Adjunctive interferon-γ immunotherapy for the treatment of... : AIDS (original) (raw)
Introduction
Cytokine therapy may potentially be of benefit in many infectious diseases, but currently has limited clinical application. Interferon-gamma (IFNγ) plays a key role in the host response to intracellular pathogens in which it is instrumental in directing the immune system towards protective Th1-type immunity. One such pathogen is Cryptococcus neoformans, which is a frequent cause of severe meningoencephalitis in HIV-infected patients [1,2]. Cryptococcal meningitis is the commonest cause of adult meningitis in Southern Africa, accounting for 63% of all microbiologically confirmed cases in the largest published series [3]. Despite optimal therapy with amphotericin B-based treatment, acute mortality remains between 24 and 37% [4–6]. There is an urgent need to improve outcomes, but drug treatment options are extremely limited, with few novel antifungal agents on the horizon.
Immunotherapy with IFNγ has been investigated as a potential adjunctive agent for the treatment of cryptococcal meningitis [7]. Animal model data support a central role for IFNγ in defence against cryptococcal infection [8–13], and IFNγ has been shown to potentiate the effect of amphotericin B both in vitro and _in vivo_[14,15]. There is also direct in-vivo evidence for the importance of IFNγ for clearance of cryptococcal infection in HIV-infected patients [16], and adjunctive IFNγ (given in addition to amphotericin B and flucytosine) has been shown to be well tolerated, with no adverse effect on CD4 cell count or viral load [7]. However, no trial sufficiently powered to investigate the efficacy of adjunctive IFNγ in the treatment of cryptococcal meningitis has previously been performed.
We performed an open-label randomized controlled trial, using the statistically powerful and clinically validated surrogate endpoint of early fungicidal activity (EFA) [17], to determine whether the addition of exogenous IFNγ to optimal antifungal therapy increased the rate of clearance of cryptococcal infection from the cerebrospinal fluid (CSF) of patients with HIV-associated cryptococcal meningitis. In view of data suggesting that the beneficial effects of IFN-γ occur very early in the treatment course [16], we compared two short-course IFNγ regimens (either 2 weeks or 3 days) with standard therapy.
Materials and methods
The study was performed at GF Jooste Hospital, a public-sector adult referral hospital in Cape Town, South Africa, and approved by the Research Ethics Committee of the University of Cape Town, the Medicines Control Council of South Africa and the London-Surrey Borders Research Ethics Committee on behalf of St. George's University of London. The study was conducted in accordance with the principles of the Helsinki Declaration and prospectively registered on the International Standard Randomised Controlled Trial Register (ISRCTN 72024361).
Participants and procedures
Between July 2007 and May 2010 sequential HIV-positive adults (age ≥21 years) with a first episode of cryptococcal meningitis, diagnosed by CSF India ink or cryptococcal antigen testing (titres ≥1:1024, Meridian Cryptococcal Latex Agglutination System; Meridian Bioscience Inc, Cincinnati, Ohio, USA) were screened for enrolment. Exclusion criteria were an alanine aminotransferase concentration more than five times the upper limit of normal (>200 IU/ml), a neutrophil count less than 500 × 106 cells/l, a platelet count less than 50 000 × 106 cells/l, pregnancy or lactation, previous serious reaction to study drugs or concomitant medication contraindicated with study drugs. Patients already receiving antiretroviral therapy (ART) were also excluded. Written informed consent was obtained from each participant, or from the next of kin for patients with altered mental status, prior to randomization. Randomization was stratified by Glasgow Coma Score (GCS) of 15 or less than 15. Patients were assigned to one of three intervention groups by means of random computer generated lists in block sizes of 8, using numbers in two sets of sealed envelopes (GCS of 15 or <15) prepared by independent persons. Study clinicians opened envelopes in sequence from the appropriate set as patients were enrolled.
The three intervention arms were amphotericin B deoxycholate (Fungizone; Bristol-Myers Squibb (Pty) Ltd, South Africa) 1 mg/kg per day by intravenous infusion over 4 h and oral flucytosine (Valeant Pharmaceuticals International Inc, Ontario, Canada) 25 mg/kg four times per day for 2 weeks (Standard therapy), standard therapy and IFNγ1b (Immukin; Boehringer Ingelheim Limited, Berkshire, UK) 100 μg by subcutaneous injection on days 1 and 3 (IFNγ two doses), and (3) Standard therapy and IFNγ1b (Immukin, Boehringer Ingelheim Limited, Berkshire, UK) 100 μg by subcutaneous injection on days 1, 3, 5, 8, 10 and 12 (IFNγ six doses).
Unless contraindicated all patients received 1 l 0.9% (normal) saline with 20 mmol KCl prior to amphotericin B to minimize nephrotoxicity, and were routinely given oral potassium and magnesium supplementation. Amphotericin B was discontinued if the serum creatinine increased to higher than 220 μmol/l despite adequate hydration, and patients were switched to fluconazole 400 mg/day. When necessary, flucytosine doses were adjusted for reduced renal function, reduced by 50% for grade 3 neutropaenia or thrombocytopaenia, and discontinued for grade 4 neutropaenia or thrombocytopaenia.
After 2 weeks all patients received fluconazole (Diflucan; Pfizer Laboratories (Pty) Ltd, South Africa) 400 mg daily for 8 weeks and 200 mg daily thereafter, unless they were taking rifampicin, in which case doses were increased by 50%. Antiretroviral therapy with stavudine, lamivudine and either efavirenz or nevirapine was initiated between 2 and 4 weeks after starting antifungal therapy, unless contraindicated, in accordance with South African national guidelines [18,19], and patients were followed up for 1-year postenrolment with particular attention paid to any clinical presentations related to cryptococcal immune reconstitution inflammatory syndrome (IRIS).
Evaluations and outcomes
Lumbar punctures with opening pressure measurements and quantitative CSF cultures were performed on treatment days 1, 3, 7 and 14. Patients with a markedly elevated opening pressure (>30 cmH2O) or symptoms of raised intracranial pressure had more frequent lumbar punctures in accordance with current guidelines [20], and quantitative cultures were also performed on these samples. CSF samples were analysed to determine the cell count and differential, protein and glucose levels. CSF IFNγ concentrations were determined using the Luminex Multianalyte System (Luminex corporation, Austin, Texas, USA) and cytokine kits (Bio-Rad Laboratories Ltd, Hertfordshire, UK), as described elsewhere [16]. Quantitative cryptococcal cultures (QCCs) were plated in serial 10-fold dilution, as previously described [17], and the dilution with the least colonies, but at least 30 colony forming units (CFUs) per 200 μl, was used to calculate CFU/ml. QCC results were verified by laboratory personnel blinded to treatment group. Cryptococcal clearance rates were calculated using a summary statistic for each patient, defined as the decrease in logCFU/ml per day using the slope of the linear regression of log CFU/ml against time for each patient, as previously described [17]. All data points were analysed except sterile cultures in the second week, if these values lessened the slope, as sterility would have been achieved before that day's lumbar puncture and this value would therefore underestimate the true slope [17].
All participants had baseline blood tests including haematology, renal and liver function, CD4+ cell counts, and HIV viral load. During the initial 2 weeks patients underwent alternate day renal function and electrolyte assessment and twice weekly haematological and liver function tests to monitor adverse events. Clinical and laboratory adverse events were graded using the NIH Division of AIDS Toxicity Table [21].
The primary outcome measure was mean rate of decrease in CSF cryptococcal CFU, also called EFA, for each treatment arm. Secondary outcome measures were mortality at 2 and 10 weeks, serious adverse events and laboratory toxicities.
Statistical analysis
Data were analysed using Stata, version 11.0 (StataCorp, College Station, Texas, USA). Variables were compared across groups using Kruskal–Wallis, _χ_2, or Fisher's exact tests as appropriate. An a-priori decision was made to use linear regression analysis to compare EFA by treatment group (using the standard treatment arm as a comparator), with adjustment for baseline fungal burden and CSF IFNγ concentration, which were found to be associated with rate of clearance in previous studies [16,17], and CD4 cell count, giving summary differences with 95% confidence intervals (95% CIs). Statistical significance was defined as P ≤ 0.05.
In an earlier trial, addition of 5FC to AmB was associated with a 74% increase in EFA [17], and standard deviations (SD) for EFA in differing treatment groups ranged from 0.13 to 0.19. Using an SD of 0.18, 27 patients per arm gave 90% power to detect a 30% or greater improvement in EFA in either of the experimental arms compared to the standard, at a 5% two-sided significance level. An initial sample size of 120 patients (40 per arm) was set to ensure at least 27 patients per arm with complete data; however, following per-protocol interim analysis in conjunction with the data safety monitoring board at 60 patients, the sample size was reduced to 90 patients as statistically significant differences in the primary endpoint were already present between treatment groups, and retention and data completeness were high.
Role of the funding source
The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. All study drugs were obtained at full cost price from their respective manufacturers.
Results
Ninety patients were randomized, but two were subsequently found to meet exclusion criteria (one previous episode of cryptococcal meningitis, one thrombocytopaenia – Fig. 1) and were excluded from analyses. Baseline clinical and laboratory characteristics were similar across groups (Table 1). At the time of screening 64 patients (71%) were known to be HIV-positive, diagnosed a median of 7 months prior to presentation with cryptococcal meningitis. All enrolled patients were HIV-infected. Overall, the median CD4 cell count was 27 interquartile range (IQR) 14–52 cells/μl, and 37% of patients had abnormal mental status, defined as seizures or GCS less than 15 prior to enrolment.
CONSORT diagram. ART, antiretroviral therapy; CM, cryptococcal meningitis; CSF, cerebrospinal fluid; IFN, interferon; TB, tuberculosis.
Baseline clinical and laboratory characteristics.
Early fungicidal activity
The rate of fungal clearance was more rapid in the two IFNγ containing arms than in the standard treatment arm (Fig. 2). The mean (SD) EFA was −0.49 (0.17) with standard treatment, −0.64 (0.27) with IFNγ two doses, and −0.64 (0.22) with IFNγ six doses. The difference in EFA was −0.15 logCFU/ml per day (95% CI −0.02 to −0.27, P = 0.02) between standard treatment and IFNγ two doses, and −0.15 logCFU/ml per day (95% CI −0.05 to −0.26, P = 0.006) between standard treatment and IFNγ six doses. Adjusting for baseline fungal burden, baseline CSF IFNγ concentration and baseline CD4 cell count did not alter the strength of association between treatment group and EFA (P = 0.01 control vs. IFNγ two doses and P = 0.008 control vs. IFNγ six doses). Further adjusting for additional factors including age, sex, weight, CSF lymphocyte count and concurrent tuberculosis (TB) treatment did not significantly change the relationship between treatment group and EFA.
Early fungicidal activity (EFA) by treatment group. The figure shows the individual patient slopes over the initial 14 days of treatment. The thick grey line is a graphic representation of the mean slope in each treatment group. Mean slope (standard deviation) is given below each plot, with a _P_-value for both unadjusted and adjusted [for baseline CD4 cell count, cerebrospinal fluid (CSF) interferon (IFN)γ and fungal burden] comparisons with control. QCC, quantitative cryptococcal culture.
Mortality
The mortality at 2 weeks was 19% (6/31) in the control group, 15% (4/27) in the IFNγ two doses group and 13% (4/30) in the IFNγ six doses, with no significant difference between groups (Table 2). At 10 weeks the mortality was 32% (10 of 31), 33% (9/27) and 27% (8/30) in the control, IFNγ two doses, and IFNγ six doses groups, respectively. The factors associated with mortality at 10 weeks in bivariate analysis were high baseline fungal burden (P = 0.02), abnormal mental status (P = 0.03), and low bodyweight at admission (P = 0.002). Most deaths in the first 2 weeks were due to cryptococcal meningitis (13/14), while the majority of those occurring between weeks 2 and 10 were due to other AIDS-related opportunistic infections (10/13, Table 3).
Outcomesa .
Safety data.
Baseline cerebrospinal fluid interferon-γ concentrations
Baseline CSF IFNγ concentration was positively associated with CSF lymphocyte count (P = 0.047) and CSF protein (P < 0.001); and negatively associated with baseline log10 fungal burden (P = 0.001). After adjustment for treatment group, baseline CSF IFNγ concentration was positively associated with increasing EFA (P = 0.02). There was no association between IFNγ concentration and CD4 cell count, abnormal mental status or CSF opening pressure (P = 0.3, 0.4 and 0.9, respectively).
Safety
All treatment arms were tolerated by the majority of participants, with eight patients (9%) having treatment interrupted or discontinued due to drug toxicities. Nearly all drug-related adverse events reported were related to amphotericin B, with anaemia being the primary problem, as previously reported [6] (see Table 3). Overall, 17 patients (18%) developed DAIDS grade 4 anaemia (Hb < 6.5 g/dl) during the first 2 weeks of treatment, with no significant difference between treatment groups. The median fall in Hb from admission to nadir value was 3.1 g/dl (IQR 2.1–4.2 g/dl with no significant variation across treatment groups. No patients developed DAIDS grade 4 renal impairment; however, eight patients had amphotericin B discontinued (two only temporarily) due to rises in creatinine, at a median of 9 days (IQR 6–11) into therapy (three in the standard therapy arm, three IFNγ two doses and two IFNγ six doses). Of note three of these patients had concurrent sepsis, and subsequently died. The remaining patients all had full resolution of renal function abnormalities. Rates of neutropaenia, thrombocytopaenia and other adverse events attributable to flucytosine or IFNγ were low.
Immune reconstitution syndromes
Cryptococcal IRIS was diagnosed in 14% of patients initiating ART (9/65), occurring at a median of 60 days (IQR 24–115 days) post-ART initiation, with no significant difference in proportions between treatment groups (Table 3). Patients who developed IRIS had lower CD4 cell counts at the time of initial cryptococcal meningitis presentation (16 vs. 36 cells/μl, P = 0.06), a less inflammatory CSF at initial cryptococcal meningitis presentation as evidenced by lower CSF lymphocyte counts (13 × 106 vs. 28 × 106/l, P = 0.1) and CSF IFNγ concentrations (19.9 vs. 59.4 pg/ml, P = 0.1), and a significantly higher baseline fungal burden (5.53 vs. 5.03 log10CFU/ml CSF, P = 0.04) and slower rate of fungal clearance (EFA −0.38 vs. −0.58 log10CFU/ml per day, P = 0.04) during initial cryptococcal meningitis presentation. This was reflected in the significantly higher fungal burden at the end of initial antifungal therapy in those who subsequently developed IRIS (1.95 vs. 0 log10CFU/ml CSF, P = 0.006). The median time to ART initiation was exactly the same in patients who did and did not subsequently develop IRIS (a median of 23 days after commencing antifungal therapy).
Long-term outcomes
Patients who survived the initial 10 weeks were followed up for a median of 1 year, and 97% commenced ART (59/61). There were three deaths during this period (two were patients who defaulted ART and regular follow-up and one died of unknown causes at home). At 6 months, 53 (60%) patients were alive, 29 (33%) were dead, and 6 (7%) were lost to follow-up, the median CD4 cell count was 131 cells/μl (IQR 75–185), and 80% had an undetectable HIV viral load. By 1-year 30 patients were known to have died (34%), eight (9%) were lost to follow-up, the median CD4 cell count was 167 cells/μl (121–222), and 83% of patients had an undetectable HIV viral load.
Discussion
The addition of short-course adjunctive IFNγ to standard amphotericin B and flucytosine treatment significantly increased the rate of clearance of cryptococcal infection from the CSF of patients with HIV-associated cryptococcal meningitis. Two doses of IFNγ were as effective as a full 2-week course, and the addition of adjunctive IFNγ was not associated with any increase in drug-related adverse events or IRIS. These data confirm the results of an earlier phase 2 study examining the safety and efficacy of adjunctive IFNγ, which showed it to be well tolerated, and suggested a trend towards improved combined mycologic and clinical success in IFNγ recipients [7].
Although this study was not powered to detect mortality differences between treatment arms, overall mortality was lower, but not statistically significant, in IFNγ-treated patients than controls at 2 weeks (14%, 8/57 vs. 19%, 6/31) and similar at 10 weeks (30%, 17/57 vs. 32%, 10/31), despite a trend towards the control group having higher CD4 cell counts and a lower proportion of patients with abnormal mental status at baseline. Of note, rate of clearance of cryptococci from the CSF, measured by EFA, has been previously shown to be independently associated with mortality in a large combined cohort analysis [22].
The patients in our study had severe cryptococcal disease, with 37% having abnormal mental status, and high fungal burdens at presentation. The mortality of 16% at 2 weeks and 31% at 10 weeks was in keeping with this, and similar to other African cohorts [4–6]. As previously described [17,22], the key predictors of mortality were high fungal burden and abnormal mental status at presentation. Low weight was also predictive of mortality. In this cohort of patients who had frequent lumbar punctures and aggressive management of raised CSF pressures as per current guidelines [20], baseline CSF opening pressure was not a predictor of mortality, in keeping with what we have previously shown in patients managed according to this study protocol [23]; in fact, survivors tended to have higher baseline CSF opening pressures that patients who died.
Despite high early mortality, long-term outcomes were good in patients who survived the acute infection. Although ART was initiated early (a median of 23 days postinitiation of antifungal treatment), the low rates of IRIS seen overall in the cohort, and absence of IRIS deaths, suggest that relatively early ART initiation is safe in patients who receive adequate amphotericin B induction therapy.
It is worth noting that many of the cases of cryptococcal meningitis included in this study could potentially have been prevented had ART been initiated soon after HIV diagnosis, particularly if combined with cryptococcal antigen screening to identify and treat patients with subclinical cryptococcal infection [24,25]. In keeping with previously reported data from South Africa [26], the majority of these ART-naive patients had been diagnosed with HIV infection many months before presentation with cryptococcal meningitis but not started treatment. Efforts to improve linkage from testing to care and quickly establish vulnerable patients on treatment are essential.
The mechanism by which exogenous IFNγ stimulates more rapid clearance of cryptococcus from the CSF has not yet been elucidated, but probably results from the direct activation of effector cells such as macrophages [27,28] and microglial cells [13], stimulating intracellular killing and favouring protective Th1 [29] rather than detrimental Th2-type immune responses [30]. Cytokines that induce a Th1 response including IL-12, and IL-18, and TNF-α have been shown to promote protection through IFNγ-dependent mechanisms [31,32], and IFNγ, produced either peripherally or in the CNS, has been shown to stimulate microglial cell activation and anticryptococcal activity [13,33].
The present study is the first to demonstrate a clear beneficial biological effect of exogenous IFNγ in the treatment of an opportunistic infectious disease. The use of a statistically powerful marker of response to therapy (EFA) enabled two dosage schedules to be examined. Very short courses of immunotherapy may be as effective, while being less expensive and more easily implemented, than longer courses – a finding that could have implications for the use of IFNγ in other infectious diseases, especially where endogenous cytokine responses have been shown to evolve rapidly [16]. At a cost of £66.67 per dose (approximately US$100 – British National Formulary price 2010), a two-dose course of adjunctive IFNγ would be economically feasible in many settings. Work is ongoing to identify patient groups in whom adjunctive IFNγ would be most beneficial, and these results need further validation in clinical endpoint studies. The study highlights the potential role of cytokine immunotherapy with IFNγ in both cryptococcal meningitis, and the broad range of other opportunistic infections requiring robust Th1-type immunity for effective host defence.
Acknowledgements
J.N.J., G.M. and T.B. are supported by the Wellcome Trust, London, UK (WT081794 and WT081667). G.M. also received SATBAT research training that was Fogarty International Center and NIH-funded (NIH/FIC 1U2RTW007373 and 5U2RTW007370). C.S. received a Fogarty International Center South Africa TB/AIDS Training Award: U2RTW007373 ICORTA. We thank Jara Llenas García and Nicky Longley for assistance with patient recruitment and follow-up.
J.N.J. designed and implemented the study, analysed the data and wrote the manuscript. G.M. implemented the study and edited the manuscript, K.R. assisted in study implementation, G.N.W. was the research nurse, T.B. assisted in study implementation, A.W. provided laboratory support, C.S. assisted in study implementation, L.G.B. and R.W. supervised implementation and critically reviewed the manuscript, T.S.H. conceptualized and designed the study, supervised implementation and helped draft the manuscript. All authors reviewed the final manuscript.
Conflicts of interest
There are no conflicts of interest.
Funding: Wellcome Trust.
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Keywords:
cryptococcal meningitis; HIV; immunotherapy; interferon-γ; randomized controlled trial; South Africa
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