Search

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried out in relation to blood sampling (anticoagulants, culture medium, filter paper), storage (temperature, time and immediate lysis) and handling (repeated thawing and freezing). The PCR was unaffected by citrate and EDTA but partly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100-fold lower sensitivity (compared to control samples frozen immediately after collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 degrees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensitivity. Repeated thawing generally resulted in 10-fold loss of sensitivity compared to blood kept frozen until DNA extraction. The presence of antimalarial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR. This may be critical in studies including individuals with low parasitaemia, mixed infections and comparison of data from different settings.

Childhood anaemia is a major public health problem in malaria holoendemic areas. We assessed the effects of antimalarial treatment in an area with drug-resistant falciparum malaria on haemoglobin levels in small children by applying the 1996 World Health Organization in vivo method for the evaluation of standard chloroquine treatment at the community level. In Fukayosi village, coastal Tanzania, 117 children aged 5-36 months with clinical malaria episodes were treated with chloroquine syrup (25 mg/kg). Early treatment failure (ETF) occurred in 20% and late treatment failure (LTF) in 22% of cases. Age > 1 year and malnutrition were protective factors against ETF. The evidence that chloroquine treatment could not prevent an exacerbation of anaemia was (i) the fact that the fall in haemoglobin level after 72 h was significantly greater in ETF than in children with LTF and an adequate clinical response, and (ii) the absence of any haematological improvement at follow-up in children receiving chloroquine alone, even in true treatment successes. In contrast, pyrimethamine/sulfadoxine administered to treatment failures improved the haemoglobin level significantly > 21 d after treatment started (mean difference 14 g/L, 95% confidence interval 2.1-27). We conclude that, when chloroquine treatment of childhood malaria is associated with a 20% ETF rate, the haemoglobin response is unsatisfactory and there is a need to change the recommended first-line treatment.

The study compared the clinical efficacy and safety of oral artemisinin and oral artesunate as well as artemisinin pharmacokinetics during and after resolution of falciparum malaria. Forty adults with symptomatic falciparum malaria were allocated at random to treatment with either oral artemisinin (500 mg single dose on day 1 followed by 250 mg twice daily for 4 d and then another 500 mg single dose on day 6) or with oral artesunate (100 mg single dose on day 1 followed by 50 mg twice daily for 5 d). Patients were admitted to hospital at the Kibaha Designated District Hospital, Kibaha, Tanzania for the duration of treatment. The patients were seen once weekly for 3 more weeks. The time to parasite clearance (PCT) after oral artesunate (26·4 ± 3·6 h) was shorter ( P = 0·002) than after artemisinin (31 ± 3·6 h). The fever subsidence time (FST) after oral artesunate (18·9 ± 4·0 h) was also shorter ( P = 0.04 ) than after artemisinin (21·8±4·6 h). Parasites were detected in 4 (20%) and 7 (35%) patients after completing treatment with artesunate and artemisinin respectively. In these patients the parasitaemia reappeared at the 3rd or 4th week of follow-up. Standard haematology, blood biochemistry and urinanalysis, performed before drug intake and again on days 6 and 14, were normal. No clinical abnormality was observed during the study period. Artemisinin plasma concentrations, determined by high performance liquid chromatography with post-column derivatization and detection by ultraviolet light, were followed up to 8 h after drug administration on days 1 and 6. Artemisinin absorption was rapid, the maximum plasma concentrations C max ) being attained at about 3 h. Artemisinin areas under the plasma concentration-time curve (AUC) and the C max values were about 6 times higher after the first dose on day 1 than on day 6. This decrease in artemisinin plasma concentration is suggestive of an increase in metabolic capacity due to pronounced autoinduction.

Chloroquine resistance of Plasmodium falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease. This association may be due to either progression of uncomplicated to severe disease following chloroquine failure or increased virulence of chloroquine-resistant parasites. The implication of this for antimalarial treatment policy is discussed.

All episodes of acute illness, in children aged 0-9 years, were registered during 3 years in a health clinic in a village of about 500 inhabitants in a malaria holoendemic area on the Tanzanian coast. Of 668 clinical episodes, 395 were diagnosed as malaria. There was no death. Only 5% of the children with malaria episodes came to the clinic after more than 3 d of symptoms. All 11 severe anaemias occurred among these children. Fever was reported in 98%, vomiting in 15%, and diarrhoea in 8% of the malaria episodes. Intermittent fever was reported in 98% of the malaria patients with more than one day of fever, compared to 4% of those with other febrile illnesses. Parasite densities > or = 10,000/microliters were found in 48% of the malaria episodes. Densities > or = 400/microliters were found in 96% of the malaria episodes and in only 8% of the other febrile illnesses. The 16 malaria episodes (4%) with densities below that level were all in children under one year of age. The ability of the rural medical aid or the doctor to differentiate malaria episodes from other febrile illnesses without microscopical examination was limited. Although very few malaria episodes were missed, substantial over-diagnosis resulted in specificity values of only 13% and 52% for their respective malaria diagnoses. It is concluded that intermittent fever was strongly associated with malaria, but a high accuracy of malaria diagnosis in febrile children requires microscopical examination.(ABSTRACT TRUNCATED AT 250 WORDS)

The susceptibilities of Plasmodium falciparum to chloroquine, mefloquine, quinine and sulfadoxine/ pyrimethamine were investigated in Mogadishu in 1988, after chloroquine resistance had spread widely in Somalia. Possible correlations of the responses to these drugs were also investigated. Standard World Health Organization in vitro micro-tests were performed. Of 76 isolates tested for chloroquine susceptibility, 58 (76%) were resistant to the drug with mean EC50 and EC99 values of 1 · 06 × 10−6 mol/litre and 10 · 44 × 10−6 mol/litre of blood, respectively, indicating a high prevalence and degree of resistance. In contrast, all isolates tested were inhibited by mefloquine 3·2 × 10−6 mol/litre of blood, quinine at 2·56 × −6 mol/litre of blood-medium mixture, and sulfadoxine/pyrimethamine at 6 · 0 0 · 075 × 10−6 mol/ litre of blood-medium mixture, indicating full sensitivity to these 3 drugs. However, a significant positive correlation was found between responses to quinine and chloroquine and between those to quinine and mefloquine; the responses to chloroquine and mefloquine were not correlated. The results may suggest that sites responsible for the correlation are shared between quinine and chloroquine but not essentially between chloroquine and mefloquine. Thus the evolution of chloroquine resistance together with increased use of quinine treatment of P. falciparum may increase the risks of development of quinine resistance, whereas the development of mefloquine resistance would not be triggered by chloroquine resistance.

The response of Plasmodium falciparum to chloroquine and mefloquine was investigated in a mesoendemic area of Somalia from 1986 to 1989. Serial in vivo field tests for chloroquine sensitivity were performed and the sensitivity in vitro to chloroquine and mefloquine was evaluated using the standard WHO in vitro microtest. Chloroquine treatment in vivo (25 mg base/kg body weight) resulted in parasite clearance in all patients within 7 d (S/RI) in 1986, while 17%, 19% and 30% RII/RIII responses were found in 1987, 1988 and 1989 respectively. There was consistent increase of parasite clearance time of the S/RI cases in all years. The sensitivity study in vitro in 1986 showed a low degree of chloroquine resistance in 3 of 29 isolates tested and a mean 50% effective dose (EC 50 ) and EC 99 of 0·34 × 10 −6 m and 1·99 × 10 −6 m , respectively. In contrast, in 1989, 12 of the 19 isolates tested were resistant to chloroquine. The mean EC 50 and EC 99 values had increased to 0·78 × 10 −6 m and 7·50 × 10 −6 m respectively. The data in vivo and in vitro indicate a rapid increase of chloroquine resistance both in frequency and degree. All isolates tested in 1986 and 1989 were fully inhibited by mefloquine at 3·2 × 10 −6 m , suggesting full sensitivity. Thus, increased resistance of P. falciparum to chloroquine did not significantly influence the sensitivity pattern of mefloquine.

118 adult Liberians from 2 villages were studied prospectively for one year with monthly blood examinations for malaria parasites. The crude parasite rate was 41.5% and the crude gametocyte rate was 6.1%. The inoculation rate varied between 0.075 in the dry season and almost 0.4 in the rainy season, which is in accordance with other data from holoendemic areas. 47.5% (56) had a titre to the Pf155/RESA antigen less than or equal to 1/50 ('low responders') and 52.5% (62) had a titre of greater than or equal to 1/250 ('high responders'). The response was not age-dependent in this adult population, which may suggest that genetic factors are determining whether the individual become a high or low responder. Antibodies against the Pf155/RESA antigen were measured in 2 surveys 8 months apart, and the mean antibody response to Pf155/RESA and its EENV sequence was constant without seasonal variation. Pf155/RESA high responders had lower parasite densities during all 3 seasons surveyed, and Pf155/RESA high responders, with high antibody reactivity against the (EENV)6 sequence from the 3' repeat region of Pf155/RESA, had significantly lower parasite densities in the rainy season of 1987. The data suggest that high titres of antibodies to the Pf155/RESA antigen, and especially to its EENV sequence, might play a role in protective immunity in adults.

Synergistic interaction between atovaquone and proguanil has been suggested as the reason for the effectiveness of Malarone®. The pharmacodynamic interactions among atovaquone, proguanil and its metabolite cycloguanil were investigated in 4 Plasmodium falciparum parasite strains by culture assays in vitro. The response parameters were determined and 2 statistical methods, log-concentration/response probit method and sum of fractional inhibitory concentrations (ΣFIC) method, were used to analyse the experimental data. Within therapeutically relevant concentration ratios, the combination of atovaquone and proguanil showed mean ΣFICs of 0.37 at EC50 (50% effective concentrations) and 0.13 at EC90, indicating high synergism. The combination of atovaquone and cycloguanil yielded corresponding mean ΣFICs of 3.70 and 2.11, indicating antagonism. The EC50 and EC90 values for proguanil alone were not influenced by RPMI-1640 medium with low concentrations of paraaminobenzoic acid and folic acid (LPLF culture medium), whereas the EC50 and EC90 values for cycloguanil were more than 10 times lower in LPLF medium than in normal RPMI-1640 medium. This confirms the hypothesis that proguanil may act on another target than dihydrofolate reductase. We conclude that the effectiveness of Malarone® is due to the synergism between atovaquone and proguanil and may not require the presence of cycloguanil.

We studied the viability of Plasmodium falciparum parasites reappearing in long-term cultures after repetitive exposure to atovaquone and proguanil. Parasites (F32 and FCR3) exposed to 100-5000 nM atovaquone for 96 hours were reduced to5% of initial parasitaemia but recrudesced after 9-15 days. Also, parasites exposed to 1000 nM atovaquone for 48, 72, 96 and 144 hours recrudesced after 9, 14, 21 and 23 days respectively. Immediately after removal of the drug, only 1-3 schizonts per 10000 red blood cells were found consistently, apparently unable to produce trophozoites and thus, possibly, adopting a "dormant state". Parasites (F32 and FCR3) exposed to 500 nM atovaquone for 72 hours reappeared after 14 days. These recrudescing parasites were then re-exposed and suppressed by atovaquone in three consecutive follow-up experiments. They reappeared after 12, 11 and 9 days respectively. No known point mutations in cytochrome b gene (cytb), associated with atovaquone resistance, were detected in any recrudescing parasites. Finally, parasites (F32) exposed to various concentrations of atovaquone and proguanil in combination for 72 hours reappeared after 9-17 days. The baseline susceptibilities of the parasites to individual drugs were similar before and after recrudescence in all experiments.

A 48 h in vitro test of the efficacy of artemisinin, dihydroartemisinin, artemether, mefloquine and chloroquine was carried out against 3 chloroquine-resistant strains of Plasmodium falciparum, strains K1 and T996 from Thailand and LS21 from India. A sigmoid Emax model was fitted to all in vitro inhibition data for each combination of drug and strain. Strains K1 and LS21 were strongly resistant to chloroquine, whereas T996 was partially resistant. Artemisinin, dihydroartemisinin and artemether were active against all strains, with complete growth inhibition at 10−7 m . Artemether and dihydroartemisinin were both more potent than artemisinin, with 50% effective (EC50) values of 0·57–1·6 n m and 0·36–3·1 n m respectively; the EC50 of artemisinin was 1·5–6·1 n m for the 3 strains. The EC50 values for mefloquine were 46–185 n m . At higher concentrations, strains K1 and LS21 were fully inhibited, while with strain T996 mefloquine did not fully inhibit even at the highest concentration, 1·28×10−6 m . It is concluded that artemisinin and its derivatives were highly effective against the 3 chloroquine-resistant strains, one of which showed borderline resistance to mefloquine.

The prevalence of antibodies against Toxoplasma gondii was studied in the population of Nyamisati village in Tanzania using the direct agglutination test, indirect immunofluorescence test, and immunosorbent agglutination test. All positive sera were positive by both direct agglutination and indirect immunofluorescence tests and were confirmed by the dye test. The seropositivity was confirmed by immunoblotting showing a distinct 32 kDa band in all the seropositive samples. The seropositivity rate was 4% (19/450) among the subjects of Nyamisati origin and 47% (15/32) among immigrants from other areas of Tanzania. Most of the infections appeared to have occurred between 5 and 15 years of age. The generally low transmission in this mainly Muslim village appeared to be related to sparse consumption of contaminated food and low prevalence of oocysts due to scarcity of felines.

The phenotypic composition of peripheral blood lymphocytes in 45 healthy adults (15 each from Bangladesh, Ethiopia and Sweden) was analysed as an indicator of the influence of environment and/or ethnic background on the human immune response. The possible interference of technical factors was minimized by highly standardized handling of samples and by use of a similar simultaneous 3-colour flow cytometry analysis technique for all samples. The percentage of CD4+ cells was lower, and the percentage of CD8+ cells was higher, in Bangladeshi and Ethiopian subjects than in those from Sweden. A higher percentage of CD57+/CD8+ T cells was also found in these 2 groups than in Swedish subjects. The percentage of gamma delta T cells was higher in Bangladeshi subjects and a difference in T cell receptor V beta expression was also noted between Bangladeshi and Swedish subjects. The data suggest that environmental or genetic factors are important bias factors to be considered in immunophenotyping studies. Possibly differences in the pattern or level of microbial challenge, as well as nutritional factors, may lead to different adaptive changes in the immune response. The potential influence of such immune adaptation on the response to vaccination or pharmaceutical therapy may be important in the development of new strategies of medical intervention in different geographical regions or ethnic groups.The phenotypic composition of peripheral blood lymphocytes in 45 healthy adult blood donors (15 each from Bangladesh, Ethiopia, and Sweden) was examined as an indicator of the influence of environment and/or ethnic background on the human immune response. A highly standardized 3-color flow cytometry analysis was performed on all samples. The percentage of CD4+ cells was lower and the percentage of CD8+ cells was higher in Bangladeshi and Ethiopian subjects than in those from Sweden. A higher percentage of CD57+/CD8+ T cells was likewise found in these 2 groups compared to Swedish subjects. The percentage of gammadelta T cells was higher in Bangladeshi subjects and a difference in T cell receptor Vbeta expression was also noted between Bangladeshi and Swedish subjects. The findings suggest that environmental or genetic factors are important bias factors to be taken into account in phenotyping studies. Possible variations in the patterns or level of microbial challenge, as well as nutritional factors, may lead to different adaptive changes in the immune response. The potential influence of such immune adaptation on the response to vaccination or pharmaceutical therapy may be essential in the development of new medical intervention approaches in different geographical regions or ethnic groups.

The causative factors of an epidemic of falciparum malaria were investigated in Balcad, Somalia, a town with previously low malaria transmission, where malaria incidence rose more than twenty-fold between 1986 and 1988. The emergence of chloroquine resistance, accelerated by high drug pressure, low herd immunity and favourable meteorological conditions were identified as major causes of the epidemic. Chloroquine resistance of grades RII and RIII was first observed in Balcad in 1987 and rapidly increased to 72% of the Plasmodium falciparum infections in 1988. In the absence of alternative treatment, resistance resulted in the accumulation of a massive infective reservoir and therefore increased malaria transmission, associated with intensive clinical symptomatology. The advent of chloroquine resistance was less violent in the area of Malable, where malaria is stable and communal immunity higher than in Balcad.

The activities of artemisinin (qinghaosu), dihydroartemisinin (dihydroqinghaosu), artemether and pyronaridine were tested in a 48 h in vitro assay against 3 chloroquine-sensitive and one chloroquine-resistant strains of Plasmodium falciparum. Growth inhibition was modelled with a sigmoid Emax model. All compounds markedly inhibited the growth of all strains although, for the chloroquine-resistant strain, merozoites were detected at concentrations as high as 10−4 m of artemisinin, dihydroartemisinin and artemether. Dihydroartemisinin, artemether and pyronaridine appeared to be more potent than artemisinin, with EC50 values of 4·7–23 n m , 0·98–6·1 n m and 4·4–18 n m respectively, while the EC50 value of artemisinin was 3–108 n m against all 4 strains.

The potential of desipramine to improve the efficacy of chloroquine against chloroquine-resistant Plasmodium falciparum in vivo in man was investigated. Fifty-three malaria patients were selected according to the criteria for the standard World Health Organization (WHO) in vivo test and were randomly divided in 2 groups. One group (n = 27) was given standard therapeutic doses of chloroquine (25 mg/kg body weight of base) and the other (n = 26) was given standard doses of chloroquine, as above, in combination with desipramine (1·3–2·8 mg/kg body weight) daily for 3 consecutive days. Standard WHO in vitro micro-tests were performed in parallel with the tests in vivo to provide chloroquine sensitivity patterns of the P. falciparum parasites. The results in vitro from both groups did not differ with regard to chloroquine sensitivity and the means of the pre-treatment parasite densities were similar. There was no apparent difference in parasite clearance in vivo between the 2 groups. This study provided no evidence for enhanced chloroquine efficacy in vivo through the use of desipramine in doses corresponding to the usual therapeutic range.

Recently, activity in vivo of norfloxacin has been reported against Plasmodium falciparum and, in vitro , ciprofioxacin has been found to be even more potent than norfloxacin. We therefore treated one patient infected with chloroquine-resistant P. falciparum at relatively low parasite density with ciprofloxacin, 200 mg intravenously twice daily for 3 d. This treatment failed to produce radical cure, although some suppressive effect was probably obtained. The growth of the P. falciparum isolate in vitro was inhibited at 12·5 μg/ml, whereas the plasma concentration recorded 1 h after infusion was only 1·2 μg/ml. It is concluded that, if ciprofloxacin is to be used for treatment of malaria, a higher dose than that normally used for bacterial infections is necessary.

Whole blood concentrations of chloroquine and desethychloroquine were determined during and after chloroquine treatment of 15 adult patients infected with P. vivax, P. ovale or P. malariae. The median of chloroquine concentrations remained practically unchanged in samples drawn three hours after initiation of treatment and in samples drawn immediately before the next dose of chloroquine. Concentrations of chloroquine remained above 1·0 μmol/litre for at least four days. The calculated sum of chloroquine and desethylchloroquine concentrations was above 1·0 μmol/litre for at least seven days. These concentrations are regarded as sufficient for treatment of P. vivax, P. ovale and P. malariae infections.

The clinical occurrence of Pneumocystis carinii and Mycobacterium tuberculosis was investigated in patients infected with human immunodeficiency virus (HIV) who had clinical pneumonia of unknown aetiology in Lusaka, Zambia. The results were compared with a similar group of patients in Stockholm, Sweden. Induced sputum samples were stained for Pneumocystis by indirect immunofluorescence using monoclonal antibody 3F6 and toluidine blue O. Mycobacterial culture and acid fast stain were performed on the specimens from Lusaka. P. carinii cysts were detected in none of 27 Lusaka patients, compared to 10 of 33 Stockholm patients. M. tuberculosis was identified in 11 of 22 Lusaka patients tested. In conclusion, P. carinii could not be incriminated as the aetiological agent of HIV-associated pneumonia in Zambia in contrast to the situation in Sweden, where Pneumocystis is the dominating aetiological agent.

The in vitro susceptibility of seven Plasmodium falciparum isolates to four schizonticidal drugs was studied in Yekepa area, northern Liberia, by the Rieckmann 24-hour micro method. The seven patients were successfully treated with the standard chloroquine regimen. The results of the individual in vitro tests all indicated full susceptibility to the four drugs. By probit analysis, the drug concentrations achieving 50% (EC 50) and 99% (EC 99) inhibition, respectively, were 0·2 and 0·7 μM chloroquine, 0·04 and 0·2 μM amodiaquine, O·01 and 0·07 μM mefloquine and 1·4 and 3·0 μM quinine.

Children two to nine years old from an area of holoendemic malaria in northern Liberia had mean HbA2 and haematocrit values significantly (P less than 0.001) lower than others from a neighbouring town where malaria is hypoendemic. After regular administration of chloroquine over two years to 38 children living in a holoendemic village, their mean HbA2 rose from 2.1%, SE +/- 0.04, to 2.6%, SE +/- 0.08 (P less than 0.001) and their mean haematocrit from 0.348, SEM +/- 0.004, to 0.382, SE +/- 0.004 (P less than 0.001), values similar to those of children from the neighbouring town. In another village where chloroquine was not given regularly, mean HbA2, haematocrit and malariometric indices were little changed at the end of the two-year period. We conclude that persistent malarial parasitaemia was the main factor in the relatively low values of the village children. Although it is not clear how malaria depresses HbA, the findings were consistent with the hypothesis that chronic malaria induces iron-deficiency.

A field study was conducted in the Malable area, in Somalia, to assess the susceptibility of Plasmodium falciparum to chloroquine and mefloquine. The in vivo response of P. falciparum to standard therapeutic regimen of chloroquine was studied in 16 children (1-12 years) using the standard WHO in vivo field test. All subjects were parasite free by day 3 and no recrudescence occurred during a 7-d follow-up. In the 24 h micro in vitro tests for chloroquine, 29 of 39 tests performed were successful. Of the 29 isolates, 3 showed distinct resistance and 2 were borderline. The drug concentration yielding 99% (EC99) inhibition was 1.64 x 10(-6) M, indicating low grade resistance. For mefloquine, 11 of 20 tests gave interpretable results and were sensitive, although with some heterogeneity. The EC50 and EC99 of 0.24 x 10(-6) M and 1.31 x 10(-6) M respectively indicate sensitivity.