Defective erythropoietin production and reticulocyte response in acute Plasmodium falciparum malaria-associated anemia.

To elucidate the relationship between falciparum malaria-associated anemia and serum erythropoietin (Epo) levels and reticulocyte response during acute malaria infection, 87 adults aged 18-65 years presenting with acute, uncomplicated malaria were examined on enrollment and for 28 days of follow-up. The 87 patients were divided into 2 groups: those with anemia (n = 45) and those without (n = 42). Serum samples were taken on admission (Day 0), then on Days 7, 21, and 28, to measure the reticulocyte count, absolute reticulocyte count, reticulocyte hemoglobin content, and erythropoietin level (Epo). The absolute reticulocyte counts for the anemic patients were significantly higher than for those without anemia on Days 0, 7, 21, and 28. The serum Epo levels for the anemic patients were significantly higher than the non-anemic group only on Day 0 (44.39 +/- 4.06 vs 25.91 +/- 4.86 mlU/ml, p < 0.001). Inadequate Epo production was found in 31.03% (27/87) of patients on Day 0, 37.93% (33/87) on Day 7, 43.67% (38/87) on Day 21, and 39.08% (34/87) on Day 28. These results indicate defective Epo production and reticulocyte response in adult patients suffering from acute P. falciparum malaria, which differs from pediatric patients. Our findings may provide the basis for further study into the choice of therapeutic strategies to treat acute P. falciparum malaria-associated anemia with recombinant human Epo to correct refractory anemia due to malaria.

Efficacy of Artequick versus artesunate-mefloquine in the treatment of acute uncomplicated falciparum malaria in Thailand.

To determine the efficacy, safety and tolerability of an alternative short-course, artemisinin-based combination therapy for acute uncomplicated Plasmodium falciparum malaria, we compared Artequick--a fixed-dosed combination of artemisinin (80 mg), piperaquine (400 mg), and primaquine (4 mg), per tablet--with a standard regimen of artesunate-mefloquine. A total of 130 patients were randomly assigned to treatment with an orally administered, once-daily, 3-day regimen of either Artequick (Group A: 3.2 mg/Kg/day of artemisinin, 16 mg/Kg/day of piperaquine, and 0.16 mg/Kg/day of primaquine) or artesunate-mefloquine (Group B: artesunate, 4 mg/Kg/day, with mefloquine, 8 mg/Kg/day). Patients receiving each regimen had a rapid clinical and parasitological response. All treatments were well tolerated, and no serious adverse effects occurred. No significant differences were found in fever- and parasite-clearance times between the two study groups. The 28-day cure rates were similarly high, at 98.5% and 100%, in groups A and B, respectively. We conclude that Artequick was as effective and well tolerated as artesunate-mefloquine and could be used as an alternative treatment for multidrug-resistant Plasmodium falciparum malaria in Southeast Asia.

Quantitation of cell-derived microparticles in plasma using flow rate based calibration.

Activation of vascular endothelium and blood cells can result in the formation of microparticles (MPs), which are membrane vesicles with a diameter < 1 microm which can play a pathogenetic role in a variety of infectious and other diseases. In this study, we validated a modified quantitative method called "flow rate based calibration", to measure circulating MPs in plasma of healthy subjects and malaria patients using FACSCalibur flow cytometry. MPs counts obtained from "flow rate based calibration" correlated closely with the standard method (R2 = 0.9, p = 0.001). The median (range) number of MPs in healthy subjects was 163/microl (81-375/microl). We demonstrated a flow rate based calibration for the quantitation of MPs in P. falciparum malaria-infected patients. The median (range) number of MPs was 2,051/microl (222-6,432/microl), n = 28 in patients with falciparum malaria. The number of MPs in plasma from patients with severe falciparum malaria was significantly higher than in uncomplicated falciparum malaria (2,567/microl (366-6,432/microl), n = 18 versus [1,947/microl (222-4,107/microl), n = 10, p < 0.01]. Cellular origin of MPs in malaria patients were mainly derived from red blood cells (35%), platelets (10%), and endothelial cells (5%). There was no significant correlation between the total number of MPs and parasitemia. Flow rate based calibration is a simple, reliable, reproducible method and more affordable to quantitate MPs.

Dose ranging studies of new artemisinin-piperaquine fixed combinations compared to standard regimens of artemisisnin combination therapies for acute uncomplicated falciparum malaria.

To determine the optimum dose of artemisinin-piperaquine combination therapies for acute uncomplicated Plasmodium falciparum malaria, we examined 7 candidate regimens in 411 patients admitted to the Bangkok Hospital for Tropical Diseases. The studies were performed from May 2005 to October 2005 and November 2005 to June 2006. We compared 3-day courses of artesunate-mefloquine, artemether-lumefantrine (Coartem) and of dihydroartemisinin-piperaquine (Artekin) as reference antimalarial treatments, with candidate regimens using 2-3 day courses of artemisinin-piperaquine, Artequick. Initially, patients receiving each of the regimens had a rapid clinical and parasitological response. All treatments were well tolerated and no serious adverse effects occurred. The 28-day cure rates were < 80% for the 2-day treatments with artemisinin-piperaquine at 2.4 mg/kg and 14.4 mg/kg, respectively, in the first study period and artemisinin-piperaquine at 3.2 mg/kg and 16.0 mg/kg, respectively, but > 98% for the 3-day regimens. These results suggest that a 3-day course of artemisinin-piperaquine at 3.2 mg/kg and 16.0 mg/kg, respectively, deserve further evaluation as an alternative treatment for multidrug-resistant P. falciparum malaria.

Efficacy of ivermectin treatment of cutaneous gnathostomiasis evaluated by placebo-controlled trial.

Previous studies have revealed that ivermectin treatment for gnathostomiasis can reduce parasitic loads in animals and make recurrent subcutaneous swelling subside in 76% of patients. Our study aimed to evaluate the efficacy of ivermectin for cutaneous gnathostomiasis treatment in a placebo-controlled trial. This study was a prospective randomized placebo-controlled study performed at The Bangkok Hospital for Tropical Diseases, Mahidol University, Thailand. Thirty patients with a serologically confirmed diagnosis of cutaneous gnathostomiasis were enrolled. Seventeen patients in the ivermectin treated group received a single dose of 12 mg ivermectin (200 microg/kg bodyweight), while 13 patients in the control group received a single dose of 40 mg of vitamin B1. The follow-up period was 1 year. Of the 17 patients, 7 (41.2%) responded to ivermectin, while no patient responded to placebo. The mean (95% Cl) time to the first recurrence of subcutaneous swelling with ivermectin and in the placebo groups were 257 (184-331) and 146 (42-250) days, respectively, (p=0.102). Although this study revealed no significant difference in the mean time to first recurrence of swelling between the ivermectin and placebo groups, there was a trend towards ivermectin efficacy against gnathostomiasis in previous animal and human studies. Further studies with different doses of ivermectin and larger sample sizes, and close monitoring for ivermectin tolerability and treatment response are necessary to confirm an efficacy of ivermectin.

Hematopoietic features and apoptosis in the bone marrow of severe Plasmodium falciparum-infected patients: preliminary study.

The mechanism of anemia in severe falciparum malaria is still not completely understood. The purpose of this study was to determine whether apoptosis in the erythroid lineage causes anemia in falciparum malaria. Bone marrow aspirated from 8 severe falciparum malaria patients, 3 normal volunteers and 5 retrospective normal bone marrow smears were investigated. By light microscopic study, 5 of 8 hyperparasitemic patients had hypocellular bone marrows and erythroid hypoplasia, whereas the other 3 patients had normal cellularity. The mean myeloid : erythroid ratio of these 5 patients was significantly (p < or = 0.05) higher than normal. Apoptosis of bone marrow nucleated cells (BMNC) could be determined from the exposure of phosphatidylserine (PS) on the cell membrane but not DNA fragmentation (180-250 bp) or ultrastructural morphology. The percentages of apoptotic BMNC and apoptotic erythroid cells in bone marrow from each patient and controls varied from low to high, and were not associated with parasitemia. This study suggests that destruction of erythroid lineage, particularly through apoptosis regulation, cannot solely account for anemia in falciparum malaria.

Tolerability of ivermectin in gnathostomiasis.

At present, no universally-accepted effective treatment for cutaneous gnathostomiasis is available. At the Hospital for Tropical Diseases, Mahidol University, albendazole 400 mg twice a day for 14 days is commonly prescribed for patients diagnosed with cutaneous gnathostomiasis. The efficacy of albendazole to induce outward migration of the parasite was less than or around 20% in 2 studies. Research for alternative, more efficacious treatment, is needed. In this prospective open-labeled study, we assessed the safety of ivermectin in 20 Thai patients diagnosed with cutaneous gnathostomiasis. Ivermectin, one time only, at dosages of 50, 100, 150, or 200 microg/kg bodyweight, was given orally to 4 groups of patients, 5 patients each group. Adverse events were recorded and laboratory tests were obtained before and after treatment. No serious adverse events occurred in this study. Forty adverse events were possibly related to ivermectin. The adverse events were malaise (35%), myalgia (30%), drowsiness (30%), pruritus (20%), nausea/vomiting (20%), dizziness (15%), diarrhea (15%), feeling of shortness of breath (10%), feeling of palpitations (10%), constipation (5%), anorexia (5%), and headache (5%). These adverse events were self-limited and not dose-related. Laboratory abnormalities were found in 3 patients (15%). Transient microscopic hematuria, pyuria, and mildly elevated liver enzymes were found in 1 patient each. Ivermectin single dose, of 50,100, 150, and 200 microg/kg bodyweight, is considered safe in Thai patients. Future trials of ivermectin on human gnathostomiasis may be performed using dosages up to 200 microg/kg bodyweight.

The efficacy and adverse effects of GPO-VIR (stavudine+lamivudine+nevirapine) in treatment-naïve adult HIV patients.

A descriptive, combined retrospective and prospective study was conducted at the Anonymous Clinic, Chon Buri Hospital, Chon Buri Province, Thailand from November 10, 2003 to January 4, 2004. A total of 83 adult HIV-treatment-naïve patients undergoing treatment with GPO-VIR (stavudine, lamivudine, and nevirapine) for at least one year were studied. The objectives of the study were to assess the efficacy of GPO-VIR by evaluating body weight changes, CD4 T-cell count changes, the occurrence of opportunistic infections, and long-term side effects, such as lipodystrophy, during treatment. Of 83 studied patients, approximately half (52.3%) of them had a body weight increase > 10% of pre-treatment body weight after 12 months treatment. After taking GPO-VIR, CD4 T-cell counts increased rapidly, by a median of 78 x 10(6) cells/l during the first three months. 39.5% of the patients attained median CD4 counts > 200 x 10(6) cells/I, and 11.6% achieved > 500 x 10(6) cells/l after 2 years of treatment. The occurrence of opportunistic infections was significantly lower after treatment with GPO-VIR (p = 0.001). Subjective assessment of lipodystrophy by physicians and patients showed that 16.8% had symptoms of lipodystrophy within 2 years of GPO-VIR treatment. There was a significant association between older age group (40-49 years) and occurrence of lipodystrophy (p = 0.043). GPO-VIR is an inexpensive and effective antiretroviral drug regimen for initiating treatment of naïve patients, but careful assessment for lipodystrophy is necessary, especially after one year of treatment.

The epidemiology of patients with severe malaria who died at the Hospital for Tropical Diseases, 1991-2004.

This study is a retrospective case series of the causes of death among patients with severe malaria. Data from the medical records of patients who were admitted to the Intensive Care Unit (ICU) of the Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand between 1991 and 2004 were analyzed. The overall hospital mortality rate was 0.2% and the ICU mortality rate was 1.8% for patients with malaria. Thirty-five patients died of malaria in the ICU during the study period, while a total of 1,866 patients were treated for malaria in the ICU during the study period. The most common complication of malaria was cerebral malaria (77.1%). The socioeconomic and demographic characteristics of those who died are examined here, as well as the cost of their treatment.

The use of flow cytometry as a diagnostic test for malaria parasites.

A total of 453 clinical blood samples were determined for malaria parasites by flow cytometric assay (FCM) and reagents from Sysmex Corporation, Japan. In this study, the FCM greatly simplified and accelerated parasite detection, with sensitivity of 91.26%, specificity 86.28% and accuracy 87.42%. Overall, the parasite counts by flow cytometric measurement correlated well with the parasitemia measured by microscopic assay (regression coefficient = 0.9409). The detection limit was 0.05-0.1% parasitemia. No evidence of malaria parasites in either blood donor volunteers or other disease patients groups was determined by FCM. However, 48 samples who had been treated with antimalarial drugs and whose parasite microscopic counts were negative, showed false-positive results. When the data of these 48 samples were analyzed, they were found to have high levels of reticulocytes, ranging from 2.0-18.9%. This finding suggested that a high reticulocyte concentration in the blood may interfere with the performance of the FCM. Further improvement, by eliminating this interference, will make the FCM one of the most promising tests for malaria diagnosis.

Evaluation of the KAT-Quick Malaria Rapid Test for rapid diagnosis of falciparum malaria in Thailand.

In recent years, several rapid diagnostic tests for falciparum malaria have been developed. KAT test results were compared with microscopy on 90 consecutive patients hospitalized at the Hospital for Tropical Diseases, Bangkok, Thailand. Fifty-one patients had P. falciparum infections while 49 had malaria due to other plasmodium species. For a geometric mean +/-SD (Min;Max;range) parasitemia of 11,481 +/- 5.0 (88;713,838;713,750), the sensitivity of the KAT test was 96% (95% CI = 86-99.5), the specificity was 92% (95% CI = 80-99), the accuracy was 94% and the reliability was 85%. These findings suggest that the KAT test is of potential interest in the diagnosis of falciparum malaria in Thailand.

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients.

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.

Clinical experience with intravenous quinine, intramuscular artemether and intravenous artesunate for the treatment of severe malaria in Thailand.

We prospectively studied 803 Thai patients admitted to the Bangkok Hospital for Tropical Diseases to assess the safety, tolerability and effectiveness of treatments for strictly defined P. falciparum malaria. Patients were assigned to one of five treatment groups: (i) a 5-day course of intravenous artesunate in a total dose of 600 mg, Group Aiv; (ii) intravenous artesunate as in Group Aiv followed by mefloquine, 25 mg/kg, Group Aiv+M; (iii) a 3-day course of intramuscular artemether in a total dose of 480 mg, Group Aim; (iv) intramuscular artemether as in Group Aim followed by mefloquine, 25 mg/kg, Group Aim+M, and (v) intravenous quinine, 200 mg/kg given in divided doses over seven days followed by oral tetracylcine, 10 mg/kg, for 7 days. When patients could take oral medications, the parenteral antimalarials were administered as oral agents. There were no major adverse effects observed with any of the five treatment regimens. With all regimens, 95 to 100% of the patients survived. Mean parasite clearance times were more rapid with the artemisinin regimens (53 to 62 hours) than with quinine (92 hours). The mean fever clearance times with intravenous artesunate (80 to 82 hours) were about a day shorter than those with intramuscular artemether (108 hours) or intravenous quinine (107 hours). Mefloquine reduced the recrudescence rate from 24 to 5% with intravenous artesunate but from 45 to 20% with intramuscular artemether; recrudescence was 4% with quinine and tetracycline. A dose and duration of therapy greater than those in this study are needed for optimal therapy with intramuscular artemether. Effective therapy for severe falciparum malaria can be provided by either intravenous artesunate followed by mefloquine or by intravenous quinine followed by tetracycline.

N-acetylcysteine in severe falciparum malaria in Thailand.

One hundred and eight patients with severe falciparum malaria underwent a placebo controlled trial with the antioxidant, N-acetylcysteine (NAC), as an adjunctive therapy along with standard intravenous artesunate therapy. Three NAC dosage regimens were used: an intravenous loading dose of 140 mg/kg followed by 70 mg/kg every four hours intravenously for up to 18 doses (Group 1); a single intravenous loading dose followed by oral NAC in the same amount as for Group 1 (Group 2); a regimen identical to Group 1 except that oral NAC was administered after the first 24 hours (Group 3). Fifty-four patients received placebo plus artesunate. Two critically ill patients died in Group 1. No patient sustained an adverse reaction to the NAC other than vomiting, and the deaths were attributed to severe disease with multiple organ involvement. The excellent results with NAC, the lack of adverse effects, and the rationale for NAC benefit supports the need for a large, double blind trial of NAC as an adjunctive therapy for severe malaria.

Management of Malaria in Thailand

The purpose of treatment for uncomplicated malaria is to produce a radical cure using the combination of: artesunate (4 mg/kg/day) plus mefloquine (8 mg/kg day) for 3 days; a fixed dose of artemether and lumefantrine (20/120 mg tablet) named Coartem (4 tablets twice a day for three days for adults weighing more than 35 kg); quinine 10 mg/kg 8-hourly plus tetracycline 250 mg 6-hourly for 7 days (or doxycycline 200 mg as an alternative to tetracycline once a day for 7 days) in patients aged 8 years and over; Malarone (in adult 4 tablets daily for 3 days). In treating severe malaria, early diagnosis and treatment with a potent antimalarial drug is recommended to save the patients life. The antimalarial drugs of choice are: intravenous quinine or a parenteral form of an artemisinin derivative (artesunate i.v./i.m. for 2.4 mg/kg followed by 1.2 mg/kg injection at 12 and 24 hr and then daily for 5 days; artemether i.m. 3.2 mg/kg injection followed by 1.6 mg/kg at 12 and 24 hrs and then daily for 5 days; arteether i.m. (Artemotil) with the same dose of artemether or artesunate suppository (5 mg/kg) given rectally 12 hourly for 3 days). Oral artemisinin derivatives (artesunate, artemether, and dihydroartemisinin with 4 mg/kg/day) could replace parenteral forms when patients can tolerate oral medication. Oral mefloquine (25 mg/kg divided into two doses 8 hrs apart) should be given at the end of the artemisinin treatment course to reduce recrudescence.