Patients studied in the cART era were older and had higher CD4 cell counts compared with those diagnosed during the historical period (data not shown). Of 240 patients 147 (61%) presented cryptococcosis revealing AIDS including 120 with cryptococcosis alone, 22 with an associated AIDS classifying disease and five with at least two other associated AIDS classifying diseases, while in the pre-cART era cryptococcosis revealed AIDS in 71/149 (48%). In recent years, cryptococcosis was more often disseminated (53% versus 30%; P < 0.0001) and meningitis more severe (33% versus 13%; P < 0.001) while the number of patients treated by antiretroviral agents was lower (36% versus 52%; P = 0.002) as compared to the historical group. When considering antifungal therapy administered in both periods, among 195 patients with meningitis at baseline, 31 (16%) received fluconazole alone and 56 patients (29%) received amphotericin B alone at the cART era. When considering pre-cART era, among the 120 patients with meningitis at baseline, 47 (39%) received fluconazole alone and 50 (42%) received amphotericin B alone.
The median duration of follow-up was 30 months (range, < 1–74 months) versus 11 months (range, < 1–101 months) in the cART and pre-cART periods, respectively. The average mortality rate per 100 person-years was 15.3 (95% CI, 12.2–18.4 per 100 person-years) in the cART era versus 63.8 (95% CI, 53.0–74.9 per 100 person-years) in the pre-cART era. The probability of death at 3 months was 18% (95% CI, 13–23%) and 21% (95% CI, 14–27%) in the cART and pre-cART periods, respectively (Fig. 3). Mortality curves diverged from 6 months onwards, probability of death at 6, 12, 24 and 48 months being 22%, 25%, 34% and 41% in the cART and 32%, 54%, 76% and 92% in the pre-cART periods, respectively. Deaths related to cryptococcosis were 43/131 (33%) in the pre-cART period and 36/94 (38%) in the cART period. Of 82 patients patients with interruption of maintenance therapy 69 (84%) were alive without relapse compared to 57/107 (53%) in the other group (P < 0.0001). In the multivariate analysis, prescription of cART was significantly associated with a longer survival (HR, 0.13; 95% CI, 0.06–0.25), after adjustment for CD4 cell count, age, HIV transmission category, AIDS stage, and baseline HIV RNA.
Basing our observations on a large number of HIV-infected patients followed long term, we first confirm that withdrawing maintenance antifungal therapy can be safely considered in patients with stable immune restoration. Secondly, we document determinants of cryptococcosis relapse and describe the evolution towards negative cryptococcal antigen during the cART era. Thirdly, we report dramatic changes in the long-term outcome, although short-term survival after the diagnosis of cryptococcosis in the cART era remained as high as in the pre-cART period .
The safety of interrupting chemoprophylaxis against various opportunistic infections in patients presenting stable immune restoration over time has frequently been documented [18,25–30]. Recommendations for HIV management have suggested similar conclusions for cryptococcosis when a sustained increase of CD4 lymphocyte count (i.e., > 100–200 cells/μl for ≥ 6 months) is reached [31,32] as documented by studies of limited sizes [15–17]. Kirk et al. showed no relapse among 39 patients with cryptococcosis whose maintenance therapy was stopped providing their CD4 cell counts were > 50 cells/μl . It was further confirmed in a randomized study including 42 patients who received 48 weeks of cART . Finally, an international observational study including patients with a CD4 cell count > 100 cells/μl reported four cryptococcosis relapses in patients with stable immune restoration . However, the alternative diagnosis of immune reconstitution inflammatory syndrome that can occur up to several months after cryptococcosis diagnosis was not excluded in three of four patients [33,34]. It is unlikely that a randomized trial will be designed given the very low rate of relapse. Thus, our report from a very large observational cohort represents the best current available data on the safety of stopping maintenance antifungal therapy.
No previous study at the cART era questioned the incidence and factors potentially associated with cryptococcal relapse. Here, we show that the relapse rate was very low, i.e., 0.9 per 100 person-years in those for whom antifungal therapy was interrupted. Based on extended follow-up of these patients, it is unlikely that this incidence of relapse is underestimated. Before the availability of cART, the estimated risk of cryptococcosis relapse in the absence of maintenance therapy was high, varying between 37% in a trial  and 50–60% in early retrospective studies [1,2]. In patients receiving daily fluconazole (100–200 mg/day) therapy, the risk varied between 2 and 4% in the literature [4,5,14] and 7.6 per 100 person-years in the present cohort. Thus, we observe here a lower risk of relapse than in the period when the best maintenance antifungal therapy was available but cART was not.
The influence of CD4 cell count on the subsequent risk of cryptococcosis relapse had not been previously documented [18–20]. Here, not only do we show that patients presenting subsequent microbiologically-confirmed relapse had a lower CD4 cell count at baseline but also that they experienced a less pronounced increase of CD4 cells during the first weeks of cART compared to patients who will not relapse. Thus, the nadir of CD4 cell count and the ability to obtain an early immune restoration significantly influence the risk of cryptococcosis relapse. Moreover, a CD4 cell count remaining < 100 cells/μl during follow-up was associated with a 5.5-fold rate of cryptococcosis relapse. We acknowledge that the cut-off for CD4 cell count was empirically estimated; nevertheless since our observation was retrospective and physicians based their decisions on no available recommendation, we believe that this might be an appropriate CD4 threshold for the decision to stop maintenance therapy. In addition, our data also suggest that clinicians should resume antifungal prophylaxis when the CD4 cell count is declining below 100 cells/μl. This emphasizes the need for careful clinical and immunological follow-up of patients for whom maintenance therapy is interrupted. Of particular importance, a serum cryptococcal antigen titre ≥ 1/512 at any time during follow-up was associated with a 3.5-fold rate of cryptococcosis relapse. In the pre-cART period, a lower initial serum cryptococcal antigen titre has been associated with a significantly lower risk of relapse . However, sequential monitoring was not classically recommended as there was no relationship with outcome [35,36]. Based on our results, monitoring cryptococcal antigen titres together with CD4 T-cell counts should help determine when to stop or to resume antifungal maintenance therapy.
Finally, another major finding disclosed here is the still high early mortality related to cryptococcosis in the period of cART as compared to an historical period despite published guidelines . Several factors might explain this observation. First, the population developing AIDS-associated cryptococcosis in Western countries has changed over time [12,13] and is diagnosed with more severe cryptococcosis as evidenced here. Second, all patients did not receive optimal therapy for cryptococcal meningitis, i.e., a combination of flucytosine and amphotericin B and management of elevated intracranial pressure-which are known to be a determinant of mycological success in patients with meningitis [37,38]. However, adjunctive therapy such as recombinant interferon-γ1b might contribute to improve the early outcome .
The dramatically improved long-term outcome and unchanged short-term evolution described here for cryptococcosis are in accordance with results reported for AIDS-associated tuberculosis  or severe Pneumocystis jiroveci pneumonia .
In conclusion, cART is associated with longer overall survival after AIDS-associated cryptococcosis. CD4 cell-count and serum cryptococcal antigen titre together with an antifungal therapy regimen should be recorded carefully during follow-up to identify patients at risk of cryptococcosis relapse.
We thank Dr Karine Sitbon and Cécile Droz for their excellent collaboration to the study; Ensemble contre le SIDA for their grant for the CryptoStop study and the fellowship for Dr Karine Sitbon and the excellent collaboration of the French Cryptococcosis Study Group including 84 clinical centers in France.
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The following members of the French Cryptococcosis Study Group enrolled patients in the present study and collected clinical data (by alphabetical order of the cities):
C. de Roux Serratrice, T. Bensaid: Aix en Provence; J.L. Schmitt, C. Raccurt: Amiens; E. Pichard, J.P. Bouchara: Angers; C. Michon, S. Blanc: Annecy; J.F. Dor, V. Blanc: Antibes; A. de la Blanchardière, H. Lefranc: Avignon; X. Lionet, F. Leturdu: Argenteuil; M. Ferrand, X. Larrouy: Bayonne; C. Drobacheff, L. Million: Besançon; O. Lortholary, C. Bouges Michel: Bobigny; J.F. Viallard, Bernard Couprie: Hôpital Haut Leveque, J.M. Ragnaud, B. Couprie, Hôpital Pellegrin, J. Beylot, B. Couprie, Hôpital St André – Bordeaux; E. Rouveix, M.E. Bougnoux: Boulogne; N. Canu, F. Laurent: Bourg en Bresse; J. Julien, M. Gavignet: Bourges; L. de St Martin, E. Moalic: Brest; S. Lefort, Brive; J.M. Korach, D. Toubas: Chalon en Champagne; B. Salles, C. Sire: Chalon sur Saone; C. Jacomet, M. Cambon: Clermont Ferrand; E. Mortier, M.L. Joly Guillou: Colombes; V. Garait, X. Fauchet, Centre hospitalier intercommunal, A.S. Lascaut, Stéphane Bretagne: Hôpital Henri Mondor – Creteil; R. Verdon: Caen; M. Duong, A. Bonnin: Dijon; A. Devidas, C. Malbrunot: Corbeil; P. de Truchis, J.L. Gaillard: Garches; J.P. Stahl, B. Lebeau, Grenoble; Y. Quertainmont, F. Botterel: Kremlin Bicêtre; I. Rivoal, M.A. de Sailly: La Roche sur Yon; J.P. Bedos, O. Eloy: Le Chesnay; P. Weinbreck, M.L. Darde: Limoges; A. Boibieux, M.A. Piens: Hôpital de la Croix Rousse, P. Mialhes, M.A. Piens: Hôpital Hôtel Dieu, C. Jourdan, M.A. Piens: Hôpital Wertheimer, M. Bret, M.A. Piens: Hôpital Edouard Herriot – Lyon; H. Tissot Dupont, A. Blancard: Hôpital de la Conception, N. Petit, A. Michel Nguyen: Hôpital Ste Marguerite – Marseille; M. Nezri, M. Bietrix: Martigues; Y. la Torre, C. Chandesris: Montargis; X. Kabbani, Montfermeil; V. le Moing, P. Rispail: Montpellier; C. Rabaud, M. Biava: Nancy; E. Billaud, O. Morin: Nantes; P. Del Giudice, M. Gari Toussaint: Fréjus; P. Dellamonica, E. Rosenthal, M. Garit Toussaint: Nice; T. Prazuck, D.M. Poisson: Orléans; J.L. Vildé, P. Yéni, O. Bouchaud, V. Descamps, C. Chochillon: Hôpital Bichat, C. Piketty, V. Lavarde: Hôpital Georges Pompidou, D. Salmon, N. Franck, A. Paugam: Hôpital Cochin, A. Compagnucci, M. Cornet: Hôtel Dieu, L. Bodard, Y. Péan: Institut Mutualiste Monsouris, C. le Maignan, V. Lavarde: Hôpital Laennec, J.P. Viard, L. Roudière, S. Challier: Hôpital Necker-Enfants malades, B. Dupont, F. Dromer: Hôpital de l'Institut Pasteur, V. Zeller, A. Datry: Hôpital Pitié Salpétrière, G. Pialoux, J.L. Poirot: Hôpital Rothschild, M.C. Meyohas, E. Frajdenrach, P. Roux: Hôpital St Antoine, J. Gilquin, A. Marmorat: Hôpital St Joseph, S. Neuville, C. Lacroix: Hôpital St Louis, I. Lecomte, P. Roux: Hôpital Tenon – Paris; G. Le Moal, C. Kaufman-Lacroix: Poitiers; C. Arvieux, S. Chevrier: Rennes; P. Aboud, L. Favennec, Hôpital Charles Nicolle, D. Benhamou, L. Favennec: Hôpital Bois Guillaume – Rouen; M.A. Khuong, N. Godineau: St Denis; T. Debord: St Mandé; B. Héry, O. Morin: St Nazaire; X. Line, X. Fiacre, Soissons; X. Martinot, J.M. Lang, J. Waller: Strasbourg; S. Chadapaud, A. Blancard: Hôpital Chalucet, J.P de Jaureguyberry, A. Blancard: Hôpital St Anne – Toulon; M. Alvarez, M.D. Linas: Toulouse; Y. Yazdanpanah, X. de Keyser: Tourcoing; D. Vittecoq, F. Botterel: Villejuif; S. Dellion, J. Breuil: Villeneuve St Georges, France.