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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, August 2006, p. 2713-2718, Vol. 50, No. 8
0066-4804/06/$08.00+0 doi:10.1128/AAC.00392-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Ingrid Lundgren,1,4
Sunny Oyakhirome,1
Bénido Impouma,1
Pierre-Blaise Matsiegui,1,2
Ayola A. Adegnika,1,2
Saadou Issifou,1,2
Jürgen F. J. Kun,2
David Hutchinson,5
Jochen Wiesner,6
Hassan Jomaa,6 and
Peter G. Kremsner1,2*
Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon,1 Department of Parasitology, Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany,2 Otto Meyerhof Centre, Heidelberg University School of Medicine, Heidelberg, Germany,3 Mayo Medical School, Mayo Clinic, Rochester, Minnesota,4 Jomaa Pharma GmbH, Hamburg, Germany,5 Institut für Klinische Chemie und Pathobiochemie, Labor Jomaa, Universitätsklinikum Giessen und Marburg, Marburg, Germany6
Received 30 March 2006/ Returned for modification 3 April 2006/ Accepted 25 May 2006
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ABSTRACT |
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2 g/dl (7/51 [14%]) are of concern. Asexual parasites and fever were cleared within median periods of 42 h and 38 h, respectively. All patients who could be evaluated were parasitologically and clinically cured by day 14 (49/49; 95% confidence interval [CI], 93 to 100%). The per-protocol, PCR-adjusted day 28 cure rate was 89% (42/47; 95% CI, 77 to 96%). Efficacy appeared to be significantly reduced in children aged 1 to 2 years, with a day 28 cure rate of only 62% for this small subgroup (5/8). The inadequate efficacy in children of <3 years highlights the need for continued systematic studies of the current dosing regimen, which should include randomized trial designs. |
INTRODUCTION |
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We previously investigated the safety and efficacy of twice-daily regimens of fosmidomycin plus clindamycin for the treatment of schoolchildren with asymptomatic infections with P. falciparum and of pediatric outpatients aged 7 to 14 years (3, 6). A twice-daily 3-day regimen of fosmidomycin plus clindamycin was shown to have satisfactory efficacy in the treatment of uncomplicated P. falciparum malaria, as demonstrated by day 14 and day 28 cure rates of 100% and 90%, respectively (6). The validity of these findings, however, was limited due to the small sample size (a cohort of 10 patients) and the partially immune study population (children aged 7 to 14 years), precluding extrapolation of these data to younger children with less parasite-specific immunity. Children of less than 5 years are at the highest risk of dying from fatal complications of either untreated or inadequately treated P. falciparum malaria (24).
This study was designed to evaluate the safety and efficacy of a 3-day twice-daily regimen of fosmidomycin plus clindamycin for the treatment of pediatric patients aged 1 to 6 years presenting with uncomplicated P. falciparum malaria. In addition, the study aimed to consolidate the results from the previous study of patients aged 7 to 14 years.
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MATERIALS AND METHODS |
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Study design. The study was an uncontrolled phase IIb study with consecutive recruitment of eligible patients. The study started in July 2002, and the last patient completed follow-up in March 2003. Patients with uncomplicated P. falciparum malaria presenting to the outpatient pediatric department of the Albert Schweitzer Hospital in Lambaréné, Gabon, were admitted to the study if they met the following inclusion criteria: age of 1 to 14 years, asexual parasitemia of between 1,000 and 100,000/µl, acute manifestation of malaria (e.g., history of fever in the preceding 24 h or a tympanic temperature of >37.5°C at baseline), body weight between 5 and 65 kg, ability to tolerate oral therapy, informed consent by the legal representative of the subject (the parents, if possible), oral agreement of the child if appropriate, and residence in the study area for a duration of at least 4 weeks. The exclusion criteria were as follows: adequate antimalarial treatment within the previous 7 days, antibiotic treatment for a concurrent infection, hemoglobin level of <7 g/dl, hematocrit of <25%, leukocyte count of >15,000/µl, mixed plasmodial infection, severe malaria, any other severe underlying disease, concomitant disease masking assessment of the treatment response, inflammatory bowel disease, and any other disease causing fever.
Study drugs and administration. Fosmidomycin was formulated as capsules containing 150 mg of active substance. Clindamycin was supplied as 75-mg capsules. The drugs were administered orally in a 3:1 fixed ratio of 30 mg/kg of body weight of fosmidomycin and 10 mg/kg of body weight of clindamycin every 12 h for a total of six doses. Each study drug administration was supervised by at least one dedicated study physician. For patients aged 1 to 2 years, capsules were opened and the contents mixed with a suitable beverage (e.g., sweet milk) to facilitate drug uptake. Subjects who vomited or rejected the study drug within 30 min received a repeat full dose. Vomiting or rejection of the second dose led to withdrawal from the study and administration of a rescue treatment.
Study flow and procedures. Patients were seen by a study physician at 12-h intervals at each drug administration and/or until two consecutive negative blood smears occurred and subsequently on days 7, 14, 21, and 28 or as otherwise indicated. At each visit during the treatment and follow-up phases, the medical history was taken, vital signs were checked, the tympanic temperature was measured, a thick blood smear was prepared from a finger prick, and adverse events were documented. Venipunctures were performed on study days 0, 2, 7, and 28 to monitor the hemoglobin level, hematocrit, and differential white blood cell count as well as liver (alanine aminotransferase) and renal (creatinine) function parameters. Urine was tested for erythrocyte and leukocyte counts, protein, bilirubin, glucose, ketone, and nitrite concentrations, and pH by using dipstick tests at the same time points. To distinguish recrudescent infections from reinfections, blood samples were kept from day 0 and the day of reappearance of asexual parasites for PCR-based genotyping of parasite strains.
End points. The primary efficacy end point for this study was the day 14 cure rate. Cure was defined as initial and sustained parasite and symptom clearance with no increase in asexual parasitemia 48 h after the initiation of treatment and the absence of microscopically detected asexual parasitemia within 120 h of the start of treatment until day 14. The primary safety end point was the incidence of adverse events after the start of treatment. Secondary end points were the parasite and fever clearance times and the PCR-corrected day 28 cure rate.
Laboratory procedures.
Dried thick blood smears were stained with 20% Giemsa solution at pH 7.2. Parasite species were identified using standard morphological characteristics, and parasitemia was counted using the volumetric Lambaréné method and expressed in parasites/µl (7, 21). Genetic analysis of parasite pfmsp-1 and pfmsp-2 genes was performed to distinguish recrudescences from new infections, comparing matched pairs of parasite isolates obtained upon admission and on the day of reappearing asexual parasitemia (14, 19). We classified a reappearing asexual parasitemia after initial clearance as reinfection if either (i) all electrophoretically separated PCR product bands detected on the day of reappearing asexual parasitemia were distinct in size from those detected on the day of admission or (ii) 50% of these bands were distinct in size in cases where sexual parasite stages comprised >5% of the overall parasite count at asexual parasite reappearance. The serum levels of alanine aminotransferase and creatinine were measured by a semiautomatic dry-slide method (Vitros; Ortho-Clinical Diagnostics). Differential blood counts were performed with an automatic high-resolution flow cytometry-based hematology analyzer (CellDyn 3000; Abbott, Santa Clara, CA).
Data management and statistical analysis. Data were captured using specifically designed concise medical record forms and subsequently entered into an electronic database. Data were then validated by complete manual review. Statistical analyses of the data were performed using Stata (version 8.2 for Mac OS X; StataCorp, College Station, TX). The Nutstat component of Epi Info was used to analyze the nutritional status of patients.
Cure rates were calculated from the number of patients with clinical and parasitological cure by day 14 or 28 divided by the total number of patients who could be evaluated (per protocol population). Fever clearance times were calculated from the start of treatment until the first of two consecutive tympanic temperature measurements remained below 37.5°C. Fisher's exact test was used to assess differences in categorical outcome variables between subgroups. The safety analysis included abnormal laboratory data and adverse events for all subjects who received at least one dose of the study drug (intention-to-treat population). Student's t test was used to compare normally distributed laboratory data. The statistical significance level was set at 5%.
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RESULTS |
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By day 14, all 49 evaluated patients remained free of malarial symptoms and asexual parasites (100%; 95% confidence interval [95% CI], 93 to 100%). From day 14 to day 28, asexual parasites reappeared in 8 of 47 evaluated patients, resulting in a crude, un-PCR-corrected cure rate of 83% (95% CI, 69 to 92%). Subsequently, PCR analysis of pairs of samples from day 0 and the day of asexual parasite reappearance showed that five of these eight asexual parasite reappearances were indeed recrudescences but that three were new infections due to inoculation(s) with different parasite strains either shortly before, during, and/or after treatment. Thus, the PCR-corrected day 28 cure rate was 89% (42/47; 95% CI, 77 to 96%).
Due to concerns with the efficiency of drug administration in patients aged 1 to 2 years, the day 28 cure rate was subsequently reanalyzed separately for patients aged 3 to 14 years and patients aged 1 to 2 years. The day 28 cure rate was 37/39 (95%; 95% CI, 83 to 99%) for patients aged 3 to 14 years but only 5/8 (62%; 95% CI, 24 to 91%) for patients aged 1 to 2 years (P = 0.029).
Treatment with fosmidomycin plus clindamycin was associated with a high cumulative gametocyte carrier rate (gametocytes detected on at least one follow-up visit) of 78% (38/49), regardless of the presence or absence of sexual parasites on the admission blood smear. In addition, there was no evidence of gametocytocidal effects of fosmidomycin plus clindamycin: in all three patients with baseline gametocytemia, asexual parasites were also detected on day 7.
The hematological response was characterized by significant recovery of the mean hemoglobin concentration from day 0 to day 28, with a mean increase of 0.9 g/dl (P = 0.002).
Safety and tolerability. There was one serious adverse event in a male patient aged 2.7 years who was diagnosed with severe anemia on study day 2. His blood hemoglobin concentration dropped from 9.2 g/dl upon admission to 4.5 g/dl on day 2, leading to hospitalization. He received parenteral quinine rescue treatment but no blood transfusion. He then made an uneventful recovery and was discharged 3 days later. This patient was retrospectively excluded from the per-protocol population due to the presence of a sign of severe malaria upon admission (schizontemia).
In total, 22 adverse clinical events in 51 patients were reported, including 21 mild and 1 moderate (the serious adverse event described above) event, of which 13, 7, and 2 events were judged to be not, possibly, or probably related to the study drugs, respectively. The majority of adverse events were reported during the treatment phase until day 7 (18/22 [82%]). The most frequent adverse events up to day 7 were gastrointestinal events (12 [all mild]), mostly abdominal pain (5).
Table 3 summarizes the evolution of laboratory parameters. Mean hemoglobin concentrations, mean leukocyte counts, and mean alanine aminotransferase concentrations dropped significantly from day 0 to day 2. A fall of 2 g/dl in the hemoglobin concentration occurred in seven patients (including the patient described above), but the hemoglobin concentration remained above 8 g/dl in all but two patients and subsequently recovered until day 28 (Table 4). The glucose-6-phosphate dehydrogenase status of these patients was not determined. The ANC in two of three patients with grade I neutropenia upon admission recovered slightly by day 2 (1,100 to 1,200/µl and 1,300 to 1,400/µl) but decreased further in the third patient (1,200 to 700/µl), resulting in grade II neutropenia on day 2 (Table 2). Seven other patients aged 6 to 9 years developed transient asymptomatic neutropenia on day 2 (six cases of grade I and one case of grade II neutropenia) (Table 2). In four more patients, the ANC dropped below 1,500/µl (grade I) between days 7 and 14. Neutrophil counts returned to normal values by day 7 for all patients with day 2 neutropenia, without intervention. Defervescence times did not differ between patients with and without neutropenia on day 2 (median times of 37 and 38 h, respectively). Neutropenia did not occur in children younger than 6 years old. A 6-year-old female patient developed a temporarily elevated plasma creatinine concentration on day 7 of 115 µmol/liter (up from 37 µmol/liter and 45 µmol/liter on days 0 and 2, respectively), with a subsequent recovery by day 28 (23 µmol/liter). No abnormal urine parameters were detected.
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DISCUSSION |
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The extension of follow-up to 28 days increased the sensitivity of the in vivo test (25), providing more precise estimates of the efficacy of an antimalarial drug. This advantage, however, is at least partly offset by the difficulty in reliably distinguishing recrudescent from new, and hence confounding, infections. We applied genetic analysis of sequence repeat-length polymorphisms of parasite pfmsp-1 and pfmsp-2 genes to estimate the PCR-corrected day 28 cure rate as 94% for patients aged 3 to 14 years and 62% for patients aged 1 to 2 years. The difference in the rates between younger and older children was statistically significant (P = 0.029).
The relatively low day 28 cure rate for a small subgroup of very young children was most likely due to lower levels of parasite-specific immunity than those in children of 3 years old. An alternative or complementary pharmacokinetic explanation for the observed lower efficacy in younger children could be related to the difficulty in administering the current capsule formulation of fosmidomycin to very young children (the powder released from the capsules was given as a slurry). Drug-induced vomiting did not occur, however, and the lack of pharmacokinetic data for these children precludes a comprehensive analysis of whether drug plasma levels were indeed affected. Pharmacokinetic studies of the concurrently developed coformulated tablet are needed to allow a meaningful examination of the relationship between pharmacokinetic and pharmacodynamic variables, i.e., the time that plasma concentrations are above the minimal parasiticidal concentrations (29), preferably in largely nonimmune patients. For the determination of fosmidomycin plasma levels, a newly developed capillary electrophoresis method may be used (8), which is expected to be superior to the bioassay used in former pharmacokinetic studies (12). The fact that all recurrent infections occurred beyond day 14 (three on day 21 and two on day 28) is a salutary reminder of the inadequacy of the day 14 cure rate used in at least one previous pivotal phase III trial (2) but is also a reason for cautious optimism for potential improvements of the efficacy of the current regimen.
Treatment with fosmidomycin plus clindamycin was associated with a large number of episodes of grade I and II neutropenia in 12 of 50 patients (24%), reminiscent of earlier findings with fosmidomycin plus artesunate (4) and fosmidomycin plus clindamycin (our unpublished data). The majority of these cases were transient and occurred shortly after the start of treatment on day 2 (n = 8). All cases resolved without further intervention until day 7. Despite a relatively high background incidence rate of asymptomatic neutropenia in this population (Table 2), possibly linked to underlying vitamin B12 and folate deficiencies (concentrations were not determined in this study), the temporal association of neutropenia episodes with the treatment phase points to a treatment-induced effect. Clindamycin has previously been associated with rare cases of transient neutropenia (28). Neutropenic patients on day 2 had comparable defervescence times to those without such episodes, suggesting that concomitant febrile bacterial infections are unlikely to be causally involved. Because of the uncontrolled design of this phase II study, it is extremely difficult at this stage to unambiguously attribute these episodes to exposure to fosmidomycin plus clindamycin in light of evidence of immunosuppression in patients with uncomplicated P. falciparum malaria (10) and sequestration or margination of neutrophils in the lungs and possibly other inner organs during malaria (23). Direct suppressive effects of antimalarials on neutrophil functions have previously been shown for chloroquine, amodiaquine, and mefloquine (15). Clearly, this is an important issue that requires particular scrutiny in future controlled studies of fosmidomycin combinations. As an interesting side note, the proportions of cases with day 0-to-day 2 falls in neutrophil counts appeared to be related to different fosmidomycin treatment regimens (with higher proportions observed with fosmidomycin plus clindamycin than with fosmidomycin plus artesunate) as well as to the severity of infection (with higher rates observed for uncomplicated malaria than for asymptomatic infection). This trend was reflected by parallel mean falls in leukocyte counts (–0.04 x 109/liter in children treated for asymptomatic infections with fosmidomycin and/or clindamycin [3], –0.8 x 109/liter in children aged 6 to 12 years treated with fosmidomycin plus artesunate [4], and –1.2 x 109/liter in children aged 7 to 14 years treated for uncomplicated P. falciparum malaria with fosmidomycin plus clindamycin [6]), suggesting that disease-treatment interactions with an impact on fluid volume (re)distributions might play an important role in determining hematological adverse drug reactions in P. falciparum malaria.
Because hemolysis is a feature of P. falciparum malaria, the relationship between drug exposure and incidence rates of important falls in hemoglobin concentrations can only be examined in the context of a controlled trial. In this study, we observed falls in hemoglobin concentration of 2 g/dl within 2 days of starting treatment in 7 of 51 patients (14%), including one patient with a drop from 9.2 g/dl upon admission to 4.5 g/dl on day 2 who required blood transfusion. Similarly high rates (16%) were reported from a recent large multicenter phase III trial comparing chlorproguanil-dapsone and sulfadoxine-pyrimethamine, two drug combinations previously associated with drug-induced hemolysis in glucose-6-phosphate dehydrogenase-deficient patients (2). Somewhat lower incidence rates were observed in previous studies of fosmidomycin plus clindamycin (4/52 [8%]) (6) and fosmidomycin plus artesunate (1/50 [2%]) for the treatment of children older than 6 years with P. falciparum malaria (4). In contrast, the hemoglobin concentration remained stable in children who previously received either fosmidomycin alone, fosmidomycin in combination with clindamycin, or clindamycin alone for asymptomatic P. falciparum infections (3). No changes of the hematological and biochemical parameters after treatment with fosmidomycin were observed in previous phase I studies conducted with 127 healthy male volunteers (13). Also, fosfomycin, a broad-spectrum antibiotic chemically related to fosmidomycin, and clindamycin have not been associated with drug-induced hemolysis until now (16, 22). It therefore seems plausible that the observed cases of hemolysis are disease rather than drug induced. However, in analogy to the potentially drug-induced cases of neutropenia in this study, this question needs to be carefully addressed in future controlled trials of fosmidomycin combinations.
The antibiotic nature of fosmidomycin and clindamycin raises the spectre of gastrointestinal side effects, particularly of serious adverse drug reactions such as pseudomembranous colitis caused by Clostridium difficile. A previous review of the available data found no evidence of clindamycin-associated pseudomembranous colitis in malaria patients who received clindamycin. The observed incidence rate of gastrointestinal adverse events of 18% (9 of 51 treated patients) until day 7 in this study was lower than those in previous studies of fosmidomycin, either alone or in combination with clindamycin, of 47% (17/36) (3) and 40% (21/52) (6), respectively, and of amodiaquine, either alone or in combination with artesunate, of 28% (28/100) and 29% (29/100), respectively (1), and nearly matched the lowest corresponding rates reported from studies with atovaquone-proguanil and fosmidomycin-artesunate, i.e., 13% (13/100) (5) and 14% (7/50) (4), respectively. This historical comparison with studies conducted at the same study site suggests that fosmidomycin plus clindamycin at least does not substantially increase the risk for gastrointestinal side effects above a disease-associated background incidence rate. A definite assessment, however, has to await controlled trials.
Fosmidomycin plus clindamycin interferes synergistically with independent functions of the P. falciparum apicoplast, a drug target that has never been exploited by commonly used antimalarials. The matching short half-lives of fosmidomycin and clindamycin might further reduce the risk of selecting for resistant parasite alleles that could be promoted by trailing subtherapeutic plasma concentration levels. However, the failure of fosmidomycin plus clindamycin to inhibit the development of sexual-stage parasites, most likely a result of its subpar efficacy against metabolically active parasite stages, may facilitate the onward transmission of resistant parasite alleles to the mosquito vector (26). In addition, the 12-hour dosing regimen may be more difficult to achieve than 24-hour dosing outside a clinical trial setting. Some of these disadvantages, however, including the excessive number of capsules in the current regimen, are expected to be addressed by coformulating fosmidomycin and clindamycin in a fixed-dose tablet. This formulation has recently become available for further clinical development.
Further work on fosmidomycin plus clindamycin should now primarily focus on the comparative evaluation of its safety and efficacy in a randomized controlled trial for the treatment of uncomplicated P. falciparum malaria in children aged 3 to 14 years. In parallel, pharmacokinetic investigation of the new tablet formulation with very young children should provide valuable information for improving the efficacy of fosmidomycin plus clindamycin in this important patient population.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the German Malaria Initiative program coordinated by the Federal Ministry of Education and Research, Germany, and the 5th Framework Programme of the European Commission (INCO-Dev contract no. ICA4-2000-10290). The funding sources had no role in the design of the study; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.
David Hutchinson is Managing Director of Jomaa Pharma GmbH. The authors have no other potential conflict of interest.
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FOOTNOTES |
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Present address: Kenya Medical Research Institute-Wellcome Trust Collaborative Research Programme, Kilifi, Kenya.
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