Therapeutic response of multidrug-resistant Plasmodium falciparum and P. vivax to chloroquine and sulfadoxine-pyrimethamine in southern Papua, Indonesia.

To determine the level of antimalarial drug resistance in southern Papua, Indonesia, we assessed the therapeutic efficacy of chloroquine plus sulfadoxine-pyrimethamine (CQ+SP) for Plasmodium falciparum infections as well as CQ monotherapy for P. vivax infections. Patients with P. falciparum failing therapy were re-treated with unsupervised quinine+/-doxycycline therapy and those with P. vivax with either unsupervised quinine+/-doxycycline or amodiaquine. In total, 143 patients were enrolled in the study (103 treated with CQ+SP and 40 with CQ). Early treatment failures occurred in four patients (4%) with P. falciparum and six patients (15%) with P. vivax. The failure rate by Day 28 for P. vivax was 65% (95% CI 49-81). After PCR correction for re-infections, the Day 42 recrudescence rate for P. falciparum infections was 48% (95% CI 31-65). Re-treatment with unsupervised quinine+/-doxycycline resulted in further recurrence of malaria in 48% (95% CI 31-65) of P. falciparum infections and 70% (95% CI 37-100) of P. vivax infections. Eleven patients with recurrent P. vivax were re-treated with amodiaquine; there were no early or late treatment failures. In southern Papua, a high prevalence of drug resistance of P. falciparum and P. vivax exists both to first- and second-line therapies. Preliminary data indicate that amodiaquine retains superior efficacy compared with CQ for CQ-resistant P. vivax.

Immunologic and psychologic therapy for patients with chronic fatigue syndrome: a double-blind, placebo-controlled trial.

PURPOSE: To evaluate the potential benefit of immunologic therapy with dialyzable leukocyte extract and psychologic treatment in the form of cognitive-behavioral therapy (CBT) in patients with chronic fatigue syndrome (CFS). PATIENTS AND METHODS: Immunologic and psychologic treatments were administered to 90 adult patients who fulfilled diagnostic criteria for CFS in a double-blind, randomized, and placebo-controlled study. A four-cell trial design allowed the assessment of benefit from immunologic and psychologic treatment individually or in combination. Outcome was evaluated by measurement of global well-being (visual analogue scales), physical capacity (standardized diaries of daily activities), functional status (Karnofsky performance scale), and psychologic morbidity (Profile of Mood States questionnaire), and cell-mediated immunity was evaluated by peripheral blood T-cell subset analysis and delayed-type hypersensitivity skin testing. RESULTS: Neither dialyzable leukocyte extract nor CBT (alone or in combination) provided greater benefit than the nonspecific treatment regimens. CONCLUSIONS: In this study, patients with CFS did not demonstrate a specific response to immunologic and/or psychologic therapy. The improvement recorded in the group as a whole may reflect both nonspecific treatment effects and a propensity to remission in the natural history of this disorder.

Assessment of pfmdr 1 gene copy number by tandem competitive polymerase chain reaction.

The pfmdr 1 gene encodes a Plasmodium falciparum homologue of the human P-glycoprotein expressed on the surface of the parasite food vacuole. Variation in copy number and specific codon mutations of pfmdr 1 have been implicated in the development of parasite resistance to antimalarial drugs. We describe here the technique of Tandem-Competitive Polymerase Chain Reaction (TC-PCR), which allows accurate measurement of pfmdr 1 copy number in parasite DNA obtained directly from small quantities (100 microliters) of red blood cells. We reliably quantified pfmdr1 in previously well characterised strains of Plasmodium falciparum with differing pfmdr1 gene copy numbers using starting amounts of between 3,000 and 40,000 gene copies. We then used TC-PCR to determine pfmdr1 gene copy number in field specimens of venous blood taken from 10 patients with malaria contracted along the Thai-Burmese border. In this region of high grade parasite resistance to mefloquine greater than 70% of samples had a copy number greater than 1 of pfmdr1 determined with a repeatability coefficient of 0.58.

The chromosomal organization of the Plasmodium falciparum var gene family is conserved.

The var gene family of Plasmodium falciparum encodes the protein PfEMP1 which is located on the surface of infected erythrocytes and is the receptor that mediates binding to ligands on endothelial cells. This family of proteins is responsible for antigenic variation and differences in binding phenotype to ligands such as CD36 and ICAM1. We have compared the organization of the var gene family in three in vitro cloned lines of P. falciparum and show that most var genes are located in the subtelomeric region of each chromosome closely linked to the repetitive sequence rep20. While most chromosomes possess var genes in the subtelomeric region, in each in vitro cloned line there are some chromosomes that have deleted subtelomeric repetitive regions which include var genes. Comparison of the location of var genes in a field isolate showed that it does not have any detectable subtelomeric deletions as all chromosomes contain var genes and rep20 sequences. We have detected three chromosomes (4, 7 and 12) that contain var gene loci in more stable central regions and the position of these genes on chromosome 4 in the cloned lines analysed is conserved. The location of most of the var gene family in the subtelomeric region of the genome of P. falciparum has important implications for the generation of antigenic diversity of the PfEMP1 protein.

Storage duration and polymerase chain reaction detection of Plasmodium falciparum from blood spots on filter paper.

To evaluate the effect of long-term storage of sample filters on the sensitivity of polymerase chain reaction (PCR) detection of malaria, 252 blood spots from patients with microscopically confirmed Plasmodium falciparum malaria were analyzed and stratified by storage duration. The spots were collected between 1996 and 2000 on filter paper and stored at room temperature. A Chelex-based method was used to extract the DNA. Unexpectedly, after the first purification, the sensitivity of the PCR from recently stored samples was low and showed progressively increased with time storage (P = 0.003, by chi-square test for linear trend). This suggested that PCR inhibitors were easier to dissolve from the more recent blood spots (< 4 years old) than from blood spots > or = 4 years old, thus leading to a time-dependent increase in PCR sensitivity. However, if DNA was purified again (when the first PCR result was negative), the cumulative sensitivity was not influenced by storage duration. This indicated that length of storage is not a critical issue providing purification is sufficient.

Phase I trial of the SPf66 malaria vaccine in a malaria-experienced population in Southeast Asia.

In preparation for a recently reported, independent field trial of SPf66 malaria vaccine efficacy in Thailand, we first established the safety and immunogenicity of two clinical lots of U.S. manufactured lots of SPf66 in a series of overlapping Phase I studies. The vaccine was produced in approved laboratories using good manufacturing practices. Two clinical lots of alum-adsorbed SPf66 were evaluated in a combined, open-label, Phase I clinical trial involving 50 healthy, malaria-experienced Karen adults and children. Volunteers were grouped by age and immunized sequentially. Group 1 had 30 adults. Group 2 had 10 children 8-15 years of age, and Group 3 had 10 children 2-6 years of age. The SPf66 vaccine was well tolerated in this malaria-experienced population. The most common side effects were erythema, induration, warmth, and tenderness at the site of injection, which typically resolved within 24-48 hr. One adult volunteer developed an acute urticarial rash following the third dose. Among adults, and to a lesser extent older children females had more local reactions than their male counterparts. Seroconversion to SPf66 by enzyme-linked immunosorbent assay occurred in 76% of volunteers receiving two or three doses. This vaccine was safe and immunogenic in malaria-experienced Karen adults and children. This study establishes the comparability of U.S.-manufactured SPf66 with that of Colombian origin, and is important for interpreting the efficacy results of U.S.-manufactured SPf66 in the same study population.

Transmission intensity and Plasmodium falciparum diversity on the northwestern border of Thailand.

Genetic analysis of the number of Plasmodium falciparum genotypes per infected person in regions of holoendemic and hyperendemic malaria suggest that in areas of lower transmission intensity, significantly fewer parasite genotypes per infected person should be found. A predominance of single clone infections in the human population could generate the controversial clonal population structure proposed for P. falciparum by Tibayrenc and others. Characterization of P. falciparum from individuals on the Thai-Burmese border, an area of hypoendemic transmission, revealed a higher number of genotypes per infected person than that predicted. Possible reasons for this observation are discussed, with particular attention paid to human migration and multidrug resistance.

Artesunate versus artemether for the treatment of recrudescent multidrug-resistant falciparum malaria.

The therapeutic efficacy and toxicity of artesunate (2mg/kg/day for five days, then 1 mg/kg/day for two days: total=12 mg/kg) was compared with that of artemether (4 mg/kg followed by 2 mg/kg/day for two days, then 1 mg/kg/day for four days: total=12 mg/kg) for the treatment of recrudescent multidrug-resistant falciparum malaria in an open randomized trial in 443 patients living on the western border of Thailand. Parasite and fever clearance times were similar in both groups; within 48 hr 94% (95% confidence interval [CI]=91-96%]) of the treated patients were aparasitemic and 93% (95% CI=89-96%) were afebrile. Symptom resolution and resolution of hepatomegaly were slightly slower in the artesunate group; adjusted hazards ratio=1.5 (95% CI=1-2.0, P < 0.01) and 2.2 (95% CI=1.4-8, P=0.04), respectively. There was no significant difference in times to resolution or development of anemia or splenomegaly between treatment groups. By day 28, 3% (95% CI=0.3-5%) of the patients treated with artesunate and 6% of those treated with artemether (95% CI = 2-9%) had recurrent infections (P=0.3). Both regimens were very well tolerated, with no significant adverse effects attributable to either derivative. Overall, these data suggest that the two oral artemisinin derivatives are safe, highly effective, and result in equivalent therapeutic responses in the treatment of drug-resistant falciparum malaria.

Efficacy of six doses of artemether-lumefantrine (benflumetol) in multidrug-resistant Plasmodium falciparum malaria.

The new oral fixed combination artemether-lumefantrine (CGP 56697) has proved to be an effective and well-tolerated treatment of multi-drug resistant Plasmodium falciparum malaria, although cure rates using the four-dose regimen have been lower than with the currently recommended alternative of artesunate-mefloquine. Two six-dose schedules (total adult dose = 480 mg of artemether and 2,880 mg of lumefantrine) were therefore compared with the previously used four-dose regimen (320 mg of artemether and 1,920 mg of lumefantrine) in a double-blind trial involving 359 patients with uncomplicated multidrug-resistant falciparum malaria. There were no differences between the three treatment groups in parasite and fever clearance times, and reported adverse effects. The two six-dose regimens gave adjusted 28-day cure rates of 96.9% and 99.12%, respectively, compared with 83.3% for the four-dose regimen (P < 0.001). These six-dose regimens of artemether-lumefantrine provide a highly effective and very well-tolerated treatment for multidrug-resistant falciparum malaria.

Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand.

Parasite genotyping by the polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in a Karen population resident on the northwestern border of Thailand where malaria transmission is low (one infection/person/year). Plasmodium falciparum infections were genotyped for allelic variation in three polymorphic antigen loci, merozoite surface proteins-1 and -2 (MSP-1 and -2) and glutamaterich protein (GLURP), before and after antimalarial drug treatment. Population genotype frequencies were measured to provide the baseline information to calculate the probability of a new infection with a different or the same genotype to the initial pretreatment isolate. Overall, 38% of the infections detected following treatment had an identical genotype before and up to 121 days after treatment. These post-treatment genotypes were considered recrudescent because of the low (< 5%) probability of repeated occurrence by chance in the same patient. This approach allows studies of antimalarial drug treatment to be conducted in areas of low transmission since recrudescences can be distinguished confidently from newly acquired infections.

Optimization of a hydrophobic solid-phase extraction interface for matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) probe surfaces derivatized with octadecanethiol (C18) can be used as hydrophobic solid-phase extraction devices to isolate and desalt biopolymers directly on the probe surface. Using quantitative MALDI, it was possible to determine the approximate amount of peptide that bound to C18 surfaces and thus to calculate a surface density. It was determined that the amount of peptide bound at the probe surface was independent of the analyte concentration in the immersion solution (from high- to sub-ng ml-1 concentrations), but rather was dependent on the immersion time of the surface as it was exposed to the analyte. The capacity of C18-derivatized probes to bind biopolymers in fixed amounts frees the analyst from the necessity for adjusting analyte concentration through multiple step procedures such as serial dilution or vacuum drying. This time savings result in an overall increase in the efficiency of the MALDI technique.

Artesunate versus artemether in combination with mefloquine for the treatment of multidrug-resistant falciparum malaria.

To compare the therapeutic efficacy of oral artesunate and artemether in combination with mefloquine for the treatment of multidrug resistant malaria, a trial was conducted in 540 adults and children on the Thai-Myanmar border. Three regimens were compared: artesunate (4 mg/kg/d for 3 d), artemether (4 mg/kg/d for 3 d), both in combination with mefloquine (25 mg/kg), and a single dose of mefloquine (25 mg/kg). The artesunate and artemether regimens gave very similar clinical and parasitological responses, and were both very well tolerated. There was no significant adverse effect attributable to the artemisinin derivatives. Fever and parasite clearance times with mefloquine alone were significantly longer (P < 0.001). After adjusting for reinfections the failure rates were 13.9% for the artesunate combination, 12.3% for the artemether combination and 49.2% for mefloquine alone (P < 0.0001; relative risk 3.8 [95% confidence interval 2.6-5.4]). Mefloquine should no longer be used alone for the treatment of multidrug resistant falciparum malaria in this area. Three-day combination regimens with artesunate or artemether are well tolerated and more effective.

Randomized comparison of mefloquine-artesunate versus quinine in the treatment of multidrug-resistant falciparum malaria in pregnancy.

Since no effective malaria prevention measures have been identified for pregnant women living on the western border of Thailand, prompt diagnosis and efficient treatment are paramount, although drug resistance in Plasmodium falciparum has narrowed the treatment options. An open randomized comparison of supervised quinine (10 mg salt/kg every 8 h) for 7 days (Q7) versus mefloquine 25 mg base/kg (total dose) plus artesunate 4 mg/kg per day for 3 days (MAS3) was conducted in 1995-97 in 108 Karen women with acute uncomplicated falciparum malaria in the second or third trimesters of pregnancy. The MAS3 regimen was more effective than the Q7 regimen: day 63 cure rates were 98.2% (95% CI 94.7-100) (n = 65) for MAS3 and 67.0% (95% CI 43x3-90x8) (n = 41) for Q7, P = 0x001. The MAS3 regimen was also associated with less gametocyte carriage; the average person-gametocyte-weeks for MAS3 was 2.3 (95% CI 0-11) and for Q7 was 46x9 (95% CI 26-78) per 1000 person-weeks, respectively (P < 0.001). MAS3 was significantly better tolerated. These evident advantages must be balanced against a possible increased risk of stillbirth with the use of mefloquine in pregnancy. Further randomized studies assessing the safety and efficacy of other artemisinin-containing combination regimens in pregnancy are needed urgently.

Artemether-lumefantrine for the treatment of multidrug-resistant falciparum malaria.

The efficacy and safety of the 6-dose regimen of artemether-lumefantrine were assessed in an open randomized trial in children and adults presenting with acute, uncomplicated Plasmodium falciparum malaria in Thailand between November 1997 and March 1998. 200 patients were enrolled in 2 centres: 150 received artemether-lumefantrine (i.e., a median total dose of 9.6 mg/kg [interquartile range 8.7-10.7] and 57.9 mg/kg of lumefantrine [52.4-64.0]) and 50 the standard combination of artesunate (12 mg/kg over 3 d) and mefloquine (25 mg/kg). All patients had rapid initial clinical and parasitological responses. The 28 d cure rates were high: 97.7% (95% confidence interval [95% CI] 93.5-99.5%) for artemether-lumefantrine and 100% (95% CI 92.5-100%) for artesunate-mefloquine. The 6-dose regimen of artemether-lumefantrine was better tolerated than, and as effective as, artesunate-mefloquine, the current standard treatment in this area of multidrug-resistant P. falciparum malaria.

Plasmodium falciparum antimalarial drug susceptibility on the north-western border of Thailand during five years of extensive use of artesunate-mefloquine.

Following a marked decline in the efficacy in vivo of mefloquine between 1990 and 1994, a combination of artesunate (4 mg/kg/d for 3 d) and mefloquine (25 mg/kg) has been used as first line treatment of uncomplicated falciparum malaria in camps for displaced persons located along the north-western border of Thailand. Antimalarial drug susceptibility of fresh isolates of Plasmodium falciparum from this population was evaluated using a radioisotope microdilution assay between 1995 and 1999. In total, 268 isolates were collected, of which 189 were from primary infections and 79 from recrudescent infections. The geometric mean 50% inhibitory concentration (IC50) values from primary infections were: dihydroartemisinin 1.2 ng/mL, artesunate 1.6 ng/mL, artemether 4.8 ng/mL, atovaquone 0.4 ng/mL, lumefantrine 32 ng/mL, chloroquine 149 ng/mL, quinine 354 ng/mL, mefloquine 27 ng/mL and halofantrine 4.1 ng/mL. A significant positive correlation was found between the susceptibility in vitro to artesunate and quinine (r = 0.43, P < 0.001), mefloquine (r = 0.46, P < 0.001), and halofantrine (r = 0.51, P < 0.001). These levels of resistance in vitro are among the highest reported and confirm continuing high level multidrug resistance in this area. Despite intensive use of the combination between 1995 and 1999 there has been a significant improvement in mefloquine sensitivity (P < 0.001) and artesunate sensitivity (P < 0.001). This supports observations in vivo that the combination of artesunate and mefloquine has reversed the previous decline in mefloquine sensitivity.

Genetic analysis of Plasmodium falciparum infections on the north-western border of Thailand.

Genetic characterization of Plasmodium falciparum infections in north-western Thailand, a region of low transmission intensity (1 infection/person each year), has found a comparable number of parasite genotypes per infected person to regions with hyperendemic malaria. Clone multiplicity and parasite diversity were found to be homogeneous across 129 infected individuals comprising a range of age-groups (1.32 parasite genotypes; n = 98), patients (aged 2-16 years) with recrudescent infections (1.54; n = 13), and pregnant women (1.61; n = 18). Individuals belonging to groups with a high risk of infection, as deduced by clinical epidemiology, did not harbour a higher number of clones per infection, nor greater parasite diversity than low-risk groups. In fact, multiple genotype infections were as common in low-risk groups, suggesting that there is frequent transmission of polyclonal infections from a single inoculum, rather than superinfection. Such a polyclonal transmission system would enable generation of extensive parasite diversity by recombination, despite the low level of transmission. However, co-infection with P. vivax was associated with fewer P. falciparum genotypes per infection.

Randomized comparison of quinine-clindamycin versus artesunate in the treatment of falciparum malaria in pregnancy.

In areas where multidrug-resistant Plasmodium falciparum (MDR-Pf) is prevalent, only quinine is known to be safe and effective in pregnant women. On the western border of Thailand, 7 days of supervised quinine (30 mg/kg daily) cures two-thirds of P. falciparum-infected women in the 2nd and 3rd trimesters of pregnancy. Artesunate is effective against MDR-Pf and the limited data on its use in pregnancy suggest it is safe. An open randomized comparison of supervised quinine (10 mg salt/kg every 8 h) in combination with clindamycin (5 mg/kg every 8 h) for 7 days (QC7) versus artesunate 2 mg/kg per day for 7 days (A7) was conducted in 1997-2000 in 129 Karen women with acute uncomplicated falciparum malaria in the 2nd or 3rd trimesters of pregnancy. There was no difference in the day-42 cure rates between the QC7 (n = 65) and A7 (n = 64) regimens with an efficacy of 100% in both, confirmed by parasite genotyping. The A7 regimen was also associated with less gametocyte carriage; the average person-gametocyte-weeks for A7 was 3 (95% CI 0-19) and for QC7 was 39 (95% CI 21-66) per 1000 person-weeks, respectively (P < 0.01). There was no difference in gastrointestinal symptoms between the groups but there was significantly more tinnitus in the QC7 group compared to the A7 group (44.9% vs 8.9%; RR 5.1; 95% CI 1.9-13.5; P < 0.001). The favourable results with quinine-clindamycin mean that there is a useful back-up treatment for women with falciparum malaria who experience quinine and artesunate failures in pregnancy. Adherence to the 7-day regimen and cost (US$18.50 per treatment) are likely to be the main obstacles to this regimen.

SPf66 malaria vaccine is safe and immunogenic in malaria naive adults in Thailand.

In preparation for an efficacy trial of malaria vaccine SPf66 in Thailand, a series of overlapping Phase I trials were conducted of US-manufactured SPf66. Here, two clinical lots were evaluated for safety and immunogenicity in a combined open-label trial. Eleven healthy, malaria naive, 18-44 year-old Thai men and women received three doses by subcutaneous injection in alternate arms at 0, 1 and 6 months. Safety was assessed by monitoring local and systemic reactogenicity and laboratory parameters. Common side effects were mild erythema, induration and tenderness at the site of injection which resolved within 24-48 h. At third immunization, two volunteers developed acute bilateral reactions with induration, erythema and pruritus limited to the sites of the second and third immunizations. Eight of 11 volunteers sero-converted by ELISA, six of whom would be classified as high responders by Colombian standards. Eight of 11 volunteers developed a lymphoproliferative response to the SPf66 antigen. Side effects were more common and antibody and lymphoproliferative responses greatest, among the four female volunteers. This initial study of SPf66 malaria vaccine in Asia constitutes an essential link between the initial Phase I study in the US and subsequent field studies in a semi-immune population in a malaria endemic area of Thailand. This study further establishes comparability of US-manufactured SPf66 with that of Colombian provenance and substantiates the validity of the subsequent negative efficacy results of SPf66 in a field trial in Thailand.