Efficacy and safety of methylene blue in the treatment of malaria: a systematic review.

BACKGROUND: Methylene blue (MB) was the first synthetic antimalarial to be discovered and was used during the late 19th and early 20th centuries against all types of malaria. MB has been shown to be effective in inhibiting Plasmodium falciparum in culture, in the mouse model and in rhesus monkeys. MB was also shown to have a potent ex vivo activity against drug-resistant isolates of P. falciparum and P. vivax. In preclinical studies, MB acted synergistically with artemisinin derivates and demonstrated a strong effect on gametocyte reduction in P. falciparum. MB has, thus, been considered a potentially useful partner drug for artemisinin-based combination therapy (ACT), particularly when elimination is the final goal. The aim of this study was to review the scientific literature published until early 2017 to summarise existing knowledge on the efficacy and safety of MB in the treatment of malaria. METHODS: This systematic review followed PRISMA guidelines. Studies reporting on the efficacy and safety of MB were systematically searched for in relevant electronic databases according to a pre-designed search strategy. The search (without language restrictions) was limited to studies of humans published until February 2017. RESULTS: Out of 474 studies retrieved, a total of 22 articles reporting on 21 studies were eligible for analysis. The 21 included studies that reported data on 1504 malaria patients (2/3 were children). Older studies were case series and reports on MB monotherapy while recent studies were mainly controlled trials of combination regimens. MB was consistently shown to be highly effective in all endemic areas and demonstrated a strong effect on P. falciparum gametocyte reduction and synergy with ACT. MB treatment was associated with mild urogenital and gastrointestinal symptoms as well as blue coloration of urine. In G6PD-deficient African individuals, MB caused a slight but clinically non-significant haemoglobin reduction. CONCLUSIONS: More studies are needed to define the effects of MB in P. falciparum malaria in areas outside Africa and against P. vivax malaria. Adding MB to ACT could be a valuable approach for the prevention of resistance development and for transmission reduction in control and elimination programs. SYSTEMATIC REVIEW REGISTRATION: This study is registered at PROSPERO (registration number CRD42017062349 ).

Predominance of dfrG as determinant of trimethoprim resistance in imported Staphylococcus aureus.

To investigate the global occurrence of trimethoprim-sulfamethoxazole resistance and the genetic mechanisms of trimethoprim resistance, we analysed Staphylococcus aureus from travel-associated skin and soft-tissue infections treated at 13 travel clinics in Europe. Thirty-eight per cent (75/196) were trimethoprim-resistant and 21% (41/196) were resistant to trimethoprim-sulfamethoxazole. Among methicillin-resistant S. aureus, these proportions were 30% (7/23) and 17% (4/23), respectively. DfrG explained 92% (69/75) of all trimethoprim resistance in S. aureus. Travel to South Asia was associated with the highest risk of acquiring trimethoprim-sulfamethoxazole-resistant S. aureus. We conclude that globally dfrG is the predominant determinant of trimethoprim resistance in human S. aureus infection.

Skin and soft tissue infections in intercontinental travellers and the import of multi-resistant Staphylococcus aureus to Europe.

Staphylococcus aureus is emerging globally. Treatment of infections is complicated by increasing antibiotic resistance. We collected clinical data and swabs of returnees with skin and soft tissue infections (SSTI) at 13 travel-clinics in Europe (www.staphtrav.eu). Sixty-two percent (196/318) SSTI patients had S. aureus-positive lesions, of which almost two-thirds (122/196) were Panton-Valentine leukocidin (PVL) positive. PVL was associated with disease severity, including hospitalization for SSTI (OR 5.2, 95% CI 1.5-18.2). In returnees with SSTI, longer travel and more intense population contact were risk factors for nasal colonization with PVL-positive S. aureus. Imported S. aureus frequently proved resistant to trimethoprim-sulfamethoxazole (21%), erythromycin (21%), tetracycline (20%), ciprofloxacin (13%), methicillin (12%) and clindamycin (8%). Place of exposure was significantly (p < 0.05) associated with predominant resistance phenotypes and spa genotypes: Latin America (methicillin; t008/CC24/304), Africa (tetracycline, trimethoprim-sulfamethoxazole; t084/CC84, t314/singleton, t355/CC355), South Asia (trimethoprim-sulfamethoxazole, ciprofloxacin; t021/CC21/318), South-East Asia (clindamycin; t159/CC272). USA300-like isolates accounted for 30% of all methicillin-resistant S. aureus imported to Europe and were predominantly (71%) acquired in Latin America. Multi-resistance to non-ß-lactams were present in 24% of imports and associated with travel to South Asia (ORcrude 5.3, 95% CI 2.4-11.8), even after adjusting for confounding by genotype (ORadjusted 3.8, 95% 1.5-9.5). Choosing randomly from compounds recommended for the empiric treatment of severe S. aureus SSTI, 15% of cases would have received ineffective antimicrobial therapy. These findings call for the development of regionally stratified guidance on the antibiotic management of severe imported S. aureus disease and put the infected and colonized traveller at the centre of interventions against the global spread of multi-resistant S. aureus.

Detection of Giardia duodenalis assemblage A and B isolates by immunochromatography in stool samples from Rwandan children.

We evaluated the performance of an immunochromatographic assay (ICA) in comparison with light microscopy and PCR for the detection of Giardia duodenalis in stool samples from 558 Rwandan children. The association of infection with clinical symptoms was similar for the three diagnostic tools. The ICA equally detected parasites of assemblages A and B and was more sensitive than light microscopy (50.4 versus 29.5% of PCR-positive samples considered true positive; p <0.0001). Hence, the ICA shows superior sensitivity compared with microscopy but still misses half of the G. duodenalis infections detected by PCR in this hyperendemic area.

Immunization of liver and renal transplant recipients: a seroepidemiological and sociodemographic survey.

Several life-threatening infections, a major risk to adult solid organ transplant (SOT) recipients on immunosuppressive therapy, can be prevented by immunization. We analyzed sociodemographic parameters and the immunization status of adult liver transplant recipients (LTX-R, n=267) and renal transplant recipients (RTX-R, n=197) SOT recipients at the Transplantation Center, Berlin, Germany. Date, number, and provider of recommended vaccines were recorded and seroprotection rates determined. The social status in both groups was similar. Most patients (89%) were not adequately informed about immunizations; and if informed, main sources were physicians (47%) and the media (40%). Vaccinations were predominantly provided by family doctors (LTX-R, 66%; RTX-R, 31%) or hemodialysis centers (RTX-R, 37%). Before transplantation, RTX-R had significantly more often received booster vaccinations against tetanus and diphtheria (P<0.005), and a primary hepatitis B immunization (55%); whereas in LTX-R, post-transplant vaccinations against hepatitis A (16%) and pneumococcal disease (13%) were more frequent. Seroprotection rates against tetanus were fairly high in LTX-R (85.3%) and RTX-R (86.8%), and considerably lower for diphtheria, hepatitis A, and influenza. Immunization rates are too low in SOT recipients. Improvement will depend on a more active role of health care providers.

Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria.

Genetic host factors play a substantial role in susceptibility to and severity of malaria, which continues to cause at least one million deaths per year. Recently, members of the toll-like receptor (TLR) family have been shown to be involved in recognition of the etiologic organism Plasmodium falciparum: The glycosylphosphatidylinisitol anchor induces signaling in host cells via TLR-2 and -4, while hemozoin-induced immune activation involves TLR-9. Binding of microbial ligands to the respective TLRs triggers the release of pro-inflammatory cytokines via the TLR/IL-1 receptor (TIR) domain and may contribute to the host response, including pro-inflammatory cytokine induction and malarial fever. In a case-control study among 870 Ghanaian children, we examined the influence of TLR-2, -4, and -9 polymorphisms in susceptibility to severe malaria. TLR-2 variants common in Caucasians and Asians were completely absent. However, we found a new, rare mutation (Leu658Pro), which impairs signaling via TLR-2. We failed to detect any polymorphisms within the TLR-9/interleukin-1 receptor domain. Two frequent TLR-9 promoter polymorphisms did not show a clear association with malaria severity. In contrast, the TLR-4-Asp299Gly variant occurred at a high rate of 17.6% in healthy controls, and was even more frequent in severe malaria patients (24.1%, p<0.05). Likewise, TLR-4-Thr399Ile was seen in 2.4% of healthy children and in 6.2% of patients (p=0.02). TLR-4-Asp299Gly and TLR-4-Thr399Ile conferred an 1.5- and 2.6-fold increased risk of severe malaria, respectively. These findings suggest TLR4-mediated responses to malaria in vivo and TLR-4 polymorphisms to be associated with disease manifestation. However some gray areas also suggest the scope for further improvements.

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.

Renal dysfunction in children with uncomplicated, Plasmodium falciparum malaria in Tamale, Ghana.

In a study performed in Tamale, in the Northern region of Ghana, cystatin C, a new and sensitive indicator of the glomerular filtration rate (GFR), was used to estimate the frequency of renal dysfunction in 78 children with uncomplicated, Plasmodium falciparum malaria. The excretion in urine of albumin, immunoglobulin G and alpha1-microglobulin was also investigated. Plasma concentrations of cystatin C were found to be elevated in 17% of the children, indicating subclinical impairment of renal function. As most (85%) of the children had glomerular as well as tubular patterns of proteinuria, it appears that both glomerulonephritis and damage to tubular cells often occur in P. falciparum malaria.

Efficacy of chloroquine in the treatment of uncomplicated, Plasmodium falciparum malaria in northern Ghana.

Chloroquine (CQ) resistance in Plasmodium falciparum contributes to growing malaria-attributable morbidity and mortality in sub-Saharan Africa. However, the extent and degree of such resistance vary considerably between endemic areas. Data on CQ resistance in northern Ghana are almost entirely lacking. The therapeutic efficacy of CQ in uncomplicated malaria was therefore assessed, in a standard, 14-day protocol, in 225 children aged <5 years in Tamale, in the Northern region of Ghana. Early treatment failure (ETF) was observed in 11% of the children and late treatment failure in 18%. High initial parasite density and young age were independent predictors for ETF. Resistant parasitological responses (RI-RIII) were seen in 57% of the cases that could be classified. More than half of these responses occurred in children fulfilling the criteria for adequate clinical response (ACR), indicating a considerable lack of agreement between parasitological and clinical outcome. During the follow-up period, haemoglobin levels increased by approximately 1g/dl not only in patients with ACR but also in those who experienced clinical failure more than 1 week post-treatment. As CQ-treatment failure occurred in >25% of the children and more than half of the parasitological responses indicated resistance, current recommendations for the treatment of uncomplicated malaria in young children in northern Ghana have to be reconsidered.

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.

Short report: increased susceptibility to Plasmodium malariae in pregnant alpha(+)-thalassemic women.

The influence of alpha(+)-thalassemia on malaria in pregnancy was assessed in a cross-sectional study of 530 women in Ghana. Plasmodial infections, alpha(+)-thalassemia, serum levels of C-reactive protein, and antimalarial drugs in urine were determined. The alpha-globin genotypes did not correlate with the prevalence of Plasmodium falciparum-infection and parasite densities. However, Plasmodium malariae tended to be more frequent in alpha(+)-thalassemic women (P = 0.05). Excluding women with residual antimalarials, a significant excess of P. malariae was observed in alpha(+)-thalassemic individuals. Febrile responses (P = 0.05) and inflammation (CRP > 0.6 mg/dl, P = 0.06) appeared to be less common in infected alpha(+)-thalassemic women and were also comparatively rare in parasitemic individuals who harbored double species infections with P. falciparum and P. malariae. Plasmodium malariae may influence the pathogenesis of falciparum malaria leading to a low prevalence of inflammation and febrile responses in alpha(+)-thalassemic women.

In vitro antiplasmodial activity of 4-phenylcoumarins from Exostema mexicanum.

The stem bark of Exostema mexicanum (Rubiaceae) is used in Latin American folk medicine as a quinine substitute for malaria treatment. Bioassay-guided fractionation of lipophilic and hydrophilic extracts from the stem bark and branches yielded two previously undescribed 4-phenylcoumarins: 4',8-dihydroxy-5,7-dimethoxy-4-phenylcoumarin (exomexin A) and 3',4'-dihydroxy-5,7,8-trimethoxy-4-phenylcoumarin (exomexin B). Together with five known derivatives the in vitro activities against a chloroquine-sensitive strain (poW) and a chloroquine-resistant strain (Dd2) of Plasmodium falciparum have been evaluated. The most lipophilic compound, 4',5,7,8-tetramethoxy-4-phenylcoumarin (O-methylexostemin) revealed the strongest antiplasmodial activity (IC50 values: 3.6 microg/ml [poW], 1.6 microg/ml [Dd2]).

Evidence for a reduced effect of chloroquine against Plasmodium falciparum in alpha-thalassaemic children.

Alpha-thalassaemia is common in malaria-endemic regions and is considered to confer protection from clinical disease due to infection with Plasmodium falciparum. In vitro, sensitivity to chloroquine (CQ) of P. falciparum infecting alpha-thalassaemic erythrocytes is reduced. We examined, in a cross-sectional study of 405 Nigerian children, associations between alpha-globin genotypes, blood concentrations of CQ, and P. falciparum parasitaemia. Of the children, 44% were alpha+-thalassaemic (36.8% heterozygous, 7.6% homozygous). CQ in blood and P. falciparum-infection were observed in 52 and 80%, respectively. CQ was more frequently found in homozygous alpha+-thalassaemic (71%) than in non-thalassaemic children (50%; odds ratio, 2.42; 95% confidence interval, 1.01-5.8). Among children with CQ in blood and despite similar drug concentrations, alpha+-thalassaemic individuals had fewer infections below the threshold of microscopy which were detectable by PCR only, and they had a higher prevalence of elevated parasitaemia than non-thalassaemic children. No such differences were discernible among drug-free children. CQ displays a lowered efficacy in the suppression of P. falciparum parasitaemia in alpha+-thalassaemic children; hence protection against malaria due to alpha+-thalassaemia may be obscured in areas of intense CQ usage. Moreover, alpha+-thalassaemia may contribute to the expansion of CQ resistance.

Impact of subpatent multi-species and multi-clonal plasmodial infections on anaemia in children from Nigeria.

Childhood anaemia in sub-Saharan Africa is often caused by Plasmodium falciparum malaria. The influence of subpatent, multi-species and polyclonal infections with malaria parasites on haematological parameters was assessed in 1996/97 in clinically healthy children in Nigeria. Of the 228 children studied, 64% were anaemic by the WHO age-dependent criteria. A univariate analysis of risk factors indicated that the prevalence of anaemia was dependent on the number of Plasmodium species detected by species-specific PCR (P < 0.0001). Furthermore, the prevalence of anaemia increased gradually with the complexity (P < 0.003) as well as with the extent of P. falciparum parasitaemia (P < 0.0001). A logistic regression analysis revealed that individuals with an enlarged spleen tended to be anaemic. The number of Plasmodium species by which an individual was infected was independently associated with anaemia (P < 0.03). ANOVA revealed that the age-corrected values for haemoglobin (Hb) and red blood cells (RBCs) were mainly influenced by the occurrence of mixed infections. Haematological parameters were also influenced by the number of different P. falciparum clones by which an individual was infected. Hb levels and RBC counts were further diminished by additional infections with P. malariae and/or P. ovale. However, the effect of multi-species infections on haematological parameters exceeded that of multi-clonal infections.

Sipandinolide: a butenolide including a novel type of carbon skeleton from Siparuna andina.

From a lipophilic extract of leaves of Siparuna andina (Monimiaceae), which exhibited antiplasmodial activity in vitro, two new compounds have been isolated: sipandinolide (1), a compound with a novel type of carbon skeleton and (-)-cis-3-acetoxy-4',5,7-trihydroxyflavanone (2). Their structures were established by spectroscopic methods; 2 showed moderate antiplasmodial activity whereas 1 was inactive.

Red cell glucose-6-phosphate dehydrogenase status and pyruvate kinase activity in a Nigerian population.

Glucose-6-phosphate dehydrogenase A- (G6PD A-) deficiency is a common enzymopathy in Africa that sporadically leads to manifest haemolytic anaemia. It is not exactly known how far the haematological status of individuals with either homozygous or heterozygous G6PD A- deficiency differs from that of individuals with normal G6PD activity. In a field study in Nigeria, we determined G6PD gene variants, the corresponding G6PD and pyruvate kinase (PK) activities, and basic haematological parameters in clinically healthy individuals, who were, in part, asymptomatically infected by malaria parasites. Red blood cell counts and haemoglobin levels were lower in G6PD A- deficient than in G6PD normal subjects. PK activities were higher in G6PD deficients, indicating a younger red cell population in these individuals. These findings suggest that G6PD A- deficiency is accompanied by chronic subclinical haemolysis. As a consequence, the reduced life span of red cells leads to an impaired diagnosis of G6PD heterozygosity when applying routine biochemical methods.

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.

Concentrations of chloroquine and malaria parasites in blood in Nigerian children.

Consumption of chloroquine (CQ) and subtherapeutic drug levels in blood are considered to be widespread in areas where malaria is endemic. A cross-sectional study was performed with 405 Nigerian children to assess factors associated with the presence of CQ in blood and to examine correlations of drug levels with malaria parasite species and densities. Infections with Plasmodium species and parasite densities were determined by microscopy and PCR assays. Whole-blood CQ concentrations were measured by high-performance liquid chromatography. Plasmodium falciparum, P. malariae, and P. ovale were observed in 80, 16, and 9% of the children, respectively, and CQ was detected in 52% of the children. CQ concentrations were >17 and <100 nmol/liter in 25% of the children, 100 to 499 nmol/liter in 14% of the children, and > or =500 nmol/liter in 13% of the children. Young age, attendance at health posts, and absence of parasitemia were factors independently associated with CQ in blood. With increasing concentrations of CQ, the prevalence of P. falciparum infection and parasite densities decreased. However, at concentrations corresponding to those usually attained during regular prophylaxis (> or =500 nmol/liter), 62% of children were still harboring P. falciparum parasites. In contrast, no infection with P. malariae and only one infection with P. ovale were observed in children with CQ concentrations of > or =100 nmol/liter. These data show the high prevalence of subcurative CQ concentrations in Nigerian children and confirm the considerable degree of CQ resistance in that country. Subtherapeutic drug levels are likely to further promote CQ resistance and may impair the development and maintenance of premunition in areas where malaria is endemic.