Increased Risk for Severe Malaria in HIV-1–infected Adults, Zambia

In areas in which malaria and HIV-1 are coendemic, adults with severe malaria who show no clinical signs of immunosuppression are likely to be infected with HIV-1.

year), HIV-1 infection was associated with severe falciparum malaria (8)(9)(10). Similarly, in Mumbai, India, where malaria transmission is low, HIV-1 prevalence was higher in persons with severe malaria than in the general population (11). Therefore, in areas of low malaria transmission, HIV-1 infection seems to be an important risk factor for severe malaria and death. In areas of high malaria transmission (>1/case/1000/year), the relationship between HIV-1 infection and severe malaria is less well established, with a small hospital-based study in Zimbabwe reporting higher risk for severe malaria and related death in HIV-1-infected adults than in HIV-1-uninfected adults (12). In all these studies, either the number of cases was small or HIV-1 testing was not performed, and CD4 cell count was not routinely done. The effect of both HIV-1 infection and malaria is important in several African countries with high malaria endemicity. We report the results of a matched case-control study exploring whether HIV-1 is an important risk factor for severe malaria in adults living Luanshya, Zambia, an area of high malaria transmission (>200/cases/year).

Study Design
We chose a case-control design for this study because severe malaria is relatively rare in areas of stable transmission (13). Each adult with severe malaria was matched with 2 controls: 1 adult who had uncomplicated malaria and 1 asymptomatic adult in the community. We matched for major confounding variables: age group (15-19, 20-29, 30-39, and 40-49 years), sex, area of residence, and seasonal variation. Controls were recruited within 4 weeks after identifi cation of case-patients.

Study Site and Patients
The study was conducted at Thompson Hospital, a government district hospital serving Luanshya district in the Copperbelt province of Zambia. In this district, 99% of the population is at risk for malaria ( Figure), and 30% of women attending voluntary counseling and testing at the antenatal care department are infected with HIV-1 (15). Luanshya is not a site of sentinel surveillance for HIV, but sentinel surveillance conducted during late 2004 and early 2005 in the adjacent district of Ndola showed an HIV prevalence rate of 30% (16). From October 2005 through April 2007, all patients 15-49 years of age who sought treatment for symptoms and signs of febrile illness were screened for falciparum malaria by thick and thin blood smear. Patients in whom severe malaria was diagnosed were enrolled after informed consent was given by the patient or by his or her legally authorized representative. We defi ned severe malaria (16) as fever (body temperature >37.5°C ) or history of fever in the previous 48 hours, Plasmodium falciparum monoinfection with a density of at least 100 parasites per 200 leukocytes (assumed to be >4,000 parasites/μL) in the absence of any other evident causes of illness, and at least 1 of the following signs: impaired consciousness (Glasgow Coma Score <10), multiple grand mal convulsions, jaundice, hypoglycemia (glycemia <2.5 mmol/L), hyperparasitemia (parasite density >100,000/μL), renal impairment, and cardiorespiratory distress. We collected information about anemia but did not include anemia in the case defi nition because it can be HIV-1 related, resulting in possible bias, and because uncomplicated and severe malaria episodes can be misclassifi ed in HIV-1-infected persons.
Patients with severe malaria were treated according to the national guidelines for severe malaria in Zambia (i.e., quinine 10 mg/kg/8 h for 7 d), with adequate supportive treatment (17). On the basis of information about areas of unstable malaria and of HIV surveillance data in Zambia, we predicted that HIV-1 increased the risk for severe malaria 5-fold and assumed a 30% HIV-1 prevalence (primary exposure) among controls. Using a 1:1 case:control ratio, we determined that we needed to recruit 30 case-control pairs to detect this risk with 80% power. Recruitment of cases continued until the required sample size was reached.
Controls with uncomplicated malaria were selected from the clinic closest to the homes of the case-patients. The fi rst patient attending the clinic and exhibiting fever or having a history of fever in the previous 48 hours, without any other disease, with a positive rapid diagnostic test, residing in the catchment area of the health center, matching sex and age group of the case-patient, and willing to participate was recruited as a control. Pregnant women were excluded. Field trips were organized to case-patients' residences, where, following the random sampling method, a community control was identifi ed and independently assessed. The fi rst healthy asymptomatic adult fulfi lling the matching criteria and willing to participate was recruited as a community control. Pregnant women were excluded. All community controls were assessed for possible confounding factors and screened for falciparum malaria by thick and thin blood smear. We sought to identify controls within 4 weeks after we recruited a case-patient.

Laboratory Tests
Laboratory technicians were blinded to patient identity and to patient-related parameters. We used microscopy to screen case-patients and community controls for malaria using Giemsa staining 10% for 5 min. A thin blood fi lm was examined to identify type of parasite; a thick fi lm was taken for quantifi cation. Computation of parasite density took into consideration the actual leukocyte count. In asymptomatic controls, we screened for P. falciparum infection by using a rapid diagnostic test, Malaria Pf immunochromatographic test (ICT) (ICT Diagnostics, Cape Town, South Africa). For each participant, a blood sample (0.5 mL) was collected for HIV-1 testing and CD4 cell count, and for hemoglobin (1 mL), by venipuncture. Neither the study staff nor the patient had access to the HIV-1 test results. HIV-1 testing followed an unlinked anonymous procedure: blood samples were sent to the hospital laboratory, where they were processed anonymously. Determine HIV1/2/O (Abbott Laboratories, Abbott Park, IL, USA) was the fi rst test. If test results were negative, the patient was considered not infected. If results were positive or indeterminate, the blood sample was tested with Unigold Recombinant HIV-1/2 (Trinity Biotech PLC, Bray, Ireland); if results were positive, the patient was classifi ed as HIV-1 infected. If results were negative, the outcome was considered indeterminate, and the sample was further tested with ELISA and Western blot. Patients, after recovering from malaria, and controls were counseled about HIV-1 and offered the opportunity for voluntary counseling and testing.
CD4 counts were determined by fl ow cytometry on a CyFlow (Cyfl ow Counter, Partec, Germany) within 5 hours after collection, and multiset software was used to obtain the absolute counts and the CD4+ lymphocyte ratio (18). A FACSCount machine (Becton Dickinson, Sparks, MD, USA) was used as a quality control to validate the accuracy of the Cyfl ow data over time and served as backup.

Ethics and Consent
The trial was approved by the ethical committee of the Institute of Tropical Medicine, Antwerp, Belgium, and by the Research Ethics Committee of the Tropical Disease Research Centre, Ndola, Zambia. The written informed consent signed by study participants or their legally authorized representatives described the purpose of the study, procedures followed, and risks and benefi ts of participation. The consent form was in both English and the local language. Patients were counseled on HIV and offered the opportunity to undergo voluntary counseling and testing in conjunction with the study; this procedure was followed to ensure adherence to the standard of counseling and testing stipulated in the guidelines for HIV/AIDS counseling in Zambia (17).

Statistical Analysis
Crude associations between the primary outcome measures and potential risk factors, including HIV-1 status and HIV-1-related immunosuppression (HIV-1+ with CD4 >350/μL and HIV-1+ with CD4 <350/μL), were described by using summary statistics, means, and count and were tested by using Wilcoxon signed rank test for matched variables.

Results
From December 2005 through March 2007, we recruited 30 case-patients with severe malaria, 30 controls with uncomplicated malaria, and 30 asymptomatic community controls who fulfi lled all inclusion criteria. Because of the low malaria incidence among adults, 5 controls with uncomplicated malaria who met the matching criteria could not be identifi ed within 28 days after admission of the casepatient to the hospital. One case-patient and corresponding matched controls were omitted because of missing laboratory results. One case-patient was recruited during March-October 2006, the low malaria transmission season. Median age was 33 years, and men and women were equally represented (Table 1). Eighty-six percent of participants lived in concrete houses. Households contained an average of 4.7 inhabitants, few of whom slept under a bed net (1.5 persons per household, 32%). Only 24% of case-patients (7/29) had slept under a bed net before admission. Impaired consciousness and hypoglycemia were the most common signs of severe malaria in HIV-1-infected case-patients (Table 2). Nineteen percent of patients, all HIV-1 infected, had anemia. All parasite densities were above the fever threshold (2,088/μL-635,500/μL), conservatively set at 2,000 parasites/μL. Seven of 13 patients with a parasite density below the geometric mean were treated with quinine only; others conservatively received some antimicrobial drugs. Six case-patients, all HIV-1-infected, had hyperparasitemia, 5 with >200,000 parasites/μL. None of the case-patients were receiving antiretroviral drugs or cotrimoxazole prophylaxis. Fifteen case-patients received concomitant antimicrobial drug treatment(s) determined by clinical symptoms: penicillin (8 patients), amoxicillin (1), gentamicin (2), metronidazole (1), fl uconazole (2), chloramphenicol (3), ciprofl oxacin (1), and cotrimoxazole (4). Clinical history, symptoms, outcome, illness duration, and other relevant parameters were similar in case-patients who received concomitant antmicrobial drugs and in those who received quinine only. Five (19%) patients died, 4 within 2 days after admission. Median length of hospital stay for successfully treated patients was 5.5 days. Casepatients were more likely to have used antimalarial treatment (45%) during the week before admission than were controls with uncomplicated malaria (28%) and asymptomatic controls (17%) ( Table 1). Three case-patients used antimicrobial agents the week before admission, compared with 2 controls with uncomplicated malaria and 1 asymptomatic control.

Risk Analysis
Case-patients differed from controls in use of other drugs during the previous week, HIV-1 infection, and CD4 count. No parasitemia was detected in any asymptomatic controls. Because of the matching, controls with uncomplicated malaria and asymptomatic controls were similar to case-patients for all other assessed risk factors (Table 1).
The extremely high proportion of low CD4 count in both case-patients and controls with uncomplicated malaria might be confounded by a temporary malaria-induced reallocation of specifi c T-cells (19). Therefore, 28 days after successful treatment, the absolute CD4 count was measured in 10 HIV-infected case-patients. During this period, the mean CD4 count increased >2-fold, from 142 (95% CI 76-269) to 320 CD4/μL (95% CI 169-607) (data not shown). However, the proportion of case-patients with CD4 count <350/μL remained substantial (70%), although slightly less than at admission (90%).

Discussion
In Luanshya, Zambia, an area where malaria is mesoendemic, HIV-1 infection is an important risk factor for severe malaria in adults, primarily in those with a low CD4 count. The increased risk for severe malaria in HIV-1-infected persons already has been reported from areas of low and unstable transmission (8-11) but never has been fi rmly established in areas of stable malaria transmission. The paucity of information results from the difficulty of obtaining it. Studies collecting relevant information retrospectively are vulnerable to considerable bias, and prospective studies are diffi cult to carry out where adults have acquired immunity and severe malaria is rare (20). In our study, recruitment of 30 persons with severe malaria from a population of 10,000 persons took 2 years. However, even prospectively, diagnosing severe malaria with certainty might be diffi cult, particularly in the presence of HIV-1 co-infection, because several opportunistic infections of AIDS patients could have clinical presentations similar to those of severe malaria. Persons with severe malaria could have had a concomitant Streptococcus pneumoniae or Salmonella enterica serovar Typhimurium infection (21) or HIV-1 immunosuppression-related meningitis, such as cryptococcal meningitis. Although, none of the case-patients we recruited were identifi ed by the treating physicians as having clinical AIDS, in the absence of a blood culture, physicians conservatively decided to concomitantly prescribe antimicrobial drugs in 15 cases. More detailed review of patients' fi les showed that most of these persons had hypoglycemia or hyperparasitemia, and none were in septic shock. Moreover, these patients recovered rapidly after treatment, and all but 1 were recruited during the high malaria transmission season, which support the argument that these persons had true cases of severe malaria.
The increased risk for uncomplicated malaria in HIV-1-infected patients with a low CD4 count is consistent with information in several cohort studies (1). Although the extremely high proportion of a low CD4 count in patients in our study might have been confounded by a temporary malaria-induced reallocation of specifi c T cells; 70% had a CD4 count <350/μL 1 month after successful treatment. Therefore, immunosuppression is likely an additional risk, but absolute CD4 count cannot be interpreted during a severe malaria episode.
Almost half of malaria cases throughout the world occur in areas where the disease is holoendemic (22). HIV-1 program managers working in areas where both diseases are prevalent should be aware that HIV-1 infection-and certainly HIV-1-related immunosuppression-are important risk factors for severe malaria. Early detection of HIV-1 infection is extremely important because comprehensive measures to prevent malaria and chemoprophylaxis with cotrimoxazole could be promptly implemented to protect against uncomplicated and severe malaria, a disease with a high fatality rate (23). Clinicians in such settings also should be aware of the strong association between severe malaria and HIV-1 so they can assess patients for other underlying diseases and offer the opportunity for voluntary counseling and testing for HIV-1 when patients have recovered from malaria.

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