[Miltefosine: a new remedy for leishmaniasis]. / Miltefosine: een nieuw geneesmiddel voor leishmaniasis.

There is a need for a safe and effective oral treatment for cutaneous and visceral leishmaniasis. Miltefosine is the first oral drug that is efficacious against different forms ofleishmaniasis, however it is not equally effective against all Leishmania species. Miltefosine is an alkylphosphocholine, originally developed for the treatment of cancer. The mechanism of action is probably based on interference with the synthesis and degradation of parasitic membrane lipids. Little is known about the pharmacokinetics ofmiltefosine; an important characteristic is its long elimination half-life of seven days or longer. The most frequent adverse effects are of gastrointestinal origin. Miltefosine should not be used during pregnancy. Over thirty leishmaniasis patients have already been treated with miltefosine in the Netherlands.

Chloroquine-treatment failure in northern Ghana: roles of pfcrt T76 and pfmdr1 Y86.

Although chloroquine (CQ) monotherapy is now generally inadequate for the treatment of Plasmodium falciparum malaria in northern Ghana--recently, 58% of 225 children failed treatment by day 14--use of the drug continues because of its low cost and wide availability. The risk factors associated with CQ-treatment failure in this region of Africa, including the T76 mutation in the chloroquine resistance transporter (pfcrt) gene and the Y86 mutation in the multidrug resistance (pfmdr1) gene of P. falciparum, have now been investigated, and genotype-failure indices (GFI) have been calculated. Treatment failure was found to be associated with young age, poor nutritional status, pfcrt T76 and pfmdr1 Y86, and early treatment failure (ETF) was also associated with high parasitaemia. The presence and concentration of 'residual' CQ in the blood of patients immediately before they were treated with CQ for the present study appeared to have no effect on outcome. Presence at recruitment of pfcrt T76 or pfmdr1 Y86 or both mutations increased the risk of treatment failure by 3.2-, 2.4- and 4.5-fold, and the risk of ETF by 9.8-, 2.7- and 10.2-fold, respectively. The pfcrt T76 GFI for clinical and all treatment failures were 2.8 and 1.4, respectively. These indices were relatively low in the younger children, those with malnutrition, and those with high parasitaemias when treated. Residual CQ did not affect the GFI substantially. Both pfcrt T76 and, to a lesser extent, pfmdr1 Y86 would be useful tools for the surveillance of CQ resistance in northern Ghana. In the current transition phase to alternative first-line treatment for P. falciparum malaria, it should be possible to provide estimates of the level of CQ resistance by monitoring the prevalences of these mutations.

Multiplicity of Plasmodium falciparum infection in pregnancy.

Little is known about the distribution and disease association of multiple Plasmodium falciparum infections in pregnant women. Genotyping of the merozoite surface protein-1 region was performed in 332 P. falciparum infected pregnant women in Ghana, and clinical and epidemiologic data were obtained. Overall, 68% of the women were infected with more than one strain (mean number of strains per carrier = 2.9). The multiplicity of infection decreased significantly with an increasing number of pregnancies, and infection with multiple P. falciparum strains was significantly associated with anemia. In logistic regression, women infected with four or more strains were 2.3 times more likely to be anemic than women harboring fewer strains. This association, however, was only observed in women with up to three pregnancies. The results suggest that with increasing gravidity and subsequent infections with multiple strains effective immune mechanisms against more and more strains develop. In pregnant women, the multiplicity of infection may be an important factor for the acquisition and maintenance of immunity against malaria.

Plasmodium falciparum pfcrt and pfmdr1 polymorphisms are associated with the pfdhfr N108 pyrimethamine-resistance mutation in isolates from Ghana.

The Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 and multidrug resistance gene analogue (pfmdr1) Y86 mutations are associated with chloroquine(CQ)-resistance. In isolates from 172 pregnant women living in the area of Agogo, Ghana, pfcrt T76 was detected in 69% and pfmdr1 Y86 in 66%. Pfcrt T76 but not pfmdr1 Y86 was more prevalent in samples from women with residual CQ in urine or serum. Parasite densities and multiplicity of infection of pfmdr wild type but not of resistant isolates were reduced by CQ. Adjusted for CQ and pyrimethamine (PYR) in urine, the P. falciparum dihydrofolate reductase (pfdhfr) N108 mutation which confers PYR-resistance was 3.1 and 3 times, respectively, more likely to be detected in isolates containing pfcrt and pfmdr1 mutations than in those comprising wild type alleles. Pfcrt, pfmdr, and pfdhfr mutations are frequent in P. falciparum from this part of Ghana which may limit the choice of drugs for the prevention of malaria in pregnancy. The association of CQ- and PYR-resistance mutations independent of recent drug use could indicate accelerated development of resistance to structurally unrelated drugs. Alternatively, it may reflect selection of resistance in persisting infections due to no longer detectable drug pressure.

Plasmodium falciparum dihydrofolate reductase alleles and pyrimethamine use in pregnant Ghanaian women.

Drug resistance in Plasmodium falciparum affects prevention of malaria in pregnancy. In a cross-sectional study of 530 pregnant Ghanaian women, P. falciparum dihydrofolate reductase (DHFR) gene mutations linked with pyrimethamine resistance were assessed and associations with pyrimethamine intake were analyzed. P. falciparum infected 69% of women without pyrimethamine use, 59% of those who had a history of pyrimethamine consumption but a negative urine test, and 53% of individuals with a positive urine test. Eighty-one percent, 43%, and 74% of the isolates contained the mutations Asn-108, Ile-51, and Arg-59, respectively. Thr-108 occurred in 8%. Pyrimethamine use was associated with increased frequencies of Asn-108 and Arg-59 but not of Ile-51 or Thr-108. In women with prophylaxis, wild-type parasites were absent and anemia tended to be more common with an increasing number of DHFR gene mutations. Pyrimethamine appears to be not adequately effective in this part of Ghana, most likely due to the predominance of resistant parasites. Selection for resistance following insufficient prophylaxis could possibly affect the efficacy of future intermittent sulfadoxine-pyrimethamine treatment.

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.

Submicroscopic Plasmodium falciparum infections in pregnancy in Ghana.

Malarial parasitaemia below the threshold of microscopy but detectable by polymerase chain reaction (PCR) assays is common in endemic regions. This study was conducted to examine prevalence, predictors, and effects of submicroscopic Plasmodium falciparum infections in pregnancy. In a cross-sectional study among 530 pregnant women in Ghana, plasmodial infections were assessed by microscopy and PCR assays. Concentrations of haemoglobin and C-reactive protein (CRP) were measured and antimalarial drugs (chloroquine, pyrimethamine) in urine were demonstrated by ELISA dipsticks. By microscopy, 32% of the women were found to harbour malaria parasites. This rate increased to 63% adding the results of the parasite-specific PCR. P. falciparum was present in all but one infection. With increasing gravidity, infection rates and parasite densities decreased and the proportions of submicroscopic parasitaemia among infected women grew. Correspondingly, anaemia, fever and evidence of inflammation (CRP > 0.6 mg/dl) were more frequent in primigravidae than in multigravidae. Antimalarial drugs were detected in 65% of the women and were associated with a reduced prevalence of P. falciparum infections and a raised proportion of submicroscopic parasitaemia. Both gravidity and antimalarial drug use were independent predictors of submicroscopic P. falciparum infections. These infections caused a slight reduction of Hb levels and considerably increased serum concentrations of CRP. Conventional microscopy underestimates the actual extent of malarial infections in pregnancy in endemic regions. Submicroscopic P. falciparum infections are frequent and may contribute to mild anaemia and inflammation in seemingly aparasitaemic pregnant women.

Anti-malarial drug use among preschool children in an area of seasonal malaria transmission in Kenya.

The aims of this study were to estimate the proportion of asymptomatic Kenyan preschool children using anti-malarial drugs, to identify factors associated with chloroquine use, and to assess the validity of frequency of febrile episodes and drug use reported by mothers or carers. Of 318 children studied, 38% (95% confidence interval [CI] = 30-47%]) tested positive for chloroquine or sulfadoxine. Of chloroquine-positive children, 15% had concentrations exceeding the estimated minimum therapeutically effective values. Among those testing negative for sulfadoxine, chloroquine-positive children were more frequently parasitemic (odds ratio = 2.6, 95% CI = 1.3-5.2), and had lower mean hemoglobin concentrations (6.1 g/L, 95% CI = 2.1-10.1) than chloroquine-negative children. Mothers over-reported the frequency of malaria or fever episodes as usually defined in medical studies, and underreported anti-malarial drug use. We conclude that anti-malarials are frequently given for treatment of malaria or malaria-associated illness, rather than prophylactically or for symptoms unrelated to malaria. Questionnaire surveys cannot replace biochemical markers to obtain information on anti-malarial drug use.

Short report: high prevalence and imbalanced age distribution of the Plasmodium falciparum dihydrofolate reductase gene Asn108 mutation in an area of low pyrimethamine usage in Nigeria.

Resistance of Plasmodium falciparum to pyrimethamine is associated with a non-silent point mutation of the parasite dihydrofolate reductase (DHFR) gene (Ser108 --> Asn108). Wide-scale use of antimalarials is thought to contribute to the emergence of drug resistance. In 131 P. falciparum-infected children in rural Nigeria, the frequency of the resistant Asn108 genotype was assessed by enzymatic restriction digestion of polymerase chain reaction-amplified DHFR sequences and compared with residual pyrimethamine blood levels. The prevalence of the Asn108 variant was 41.2%. In 18.3% of the isolates, both the Asn108 and the wild-type alleles were present. In contrast to the high prevalence of resistant genotypes, residual pyrimethamine blood levels were detected in only 4%. Furthermore, age was found to be a determinant of the parasite genotype since the proportion of Asn108 variants decreased with age (P < 0.05). These findings indicate that additional, unidentified factors, rather than selection by residual drug levels alone, might be responsible for the emergence of pyrimethamine-resistant parasite genotypes.

The development of an immunoassay for the detection of artemisinin compounds in urine.

We have produced monoclonal antibodies against artelinic acid and investigated the reactivity with artemisinin drugs and metabolites. Antibody F170-10 is fairly specific for artelinic acid but does bind artemisinin and artemether (3-5% cross-reactivity). Dihydroartemisinin, artesunate, and metabolites of artemisinin showed less reactivity. With this antibody, an inhibition ELISA has been set up to detect artemisinin compounds in urine. In healthy subjects who received a single oral dose of artemisinin, artemether, artesunate or dihydroartemisinin, ELISA reactivity in urine was found. This reactivity in urine paralleled the plasma concentrations of artemether and dihydroartemisinin. The results show that this immunoassay for artelinic acid can be used to detect artemisinin compounds in urine for about 8 hr after intake. With a more sensitive test, this simple method as a urine dipstick may be become useful for drug use and compliance studies in malaria-endemic areas where the artemisinin derivatives are increasingly used.

Urocanic acid isomers are good hydroxyl radical scavengers: a comparative study with structural analogues and with uric acid.

UV-exposure of the epidermis leads to the isomerisation of trans-UCA into cis-UCA as well as to the generation of hydroxyl radicals. This study shows by means of the deoxyribose degradation test that UCA isomers are more powerful hydroxyl radical scavengers than the other 4-(5-)substituted imidazole derivatives, such as histidine, though less powerful than uric acid. UCA, present in relatively high concentrations in the epidermis, may well be a major natural hydroxyl radical scavenger.

Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication.

Registration in Europe of several artemisinin drugs for the treatment of malaria can soon be expected. Artemisinin is isolated from the herb Artemisia annua, in use in China more than 2000 years as a herbal tea against fever. Artemisinin drugs are being used extensively in South-East Asia and increasingly in Africa. Active derivatives have been synthesized - artemether, arteether and artesunate - which are used for oral, intramuscular, rectal and intravenous administration. The origin, mechanism of action, efficacy and safety in patients, the pharmacokinetics and the position of this group of compounds among existing antimalarials are discussed in this review.

Sensitive ELISA dipstick test for the detection of chloroquine in urine under field conditions.

OBJECTIVE: To evaluate a new enzyme-linked immunosorbent assay (ELISA) dipstick test for detecting chloroquine (CQ) in urine in a malaria-endemic region of north-western Namibia. METHOD: Urine samples from 92 patients attending the outpatient department of Kamhaku Hospital with suspected malaria infection were tested for CQ with both the Dill-Glazko test and the ELISA dipstick test. Results were compared to the history of CQ intake as documented in the patients' health passes. RESULTS: The dipstick test proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 120 nmol/l. It showed high agreement with the history of CQ intake within the last 6 months. The specificity in a negative control group was 100%. The Dill-Glazko test was far less sensitive and specific with a lower detection limit of 150 micromol/l. CONCLUSION: The dipstick test can be used in pharmacological studies to evaluate the use of CQ, and as an inclusion criterion for in vivo and in vitro sensitivity tests, whereas the Dill-Glazko test is appropriate to test compliance during and a few days after CQ intake.

Decreased antitoxic activities among children with clinical episodes of malaria.

Healthy Gambian children, children with clinical Plasmodium falciparum malaria, and children with asymptomatic P. falciparum infections were studied to investigate whether antitoxic activities may contribute to protection against malarial symptoms. Markers of inflammatory reactions, soluble tumor necrosis factor receptor I, and C-reactive protein were found in high concentrations in children with symptomatic P. falciparum malaria compared with levels in children with asymptomatic P. falciparum infections or in healthy children, indicating that inflammatory reactions are induced only in children with clinical symptoms. Concentrations of soluble tumor necrosis factor receptor I and C-reactive protein were associated with levels of parasitemia. We detected antitoxic activities in sera as measured by their capacity to block toxin-induced Limulus amoebocyte lysate (LAL) activation. Symptomatic children had decreased capacity to block induction of LAL activation by P. falciparum exoantigen. The decreased blocking activity was restored in the following dry season, when the children had no clinical malaria. Symptomatic children also had the highest immunoglobulin G (IgG) reactivities to conserved P. falciparum erythrocyte membrane protein 1 and "Pfalhesin" (band #3) peptides, indicating that such IgG antibodies are stimulated by acute disease but are lost rapidly after the disease episode. Half of the children with symptomatic infections had low levels of haptoglobin, suggesting that these children had chronic P. falciparum infections which may have caused symptoms previously. Only a few of the children with asymptomatic P. falciparum infections had high parasite counts, and antitoxic immunity in the absence of antiparasite immunity appears to be rare among children in this community.

Efficacy and tolerance of artemisinin in short combination regimens for the treatment of uncomplicated falciparum malaria.

Two oral regimens comprising a single dose of 20 mg/kg of artemisinin followed by three days of quinine, 10 mg/kg three times a day (AQ), or doxycycline, 4 mg/kg once a day (AD), were compared with a standard seven-day course of oral quinine, 10 mg/kg three times a day (Q), in the treatment of uncomplicated falciparum malaria. Of 161 treated patients, 157 could be included in the analysis. The mean +/- SD parasite clearance time was 43 +/- 14 hr for AQ and 41 +/- 19 hr for AD, and significantly longer for quinine: 66 +/- 24 hr (P = 0.0001). Treatment failure occurred in one Q and in 3 AD patients. The recrudescence rate was 16% for Q, 28% for AQ, and significantly worse for AD: 67% (P = 0.0001). Adverse effects were mainly limited to cinchonism. The conclusion is that a seven-day course of quinine is still effective in the initial treatment of uncomplicated falciparum malaria in Vietnam, but one should pay attention to possible recrudescence. The addition of a single 20 mg/kg per os dose of artemisinin allows for shortening the duration of treatment, with faster parasite clearance, comparable efficacy, and better tolerance, but with no reduction of recrudescence. The combination of artemisinin with three days of doxycycline is also not effective in preventing recrudescence.

Response of childhood malaria to chloroquine and Fansidar in an area of intermediate chloroquine resistance in Côte d'Ivoire.

The parasitological, clinical and haematological responses to chloroquine treatment were studied in children during a 28-day follow-up in an area of Côte d'Ivoire with intermediate chloroquine resistance. The parasitological, clinical and haematological responses to Fansidar were also investigated in patients who returned to the health centre within 28 days with symptoms of malaria. Of 82 children aged 0-9 years who completed the study, only 67% were parasite-negative on thick blood film on day 7, which decreased to 21% by day 28. While chloroquine treatment still produced clinical remission at day 7 in 95% of the children, 35% had recurrent fever with concomitant parasitaemia before day 28. All fever cases subsequently treated with Fansidar remained parasite-negative over a period of 28 days. On day 28 the haematocrit levels were higher in those children who responded successfully to treatment with either chloroquine or Fansidar than in the children who were still parasite-positive but without fever (two-tailed t-test, P = 0.02). The rate of resistance to chloroquine was most pronounced among the younger children (< 5 years), 18% of whom showed clinical failure by day 14. This study underlines the importance of monitoring the durability of response to chloroquine treatment for at least 14 days in young children in Côte d'Ivoire.

In situ localization of chloroquine and immunohistological studies in UVB-irradiated skin of photosensitive patients.

Chloroquine can prevent photosensitivity reactions, but its mechanism of action is poorly understood. To investigate if the drug may interfere with inflammatory or immunological mechanisms of the UV-induced erythema of photosensitive patients, we studied the localization of chloroquine in the skin and its effect on the epidermal/dermal expression of IL-1, TNF-alpha, IL-6 and ICAM-1 and the occurrence of different lymphoid cells in normal skin and UVB-induced erythema in 8 patients with photosensitive discoid and systemic lupus erythematosus and 4 patients with polymorphic light eruption (PMLE), before and during chloroquine treatment. Using a specific monoclonal antibody against chloroquine, we found a strong granular staining pattern of mainly keratinocytes in all biopsy specimens from normal and erythematous skin during chloroquine treatment. In non-irradiated skin, T lymphocytes, macrophages and HLA-DR expressing cells were sparsely distributed within the dermis in similar amounts before and during chloroquine treatment. In UVB-induced erythema an increase in the number of these cells, mainly located in the dermal perivascular area, was seen before medication. During chloroquine treatment such cellular infiltration was reduced. ICAM-1 expression was detected on the endothelium of dermal vessels but not on keratinocytes. The accumulation of chloroquine in the epidermis and the decreased cellular infiltration in erythematous skin during chloroquine treatment indicate a local anti-inflammatory effect. This effect may be due to either unspecific UV-protective properties of the drug or to some specific downregulating action by chloroquine on keratinocyte function.

Investigation of chloroquine intake in Biombo region, Guinea Bissau.

The use of chloroquine (CQ), during the dry season was determined for 236 patients from 5 villages in Biombo, Republic of Guinea Bissau, West Africa. The antimalarial drug was measured in whole blood (dried samples) using High Performance Liquid Chromatography (HPLC) and in urine samples by Enzyme Linked Immunosorbent Assay (ELISA). The results showed that CQ consumption is low.