Cryptococcal infection is a common opportunistic infection among severely immunosuppressed HIV-infected patients. In highly endemic countries like Thailand, its prevalence among AIDS patients ranges from 18% to 36%1,2 and its yearly incidence among HIV-infected individuals without highly active antiretroviral therapy (HAART) is estimated at 8.8 per 100 person-years.3 In Cambodia, it was a leading cause of mortality among AIDS patients hospitalized in Phnom Penh in the late 1990s.4,5 Latex agglutination tests for detection of capsular serum cryptococcal polysaccharide (CPS) are used to diagnose cryptococcosis. The sensitivity and specificity of these tests in patients with culture-proven cryptococcal meningitis (CM) are higher than 95%,6 provided that an appropriate cutoff for the titer (usually ≥1:8) and pronase pretreatment of the samples are used. In resource-limited countries, where serum cryptococcal antigen testing is not routinely available, diagnosis of cryptococcal infection may be difficult because of the atypical presentation of the disease and coinfection with other opportunistic agents. In this context, we estimated the prevalence and the determinants of cryptococcal antigenemia and evaluated the clinical contribution of systematic cryptococcal antigenemia detection in a large prospective cohort of Cambodian HIV-infected patients.
PATIENTS AND METHODS
The study took place within the HAART access program at Prea Bat Norodom Sihanouk Hospital (program supported by Médecins Sans Frontières, France) and Calmette Hospital (program supported by Médecins Du Monde, France), Phnom Penh, Cambodia between April and November 2004. All HIV-infected adult patients seen for the first time in consultation at either participating center were invited to participate in the study whether or not they required hospitalization. Those with a CD4+ T-cell (CD4) count <200 cells/mm3 and no prior history of cryptococcosis, fluconazole prophylaxis, or antiretroviral therapy were eligible for the study. After signing an informed consent form, patients answered a questionnaire on sociodemographic characteristics and occupations potentially associated with exposure to Cryptococcus neoformans. The clinical signs of meningoencephalitis were categorized as neck stiffness, altered mental status, and neurologic deficit. All patients were tested for serum CPS during the consultation (outpatients) or within 24 hours of hospitalization (inpatients). A blood sample was tested on the same day for the presence of CPS at the Institut Pasteur du Cambodge. All patients with positive serum CPS and all patients with signs suggestive of meningoencephalitis had cerebrospinal fluid (CSF), blood, and urine samples drawn for India ink staining examination (CSF and urine), CPS detection (CSF), and culture (CSF, blood, and urine). Chest radiography was performed in patients with positive serum CPS or with respiratory symptoms.
This study was approved by the National Ethics Committee for Health Research of the Cambodia Ministry of Health.
Serum CPS was detected using the Latex Agglutination System (CALAS; Meridian Bioscience Europe, Nice, France) according to manufacturer's instructions. Samples were serially diluted and considered positive for agglutination reactions coded 2+ or more at a dilution ≥1:8. The final titer corresponded to the highest dilution associated with a 2+ or greater agglutination reaction. All samples that tested 2+ at a dilution of 1:2 or 1:4 were retested with another CPS detection test (Pastorex Crypto Plus, Biorad, Marnes-La-Coquette, France; Premier, Meridian Bioscience Europe). Samples positive with any other test were considered positive for the study. Samples negative with all the other tests were considered negative.
All cultures were systematically performed on Sabouraud-chloramphenicol media at 37°C and room temperature for 15 days. Positive cultures were identified as C. neoformans by a urease positive test result and by the presence of a capsule after India ink staining. Serotyping was done using the Crypto-check kit (Iatron Laboratories, Tokyo, Japan) and confirmed at the French National Reference Center for Mycoses and Antifungals (Institut Pasteur, Paris, France).7 In addition, appropriate media for the detection of bacterial pathogens were used on the same samples. CD4 cell counts were performed using single-platform technology FACSCount (Becton Dickinson; Franklin Lakes, NJ).
We used the Centers for Disease Control and Prevention (CDC) classification to determine the AIDS stage.8
Cryptococcal infection was defined by a positive CPS agglutination test (in serum or CSF), positive C. neoformans culture (in blood, CSF, or urine), or positive India ink direct examination of CSF.9 CM was defined by positivity of at least 1 test on a CSF sample (direct, culture, or CPS detection). Isolated positive cryptococcal antigenemia (IPCA) was defined as positive serum CPS detection with negative CPS detection in CSF, India ink staining, and cultures (CSF, blood, and urine) and no pulmonary cryptococcosis. This latter condition refers to patients presenting with a positive serum cryptococcal antigen dilution (≥1:8) without evidence of clinical disease.
Patients diagnosed with CM received amphotericin B (0.7 mg/kg/d for 2 weeks) during hospitalization, followed by fluconazole (400 mg/d for 8 weeks) after hospital discharge according to the national protocol (flucytosine was not available in Cambodia because of its high cost). Patients diagnosed with IPCA received ambulatory treatment with fluconazole (200 mg/d for 12 weeks). All patients with cryptococcal infection received maintenance therapy (200 mg/d of fluconazole) after completion of the initial phase of treatment. All patients with an indication for HAART were treated accordingly through the local programs.
Statistical analyses were performed using SAS 8.02 (SAS Institute, Cary, NC) and STATA 7 (Stata Statistical Software; Stata Corporation, College Station, TX). Variables were compared across groups using the Mann-Whitney U test for continuous variables and the χ2 or Fisher exact test for categoric variables. For the body mass index (BMI), a cutoff of 15.4 kg/m2, corresponding to the lowest quartile, was chosen to define categories. Statistical significance was defined as P < 0.05. Multivariate analysis was performed using logistic regression to determine factors associated with positive serum CPS detection among patients with no symptoms highly suggestive of meningoencephalitis (ie, no neck stiffness, no severe neurologic signs).
Between April and November 2004, 441 HIV-infected patients aged 18 years or older with a median CD4 count of 31.5 (interquartile range [IQR]: 9 to 125) cells/mm3 were seen in the 2 hospitals participating in the study. Approximately one fourth of the patients did not meet the inclusion criteria for the study and were not enrolled: CD4 count ≥200 cells/mm3 (n = 74), previous history of follow-up in the same center (n = 33), pregnancy (n = 3), current primary prophylaxis by fluconazole (n = 2), past history of cryptococcosis (n = 1), and refusal of blood puncture (n = 1). Thus, 327 patients were enrolled in the study (Table 1). The median age was 35 (IQR: 31 to 40) years, with a slight excess of men compared with women (M/F ratio = 1.2). Approximately half (55.2%) of the patients were from Phnom Penh, and the others were from the countryside. A total of 49.9% (163 of 327) of participants were inpatients, with a median duration of symptoms before hospital admission of 20 (IQR: 10 to 35) days. Patients were severely immunosuppressed, as shown by a low (≤50) Karnofsky index in 19.6%, a low (16.9) median BMI, a high (57.5%) percentage of CDC stage C3 disease, and a low (24 cells/mm3) median CD4 count.
The prevalence of positive serum CPS detection was 17.7% (58 of 327) of patients in the study population. The prevalence of serum CPS detection was also determined in the group of patients without symptoms highly suggestive of meningoencephalitis (ie, no neck stiffness, no severe neurologic signs), and 10.8% (32 of 295) of patients had a positive test result. Factors associated with positive serum CPS detection in these 295 patients are described in Table 2. In multivariate analysis, male gender (marginally significant), countryside residence, CD4 count ≤50 cells/mm3, headache, and low BMI were all independently associated with positive serum CPS detection. After microbiologic investigations of patients with positive serum CPS or symptoms suggestive of meningoencephalitis, 59 cryptococcal infections were diagnosed. Of these, 41 were CM, 2 were pulmonary cryptococcosis, 10 were IPCA, and 6 could not be properly classified (2 patients died on the day of enrollment, and 4 did not have the full set of examinations, including 2 with a positive culture for C. neoformans in blood and urine but no lumbar puncture). All infections except 1 were diagnosed in patients with a CD4 count <100 cells/mm3. C. neoformans was found in the blood of 47.4% (18 of 38) of patients with CM and in the urine of 8.1% (3 of 37). There was no case of cryptococcal fungemia in the absence of meningitis or pneumonia. All strains of C. neoformans were serotype A. The median CPS serum dilution was higher among patients with CM compared with patients with IPCA (1:2048 vs. 1:16, respectively; P < 10−4), and serum CPS dilution >1:64 had a 97.4% (37 of 38 patients) positive predictive value for CM (Fig. 1). Of note, 1 patient with CM had negative serum CPS. Overall, the sensitivity of serum CPS detection at a titer ≥1:8 or >1:64 for the diagnosis of CM was 97.6% (40 of 41 patients) and 90.2% (37 of 41 patients), respectively. The prevalence of cryptococcal infection declined with increasing CD4 count levels, from 22.5% (51 of 227) among patients with a CD4 count ≤50 cells/mm3 to 12.7% (7 of 55) among patients with a CD4 count between 51 and 100 cells/mm3 to 2.2% (1 of 45) among patients with a CD4 count between 101 and 200 cells/mm3. Without serum CPS screening, 17 (28.8%) of 59 cryptococcal infections would not have been diagnosed on the day of consultation: 10 cases of IPCA, 4 cases of asymptomatic CM, and 3 cases of CM with negative CSF India ink staining and culture (but with positive CSF CPS testing).
Patients with CM (n = 41) were hospitalized for a median duration of 21 days. The cumulative mortality was 4 (9.8%) of 41 patients at week 3 and 15 (36.6%) of 41 patients at week 12. Ten patients diagnosed with IPCA were treated with oral fluconazole (200 mg/d) for 12 weeks. Of these, 2 died because of respiratory coinfections with Pneumocystis jiroveci and Mycobacterium tuberculosis. At week 12, serum CPS titers had declined among the 8 surviving patients with IPCA compared with baseline from 1:1024 to 1:16 in 1 patient, 1:32 to 1:8 in 1 patient, 1:16 to 1:8 in 1 patient, and 1:16 to negative in 5 patients.
This study has shown a high prevalence (18.0%) of cryptococcal infection among severely immunosuppressed HIV-infected patients in Cambodia. These figures are in accordance with those previously reported from other Southeast Asian countries.3-5 Serum CPS detection has been highly valuable in this study to allow early diagnosis of cryptococcal infection among patients with low CD4 cell counts. Among the 295 patients without signs highly suggestive of meningoencephalitis, male gender (marginally significant), countryside residence, CD4 count ≤50 cells/mm3, headache, and low BMI were all independently associated with positive serum CPS detection. We did not consider headache to be a specific symptom of CM, because this complaint was common in the study population as a whole (187 [57.2%] of 327 patients) and not specific for meningitis. Although men are known to be at increased risk of cryptococcal infection compared with women,2,10-13 this study suggests that this association is independent of occupation, residence, and degree of HIV-related immunosuppression, suggesting a gender-related biologic effect. Countryside residence may be associated with higher exposure to birds, and studies in suburban areas of Thailand have reported a high isolation rate of C. neoformans from chicken feces.14
Of the 59 patients with cryptococcal infection, 17 (28.8%) would not have been diagnosed on the day of consultation without the agglutination test performed on serum. Had the patients with IPCA not been treated as the result of CPS testing, they would likely have developed CM, as reported previously.15 As recommended by the Infectious Disease Society of America's cryptococcal disease systematic review and practice guidelines, such patients should probably be treated with antifungal therapy, although no retrospective or prospective studies have been conducted to investigate treatment options.16 Early diagnosis of cryptococcal infection, as in IPCA, allows much safer and less expensive treatment with oral fluconazole compared with the intravenous amphotericin B used in CM. One may argue that among these patients with CD4 counts <100 cells/mm3, chemoprophylaxis with fluconazole would be prescribed anyway, regardless of the results of serum CPS detection. Indeed, prophylaxis with fluconazole at CD4 counts <100 cells/mm3 is recommended in Cambodia. Positive serum CPS detection triggers more in-depth clinical and microbiologic investigations to rule out meningitis or disseminated cryptococcosis, however, allowing treatment adjustment in patients with more severe forms of infection according to current therapeutic recommendations. Furthermore, these patients may be better monitored after HAART initiation, because preexisting cryptococcal infection puts them at risk of immune reconstitution infection syndrome.17 Of note, elevated (titer >64) serum CPS levels were highly suggestive of CM (positive predictive value of 97.4%), as shown elsewhere.18 In laboratories where India ink staining or culture cannot be performed routinely, as is the case in most Cambodian sites at the present time, CPS detection may prove to be a useful alternative.
This study has demonstrated the high prevalence of cryptococcal infection among HIV-infected patients in Cambodia. Systematic testing of serum CPS proved clinically valuable for early detection, full investigation, and appropriate treatment of cryptococcal infection in patients with CD4 counts <100 cells/mm3.
The authors thank Chheang Rattanak for the microbiologic examinations, Chanthy Leng and Duong Chan Min for study monitoring, and all coinvestigators (Khuon Pichith, Saem Thavary, Limsreng Setha, Pierre-Régis Martin, Mao Heng, Hoy-Sereivathanak, and Som Kolayan). They also thank Suna Balkan, Médecins Sans Fontières Paris, and Anne Badrichiani for their collaboration in this project; Loïc Chartier for the data management; Dorothée Raoux for serotyping the Cryptococcus neoformans isolates (CNRS URA3012, Institut Pasteur, Paris, France); and Yazdan Yazdanpanah and Jean-François Delfraissy, Director of ANRS, for helpful suggestions on the study design. The authors also thank the Diflucan Partnership Program (Pfizer) for access to their fluconazole donation program.
1. Chariyalertsak S, Sirisanthana T, Saengwonloey O, et al. Clinical presentation and risk behaviors of patients with acquired immunodeficiency syndrome in Thailand, 1994-1998: regional variation and temporal trends. Clin Infect Dis
2. Amornkul PN, Hu DJ, Tansuphasawadikul S, et al. Human immunodeficiency virus type 1 subtype and other factors associated with extrapulmonary cryptococcosis
among patients in Thailand with AIDS
. AIDS Res Hum Retroviruses
3. Chariyalertsak S, Supparatpinyo K, Sirisanthana T, et al. A controlled trial of itraconazole as primary prophylaxis for systemic fungal infections in patients with advanced human immunodeficiency virus infection in Thailand. Clin Infect Dis
4. Chhin S, Rozycki G, Pugatch D, et al. Aetiology of meningitis in HIV
-infected patients in a referral hospital in Phnom Penh, Cambodia
. Int J STD AIDS
5. Pichith K, Chanroeun H, Bunna P, et al. Clinical aspects of AIDS
at the Calmette Hospital in Phnom Penh, Kingdom of Cambodia
. A report on 356 patients hospitalized in the Medicine “B” Department of the Calmette Hospital. Sante
6. Temstet A, Roux P, Poirot JL, et al. Evaluation of a monoclonal antibody-based latex agglutination test for diagnosis of cryptococcosis
: comparison with two tests using polyclonal antibodies. J Clin Microbiol
7. Dromer F, Gueho E, Ronin O, et al. Serotyping of Cryptococcus neoformans
by using a monoclonal antibody specific for capsular polysaccharide. J Clin Microbiol
8. Centers for Disease Control and Prevention. 1993 Revised classification system for HIV
infection and expanded surveillance case definition for AIDS
among adolescents and adults. MMWR Recomm Rep
9. Dromer F, Mathoulin-Pelissier S, Launay O, et al. Determinants of disease presentation and outcome during cryptococcosis
: the CryptoA/D study. PLoS Med
10. Dromer F, Mathoulin S, Dupont B, et al. Individual and environmental factors associated with infection due to Cryptococcus neoformans
serotype D. French Cryptococcosis
Study Group. Clin Infect Dis
11. Dromer F, Mathoulin-Pelissier S, Fontanet A, et al. Epidemiology of HIV
in France (1985-2001): comparison of the pre- and post-HAART eras. AIDS
12. Sorvillo F, Beall G, Turner PA, et al. Incidence and factors associated with extrapulmonary cryptococcosis
among persons with HIV
infection in Los Angeles County. AIDS
13. Hajjeh RA, Conn LA, Stephens DS, et al. Cryptococcosis
: population-based multistate active surveillance and risk factors in human immunodeficiency virus-infected persons. Cryptococcal Active Surveillance Group. J Infect Dis
14. Kuroki M, Phichaichumpon C, Yasuoka A, et al. Environmental isolation of Cryptococcus neoformans
from an endemic region of HIV
in Thailand. Yeast
15. Feldmesser M, Harris C, Reichberg S, et al. Serum cryptococcal antigen in patients with AIDS
. Clin Infect Dis
16. Saag MS, Graybill RJ, Larsen RA, et al. Practice guidelines for the management of cryptococcal disease. Infectious Disease Society of America. Clin Infect Dis
17. Lortholary O, Fontanet A, Memain N, et al. Incidence and risk factors of immune reconstitution inflammatory syndrome complicating HIV
in France. AIDS
18. Desmet P, Kayembe KD, De Vroey C. The value of cryptococcal serum antigen screening
patients in Kinshasa, Zaire. AIDS
Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
AIDS; Cambodia; cryptococcal antigenemia; cryptococcosis; Cryptococcus neoformans; HIV; screening