity or cytokine-enhanced antibody-dependent killing by macrophages [16, 29, 37, 38]. Thus, persons with diminished Th1 responses would be expected to exhibit greater cryptococcal loads for longer durations. Because cryptococcal antigen persists for months [39, 40], the initially ineffectual inflammatory response in CSF from patients at risk for IRIS appears to transform in the setting of immune restoration into an exaggerated inflammatory response directed at the persisting antigen burden.
These data advance our understanding of CM-IRIS pathophysiology by characterizing the inflammation in the CSF, at the site of the exaggerated response. A robust Th1 T-cell response was evident in the CSF at the time of IRIS, with a 3-fold increase in IFN-γ compared to initial CM. Also present at elevated levels at the time of IRIS were pro-inflammatory cytokines, such as IL-6 and TNF-α, which are typically produced by macrophages and antigen presenting cells, as well at T-cells. In contrast, IL-17 levels at the time of IRIS were similar to initial levels (P=.099), suggesting that CM-IRIS pathogenesis is not driven by a Th17 response. Also, levels of Th2 cytokines such as IL-4, IL-5, and IL-10 were negligible at the time of IRIS. Overall, our results suggest that pro-inflammatory cytokine responses, including Th1 cytokines, are involved in IRIS pathogenesis.
Chemokines present in CSF at the time of IRIS included CCL2, CCL11, and VEGF. CCL2 (MCP-1), a chemotactic factor for dendritic cells, monocytes, and T-cells, did not appear to contribute to IRIS pathogenesis. Levels of CCL2 (MCP-1) decreased at the time of IRIS compared to initial levels at the time of CM diagnosis and were comparable to HIV-infected persons without IRIS receiving ART . In contrast, levels of TNF-α were elevated at the time of IRIS. TNF-α induces VEGF-A which increases vascular permeability, stimulates chemotaxis of macrophages and CD4+CD45RO+ memory T-cells , and VEGF has co-stimulatory activity for IFN-γ-secreting Th1 memory T-cells . Lastly, granulocyte-colony stimulating factor (G-CSF) was elevated in CSF at the time of CM-IRIS. In cryptococcosis, G-CSF levels may correlate with CSF clearance of cryptococcus . Whereas G-CSF is normally considered a hematopoietic growth factor, G-CSF is also produced by macrophages and increases the innate antifungal activity of neutrophils and macrophages [43, 44]. Overall, our results revealed increasing CSF inflammation (cytokines, WBCs, and protein) coincident with the development of IRIS. These results differ from those of a pioneering but smaller study from Cape Town in which levels of CSF cytokines did not differ between subjects with CM and CM-IRIS .
Whether CSF cytokine profiles can be used to clinically risk-stratify HIV-infected patients starting ART remains an open question. Our results suggest that widely available CSF parameters such as protein and WBC are generally informative of IRIS risk, yet we were unable to identify a particular cut point as an ideal diagnostic threshold. However, those with more prominent CSF inflammation (WBC >25 cells/μL, protein >50 mg/dL) at time of initial CM diagnosis only infrequently (25.5%) developed IRIS on ART whereas 71% of those below both thresholds developed IRIS. Thus, high risk persons may be identifiable before starting ART. Potential theoretical strategies to reduce IRIS risk could include more aggressive management of increased intracranial pressure, more potent antifungal therapy, altering the timing of ART, or prophylactic use of anti-inflammatory medications in patients at higher risk of IRIS. However, the association between decreased inflammation and the development of IRIS may create problems in preventative anti-inflammatory therapy. For instance, although TNF-α levels were increased at time of IRIS, low levels of TNF-α at initial CM were associated with increased IRIS risk. Thus, prophylaxis with accessible anti-TNF agents, such as chloroquine or thalidomide, could slow cryptococcal clearance and inadvertently increase the risk of IRIS and death.
We also identified a distinct difference in CSF cytokine profiles between cases of CM-relapse and CM-IRIS at the time of these events. CM-relapse was associated with persistent viable organisms and a lack of inflammation in CSF whereas CM-IRIS showed no organisms but a more robust inflammatory profile. CM-relapse was characterized by the lack of pro-inflammatory cytokines in the CSF similar to the cytokine profile observed with initial CM. However, relapse and IRIS are not always distinct or completely separate. Two of the five cases of CM-relapse later developed culture-negative paradoxical-IRIS. IRIS is an immunologic event that can occur in the presence of live or dead organisms. While the presence of live organisms, as seen in relapse, influences clinical management, in contrast, whether the cryptococcal antigens driving the inflammatory response in IRIS derive from live organisms, dead intact organisms or cellular debris, may likely be inconsequential from an immunologic perspective. Thus, some persons could have IRIS with low-level positive cultures.
The diagnostic value of positive CSF cultures within the first few weeks of ART and the development of IRIS remains unsettled. Within the expected treatment course, 50% of patients treated with amphotericin B in Uganda show positive CSF cultures at 2 weeks , and a minority of CSF cultures can remain positive up through 8–10 weeks . Clinical considerations in suspected IRIS events early in ART would suggest anti-fungal regimens should be intensified while awaiting CSF culture results to exclude relapse, especially if anti-inflammatory therapies are given. The degree of inflammation in the CSF, evident by increasing CSF WBCs coupled with decreasing CRAG titers at the time of recurrent symptoms, is a useful initial diagnostic tool to differentiate IRIS from relapse. However, culture is essential. Cryptococcal cultures were more informative than CRAG titers alone for distinguishing IRIS from relapse, as 25% of persons with IRIS had negative CSF cultures but a <4-fold decrease in CRAG titer.
Overall, our results indicate that a paucity of initial inflammation at the time of CM diagnosis was associated with increased risk for subsequent development of exaggerated inflammatory responses associated with CM IRIS. Future studies are needed to determine whether our findings can be validated in other populations and to determine predictors of the response to medical treatment of CM-IRIS. In characterizing the pathophysiology of CM-IRIS at the site of inflammation, this study is a first step toward selecting rational therapeutic strategies for CM-IRIS.
We thank Dr. Abdu Musubire, Ms. Jane Ndyetukira and Ms. Irene Namugga for patient care. We wish to thank Dr. Henry Kajumbula for laboratory support as well as Dr. Chandy John for assistance with cytokine profiling. We thank Jonathan C. Jeschke for the graphic design of figure 1.
Financial Support: This work was supported by the NIH National Institute of Allergy and Infectious Diseases (R03AI078750-01; K12RR023247-04; K23AI073192-01A2; T32AI055433-05; L30AI066779), Minnesota Medical Foundation through the Robert and Mabel Bohjanen Immune Reconstitution Research Fund, Tibotec REACH Initiative, University of Minnesota Translational Research Grant, University of Minnesota Dean’s Grant in Aid and the Office of International Programs and the Veterans Affairs Research Service. The funding sources had no role in the design, conduct of the study, or decision to seek publication.
Conflicts of Interest: No conflicts of interest exist.
Financial Disclosures: None
This was presented as an abstract at the 2009 Infectious Diseases Society of America annual meeting.
Author Contributions: Dr. Boulware has full access to all the data in the study and takes responsibility for the integrity and accuracy of the data and data analysis.Study Concept and Design: Bohjanen, Boulware, Janoff
Acquisition of Clinical Data: Meya, Kambugu
Acquisition of Laboratory Data: Bonham, Park, Wiesner
Statistical Analysis: Bonham, Boulware
Interpretation of data: Boulware, Bohjanen, Wiesner, Janoff
Drafting the manuscript: Bonham, Boulware, Bohjanen, Janoff
Critical revisions for intellectual content: Bohjanen, Boulware, Wiesner, Janoff
Obtaining funding: Bohjanen, Boulware
Administrative, technical, or material support: Bohjanen, Kambugu, Park