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Paucity of initial cerebrospinal fluid inflammation in cryptococcal meningitis is associated with subsequent immune reconstitution inflammatory syndrome

  1. David R. Boulware1,2,
  2. Shulamith C. Bonham1,2,
  3. David B. Meya1,4,
  4. Darin L. Wiesner2,3,
  5. Gregory S. Park2,
  6. Andrew Kambugu1,4,
  7. Edward N. Janoff5 and
  8. Paul R. Bohjanen1,2,3
  1. 1Division of Infectious Diseases & International Medicine, Department of Medicine
  2. 2Center for Infectious Diseases and Microbiology Translational Research
  3. 3Department of Microbiology, University of MinnesotaMinneapolis, Minnesota
  4. 4Infectious Disease Institute, Makerere UniversityKampala, Uganda
  5. 5Mucosal and Vaccine Research Program Colorado, Division of Infectious Diseases, University of Colorado Denver School of Medicine, Denver Veterans Affairs Medical CenterDenver, Colorado
  1. Reprints or correspondence: David Boulware, MD, MPH, DTM&H, MMC 250, 420 Delaware St SE, Minneapolis, MN 55455 (boulw001{at}umn.edu).
 
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Abstract

Background. Cryptococcal meningitis (CM)-related immune reconstitution inflammatory syndrome (IRIS) complicates antiretroviral therapy (ART) in 20%–40% of ART-naive persons with AIDS and prior CM. Pathogenesis is unknown.

Methods. We compared initial cerebrospinal fluid (CSF) cultures, inflammatory markers, and cytokine profiles in ART-naive patients with AIDS who did or did not subsequently develop IRIS after starting ART. We also compared results obtained at IRIS events or CM relapse.

Results. Of 85 subjects with CM, 33 (39%) developed CM-related 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-γ, interleukin-6, interleukin-8, and tumor necrosis factor-α, compared with subjects not developing IRIS (P<.05, for each). Initial CSF white blood cell counts ⩽25 cells/µL and protein levels ⩽50 mg/dL were associated with development of IRIS (odds ratio, 7.2 [95% confidence interval, 2.7–18.7]; P < .001). Compared with baseline levels, we identified CSF elevations of interferon-γ, tumor necrosis factor-α, granulocyte colony-stimulating factor, vascular-endothelial growth factor, and eotaxin (CCL11) (P < .05, for each) at the time of IRIS but minimal inflammatory changes in those with CM relapse.

Conclusion. Patients who subsequently develop CM-related IRIS exhibit less initial CSF inflammation at the time of CM diagnosis, compared with 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 the care of patients with human immunodeficiency virus (HIV)/AIDS. IRIS is characterized by a clinical “paradoxical reaction” most often without detectable or viable organisms. IRIS commonly occurs during the first 6 months of antiretroviral therapy (ART) in HIV-infected persons with CD4 counts <200 cells/µL and opportunistic infections, and IRIS associated with cryptococcal meningitis (CM) is emerging as a major problem, especially in resource-limited regions [1–3]. Overall, CM is the most common fatal opportunistic infection in patients with AIDS worldwide [4] and is the initial AIDS-defining illness in 10%-20% of patients with AIDS in Africa [5–7]. Clinical manifestations of CM-related 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 being developed [8–12].

One limitation of providing appropriate medical care for CM-related IRIS is the lack of understanding of IRIS pathophysiology. IRIS is proposed to occur because of 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, because most studies have involved evaluation of inflammation in peripheral blood. Thus, we directly measured markers of inflammation in the target organ system, ie, the cerebrospinal fluid (CSF), in persons with and without CM-related 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-γ (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].

Figure 1.
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Figure 1.

Immunology of Cryptococcus neoformans. The human immune response to C. neoformans depends on coordinated interaction of antigen presenting cells with a type-1 CD4 T-helper (Th1) cell response. A, The immune response begins with innate defense in the submucosa through alternative complement opsonization of C. neoformans, which allows for efficient phagocytosis by neutrophils, macrophages, and dendritic cells [17–20]. B, Dendritic cells localize the pathogen to the endolysosome through phagocytosis where the organism is degraded and processed and antigenic peptides are presented on surface major histocompatibility complex class-II (MHC-II) to T cells in lymphatic tissue [20, 21]. C and D, When T cells have a matching T cell receptor for the peptide, appropriate cytokine signals from dendritic cells allow naive Th0 T cells to differentiate into effector T cells [22]. E, These effector T cells are important in the classical activation of macrophages via interferon-γ (IFN-γ) for fungal clearance. Macrophages require IFN-γ stimulation to up-regulate antimicrobial peptides, reactive oxygen species (ROS), and proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), thereby sufficiently killing the phagocytosed, facultative intracellular fungal cells [22]. In the absence of an effective Th1 T cell response, humoral immunity via immunoglobulin (Ig) G1, IgG2a, and IgG2b directed toward GXM is ineffective alone [23, 24], and the adaptive arm of the immune system remains paramount to cryptococcal defense. Observation of delayed-type hypersensitivity responses, requirements for cell-mediated immunity, and IFN-γ dependence all support that a Th1 response is essential to cryptococcal immunity [22, 25, 26]. In human clinical studies, the IFN-γ (Th1 cytokine) present in the CSF is associated with the rate of fungal culture clearance [29, 37, 38].

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-naive patients during their initial CM as well as at the time of CM-related IRIS or CM relapse to better understand the immune pathways associated with IRIS, so we can develop rational strategies to predict, prevent, and treat CM-related IRIS.

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Methods

Study participants. A prospective cohort of 199 HIV-infected, ART-naive Ugandans with a first episode of CM was recruited at Mulago Hospital, Kampala, Uganda, over a 26- month cumulative period from July 2006 through July 2009. Of these, 170 had prospective clinical data collected and 130 persons had CSF samples stored. CM treatment and outcomes were previously described in subjects recruited in 2006 [30]. All subjects received CM induction therapy with amphotericin (0.7–1.0 mg/kg/day for 14 days), followed by fluconazole at 400 mg, according to Department of Health and Human Services- Infectious Diseases Society of America guidelines [31]. In 2009, fluconazole (800 mg) 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 samples 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 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-related IRIS was defined according to the International Network for the Study of HIV-associated IRIS definition [35] (http://www.inshi.umn.edu/definitions; Table 1), with 3 complex cases decided by expert physician opinion. Written informed consent and Institutional Review Board approval were obtained from the University of Minnesota and the Uganda National Council of Science and Technology.

Table 1.
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Table 1.

International Network for the Study of HIV-Associated IRIS Consensus Case Definition for Paradoxical Cryptococcal Immune Reconstitution Inflammatory Syndrome

Laboratory. CSF clinical laboratory parameters, including protein level, cell count, and glucose level, 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 Pathologists-accredited laboratory. Quantitative CSF cultures were performed using a calibrated loop with 10 µL of CSF cultured on Sabouraud agar. Visible colonies were counted by 2 technicians, with the mean colony count recorded. CSF supernatant was frozen at −80°C, and then shipped on dry ice (−20°C) to Minnesota. Multiplex cytokine profiling of 27 cytokines/chemokines were measured on 72 specimens with use of the Luminex platform (Human 27-Plex Panel; Bio-Rad).

Statistical analysis. To identify baseline predictors for developing future IRIS, we compared the clinical CSF parameters and cytokine profiles between groups with use of the nonparametric Mann-Whitney U test (SPSS, version 17.0.1; SPSS). Differentially expressed cytokines by univariate analysis with P values <.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 (CI) indicates risk. For a 25-person sample size with IRIS, we estimated >90% power to detect an effect size ⩾1 standard deviation difference in means between groups.

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Results

Cohort demographic characteristics. Of 170 patients with CM and baseline CSF results, 85 (50%) survived to initiate ART at a median of 5 weeks after CM diagnosis (Figure 2). Thereafter, 33 (39%) developed paradoxical CM-related IRIS with CNS manifestations at a median of 8 weeks after ART initiation (interquartile range [IQR], 4–17 weeks). Another 9 (10%) developed probable cryptococcal-related IRIS with non-CNS manifestations, such as lymphadenitis or pneumonitis, which developed newly while receiving 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 tuberculosis, and 16% were currently receiving tuberculosis treatment. Residual neurologic deficits were common, with 16% having visual impairment and 5% having gross hearing loss after hospital discharge.

Figure 2.
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Figure 2.

Cryptococcal meningitis cohort profile, 2006–2009. CNS, central nervous system; HIV, human immunodeficiency virus. aMissing clinical data because of patient leaving against medical advice or missing cerebrospinal fluid (CSF) sample because of processing at external laboratories with incomplete diagnostic analysis and without CSF storage. bThirteen specimens had inadequate volume for CSF storage and did not have cytokine profiling performed. cOne person had both relapse and immune reconstitution inflammatory syndrome (IRIS). dTwenty-seven cryptococcal meningitis- related IRIS CSF specimens were stored.

Baseline demographic and clinical laboratory parameters were similar among those who did or did not develop CM-related IRIS (Table 2). 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 ART initiation, the mean CD4 count was 30 cells/µL. The cohort's median quantitative culture result was 1.33×104 colony-forming units (CFU)/mL of CSF (IQR, 1.7×103 to 3.65×104CFU/mL) with a median CRAG titer of 1:1024 (IQR, 1:256 to 1:1024).

Table 2.
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Table 2.

Cerebrospinal Fluid (CSF) Profiles in Persons with Cryptococcal Meningitis (CM) at Initial Diagnosis and at the Time of Diagnosis of Immune Reconstitution Inflammatory Syndrome (IRIS)

The clinical IRIS events presented with signs and symptoms of headache (85%), photophobia (32%), vomiting (28%), meningismus (25%), papilledema (25%), and seizures (12%). Lumbar puncture revealed elevated intracranial pressure (median, 305 mm CSF [IQR, 178–450 mm CSF]), elevated CSF white blood cell (WBC) count (median, 31 cells/µL [IQR, 5–85 cells/µL]), elevated protein level (median, 80 mg/dL [IQR, 58–123 mg/dL]), and sterile cultures, except for 1 subject who had a CSF quantitative culture of 70 CFU/mL at 5 weeks after ART initiation (7 weeks of antifungal therapy). In this subject, CSF WBC counts increased from 5 to 75 cells/µL, and protein levels increased from 20 to 40 mg/dL. Overall, the median decrease in CSF CRAG from baseline to the time of IRIS was 8-fold (3 log2 [IQR, 2–9 log2]), but 25% of persons with IRIS had negative cultures accompanied by a <4-fold decrease in quantitative CRAG titer. Four subjects with CM-related IRIS presented with seizures, which on computed tomography and/or postmortem exam were associated with cryptococcoma(s). The CD4 counts at IRIS events (median, 80.5 cells/µL [IQR, 47–167 cells/µL) were similar to control cohort CD4 counts at 8–12 weeks (median, 77 cells/µL [IQR, 41–121 cells/µL]; P = .6).

CSF cytokine levels in CM. Seventy-two CSF samples were evaluated (13 had insufficient volume collected for analysis), including 27 from subjects with CM-related IRIS. Persons who subsequently developed CM-related IRIS after starting ART had less inflammation present in their CSF at the time of their initial CM diagnosis. CSF WBC counts and protein levels were lower in persons who developed IRIS than in those who did not develop IRIS after starting ART (P = .012 and P = .004, respectively). CSF levels of interleukin (IL)-6, IL-8, IFN-γ, tumor necrosis factor-α (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 levels were comparable (P > .2).

Figure 3.
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Figure 3.

Initial cerebrospinal fluid (CSF) profiles with cryptococcal meningitis in persons who do and do not develop immune reconstitution inflammatory syndrome (IRIS). This displays the initial CSF profile (only) in antiretroviral therapy (ART)-naive persons with a first episode of cryptococcal meningitis (CM), compared with their later risk of cryptococcal-related IRIS with central nervous system manifestations after initiating human immunodeficiency virus therapy. Persons with “Future IRIS” after starting human immunodeficiency virus therapy had less initial inflammation in their CSF, compared with persons with uneventful immune reconstitution with ART (“No IRIS”). The pattern of inflammation is consistent with a decreased Th1 response in persons with future IRIS. Th1 immune responses are necessary for cryptococcal clearance; thus, persons with a highly dysfunctional immune system may be predisposed to IRIS by failing to effectively clear antigen, even though they have similar CD4 counts and similar burden of initial CM infection at time of initial cryptococcal meningitis (eg, similar cryptococcal antigen (CRAG) titer and quantitative culture). The y-axis units for white blood cells (WBC) are cells/µL; protein is mg/dL; and cytokines and chemokines are pg/mL. Other CSF cytokines with low but statistically different levels were interleukin (IL)-4 (median, 1.4 pg/mL overall; P = .047) and IL-1β (median, 2.3 pg/mL overall; P = .035). G-CSF, granulocyte colony-stimulating factor; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α.

IRIS predictors at initial CM diagnosis. For the initial, baseline pre-ART CSF specimen, each 2-fold increase in IL-8 level was associated with increasing protection from IRIS (OR, 0.32 [95% CI, 0.17–0.62]; P = .001) after initiating ART. Conversely, increasing levels of IL-1β were associated with increased risk of IRIS (OR, 2.1 [95% CI, 1.2–3.6]; P = .012) per 2-fold change, as were possibly CCL2 levels (monocyte chemotactic protein-1 [MCP-1]) (OR, 1.56 [95% CI, 0.99–2.4]; P = .053).

The combination of initial CSF WBC count ⩽25 cells/γL and CSF protein level ⩽50 mg/dL was more highly associated with increased risk of IRIS (OR, 7.2 [95% CI, 2.7–18.7]; P < .001), with a diagnostic sensitivity of 69% (27 of 39), specificity of 76% (35 of 46), positive likelihood ratio of 2.90, and negative likelihood ratio of 0.40. The overall proportion of subjects correctly classified by CSF WBC count and protein level was 73% (62 of 85). Thus, initial CSF parameters, including low levels of proinflammatory cytokines, low protein levels, and low cellular response, were associated with the subsequent development of IRIS.

Changes in inflammation with CM-related IRIS. At time of CM-related IRIS, CSF WBC counts, CSF protein levels, and CSF levels of multiple proinflammatory cytokines were increased, compared with baseline CSF levels from the same subjects at the time of CM diagnosis (Table 2). We observed 3-fold increases in median IFN-γ, TNF-α, granulocyte colonystimulating factor levels as well as 2-fold elevations in vascularendothelial growth factor (VEGF) and eotaxin (CCL11) levels (P < .05) at the time of IRIS, compared with levels at 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.

CM relapse. In contrast to culture-negative CM-related IRIS, CSF parameters in 5 persons with culture positive CM relapse showed limited signs of inflammation. Compared with paradoxical CM-related IRIS, CSF from patients with CM culture-positive relapse had lower levels of the proinflammatory 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 those at initial CM diagnosis. The WBC counts in subjects with CM relapse were lower (mean, 12 cells/µL [range, 5–30 cells/µL]) than in those with CM-related IRIS (mean ± standard deviation, 68±66 cells/µL; P = .05).

Definite CM relapse was diagnosed in 4 persons with positive CSF cultures after 12–25 weeks of ART. No clinical features differentiated relapse from IRIS at event presentation. In 3 subjects, CSF quantitative cultures grew 30,000–38,000 Cryptococcus neoformans CFU/mL, with 2-fold increases in CSF CRAG titer to 1:2048, and 2 of these subjects experienced virologic failure. A fourth subject had 3 separate episodes of recurrent meningitis at 12, 24, and 28 weeks of ART. The first episode was culture negative (ie, CM-related IRIS) and was managed with therapeutic lumbar punctures to control pressure. The second episode occurred at 12 weeks, and the patient received corticosteroids for presumed refractory CM-related IRIS; however, the CSF quantitative culture was 900 CFU/mL (ie, CM relapse). This patient subsequently developed multiple intraparenchymal, cystic brain cryptococcomas causing obstructive hydrocephalus, but CSF culture was negative at 28 weeks. The patient died, and postmortem examination revealed intraparenchymal 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 2 weeks of amphotericin with a complete clinical response followed by 4 weeks of fluconazole (400 mg) before presenting with recurrent meningitis. The CSF quantitative culture decreased from 25,000 to 5000 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 CSF findings in patients with CM relapse.

A sixth case patient failed to improve with antifungal therapy and was classified as experiencing CM treatment failure. This subject received corticosteroids prior to receiving a diagnosis of CM. 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 sixth cranial nerve palsy developed, but the patient refused further lumbar punctures and died at 4 weeks of ART.

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Discussion

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 with 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 patients who did not experience IRIS, persons with future IRIS had lower CSF levels of the Th1-associated cytokine IFN-γ and of the proinflammatory 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-related 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 with that at initial CM. Also present at elevated levels at the time of IRIS were proinflammatory 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-related 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 proinflammatory 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 with initial levels at the time of CM diagnosis, and were comparable to those in HIV-infected persons without IRIS receiving ART [41]. In contrast, levels of TNF-α were elevated at the time of IRIS. TNF-α induces VEGF-A, which increases vascular permeability and stimulates chemotaxis of macrophages and CD4+CD45RO+ memory T cells [42]; VEGF also has costimulatory activity for IFN-γ-secreting Th1 memory T cells [42]. Lastly, granulocyte colony-stimulating factor was elevated in CSF at the time of CM-related IRIS. In cryptococcosis, granulocyte colony-stimulating factor levels may correlate with CSF clearance of Cryptococcus [38]. Whereas granulocyte colony-stimulating factor is normally considered to be a hematopoietic growth factor, it 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 statistically differ between subjects with CM and those with CM-related IRIS [1].

Whether CSF cytokine profiles can be used to clinically riskstratify HIV-infected patients starting ART remains an open question. Our results suggest that widely available CSF parameters, such as protein and WBC levels, 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 count >25 cells/µL, protein level >50 mg/dL) at the time of initial CM diagnosis only infrequently (25.5%) developed IRIS while receiving ART, whereas 71% of those with measurements 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 antiinflammatory 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-related IRIS at the time of these events. CM relapse was associated with a persistent lack of viable organisms and lack of inflammation in CSF, whereas CM-related IRIS showed no organisms but a more robust inflammatory profile. CM relapse was characterized by a lack of proinflammatory 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 5 patients with CM relapse later developed culture-negative paradoxical IRIS. IRIS is an immunologic event that can occur in the presence of live or dead organisms. Although the presence of live organisms, as seen in relapse, influences clinical management, 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 [30], and a minority of CSF cultures can remain positive up through 8–10 weeks [39]. Clinical considerations in suspected IRIS events early in ART would suggest that antifungal 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 WBC counts coupled with decreasing CRAG titers at the time of recurrent symptoms, is a useful initial diagnostic tool to differentiate IRIS from relapse. However, CSF 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-related 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-related IRIS. In characterizing the pathophysiology of CM-related IRIS at the site of inflammation, this study is a first step toward selecting rational therapeutic strategies for CM-related IRIS.

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Acknowledgments

We thank Dr Abdu Musubire, Ms Jane Ndyetukira, and Ms Irene Namugga for patient care. We 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.

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Footnotes

  • Presented in part: 2009 Infectious Diseases Society of America annual meeting.

  • Potential conflicts of interest: none reported.

  • Financial support: NIH National Institute of Allergy and Infectious Diseases (R03AI078750-01, K12RR023247-04, K23AI073192-01A2, T32AI055433-05, and 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.

  • Received February 18, 2010.
  • Accepted April 14, 2010.
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  1. J Infect Dis. (2010) 202 (6): 962-970. doi: 10.1086/655785
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