Residual Parasitemia Research Articles

High prevalence of persistent residual parasitemia on days 3 and 14 after artemether-lumefantrine or pyronaridine-artesunate treatment of uncomplicated Plasmodium falciparum malaria in Nigeria.

Microscopic evaluation of parasite clearance is the gold standard in antimalarial drug efficacy trials. However, the presence of sub-microscopic residual parasitemia after artemisinin-based combination therapy (ACT) needs to be investigated. One hundred and twenty (AL: n = 60, PA: n = 60) days 3 and 14 dried blood spots, negative by microscopy were analysed for residual parasitemia using nested PCR. Isolates with residual parasitemia on days 3 and 14 were further genotyped with their corresponding day-0 isolates using merozoite surface proteins msp-1, msp-2, and glurp genes for allelic similarity. Persistent PCR-determined sub-microscopic residual parasitemia at day 3 post ACT treatment was 83.3 (AL) and 88.3% (PA), respectively (ρ = 0.600), while 63.6 and 36.4% (ρ = 0.066) isolates were parasitemic at day 14 for AL and PA, respectively. Microscopy-confirmed gametocytemia persisted from days 0 to 7 and from days 0 to 21 for AL and PA. When the alleles of day 3 versus day 0 were compared according to base pair sizes, 59% of parasites shared identical alleles for glurp, 36% each for 3D7 and FC27, while K1 was 77%, RO33 64%, and MAD20 23%, respectively. Similarly, day 14 versus day 0 was 36% (glurp), 64% (3D7), and 32% (FC27), while 73% (K1), 77% (RO33), and 41% (MAD20), respectively. The occurrence of residual parasitemia on days 3 and 14 following AL or PA treatment may be attributable to the presence of either viable asexual, gametocytes, or dead parasite DNAs, which requires further investigation.

Scientific Findings of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

For a decade, the Southern and Central Africa International Center of Excellence for Malaria Research has operated with local partners across study sites in Zambia and Zimbabwe that range from hypo- to holoendemic and vary ecologically and entomologically. The burden of malaria and the impact of control measures were assessed in longitudinal cohorts, cross-sectional surveys, passive and reactive case detection, and other observational designs that incorporated multidisciplinary scientific approaches: classical epidemiology, geospatial science, serosurveillance, parasite and mosquito genetics, and vector bionomics. Findings to date have helped elaborate the patterns and possible causes of sustained low-to-moderate transmission in southern Zambia and eastern Zimbabwe and recalcitrant high transmission and fatality in northern Zambia. Cryptic and novel mosquito vectors, asymptomatic parasite reservoirs in older children, residual parasitemia and gametocytemia after treatment, indoor residual spraying timed dyssynchronously to vector abundance, and stockouts of essential malaria commodities, all in the context of intractable rural poverty, appear to explain the persistent malaria burden despite current interventions. Ongoing studies of high-resolution transmission chains, parasite population structures, long-term malaria periodicity, and molecular entomology are further helping to lay new avenues for malaria control in southern and central Africa and similar settings.

Different Plasmodium falciparum clearance times in two Malian villages following artesunate monotherapy

BackgroundArtemisinin resistance described as increased parasite clearance time (PCT) is rare in Africa. More sensitive methods such as qPCR might better characterize the clearance phenotype in sub-Saharan Africa. MethodsPCT is explored in Mali using light microscopy and qPCR after artesunate for uncomplicated malaria. In two villages, patients were followed for 28 days. Blood smears and spots were collected respectively for microscopy and qPCR. Parasitemia slope half-life was calculated after microscopy. Patient residual parasitemia were measured by qPCR. ResultsUncorrected adequate clinical and parasitological responses (ACPR) observed in Faladje and Bougoula-Hameau were 78% and 92%, respectively (p=0.01). This reached 100% for both after molecular correction.Proportions of 24H microscopy positive patients in Faladje and Bougoula-Hameau were 97.2% and 72%, respectively (p<0.0001).Slope half-life was 2.8h in Faladje vs 2H in Bougoula-Hameau (p<0.001) andProportions of 72H patients with residual parasitemia were 68.5% and 40% in Faladje and Bougoula-Hameau, respectively (p=0.003). The mean residual parasitemia was 2.9 in Faladje vs. 0.008 in Bougoula-Hameau (p=0.002).Although artesunate is efficacious in Mali, the longer parasite clearance time with submicroscopic parasitemia observed may represent early signs of developing P. falciparum resistance to artemisinins.

Artemisin Combined Therapy in malaria patients: Do we need to search for more?

Introduction: Malaria is a vector borne disease highly prevalent in the topical developing countries. Two main species of plasmodium causing majority of diseases manifestations are P. Vivax and P. falciparum. The approach to antimalarial selection is determined based on the location of the patient. For treatment of uncomplicated P. falciparum malaria, according to WHO guidelines first line therapy mainly includes Artemisin Combined Therapy. Concerns about the emergence of resistance to artemisin derivatives have increased recently. The main aim of the present study is to investigate the effect of using a combination of intravenous artesunate along with oral doxycycline, as a novel ACT for Malarial infections – falciparum vs. vivax. Methodology: Prospective observational study was carried out at V S General Hospital in Ahmedabad, Gujarat, India. A full history of current illness was taken followed by examining the serial peripheral smear reports of the patients till the malarial parasites are not seen on two consecutive occasions. Samples were taken at least 6 hours apart by the capillary method and oral temperature was measured every 6 hours. We excluded other associated viral fevers such as dengue, pediatric age group (<12 years) and Co-Morbid illnesses like hepatic or renal dysfunction. Data analysis was done using SPSS software version 20. Results: Student t test showed that PCT was significantly more in falciparum (mean= 74.53 hours) patients as compared to vivax patients (mean= 51.89 hours). (p<0.001). Also duration of stay was significantly more in patients having falciparum (mean =3.63 days) as compared to patients having vivax (mean = 2.32 days) (p<0.001) Multivariate analysis by linear regression showed that species of the parasite was the most significant independent predictor (B=12.552) of the time to parasite clearance and other significant variable was Grade of parasitemia at 0 hour (B= 12.798). We also found that patients with residual parasitemia was 83.78 %, 40.54% and 10.81% in vivax group whereas it was 100%, 88.15% and 46.05% in falciparum respectively at 24, 48 and 72 hours. Conclusion: The study shows that PCT and residual parasitemia is very high in falciparum patients as compared to previous reports of different studies and also as compared to vivax group patients. ACT resistance is a grave concern for falciparum and more studies should be done to understand pathophysiology and its prevalence in India. We strongly suggest that a continues monitoring needs to be implemented in health policy to understand the dynamicity of emerging resistance

Molecular Detection of Residual Parasitemia after Pyronaridine–Artesunate or Artemether–Lumefantrine Treatment of Uncomplicated Plasmodium falciparum Malaria in Kenyan Children

Artemisinin resistance is rapidly rising in Southeast Asia and may spread to African countries, where efficacy estimates are currently still excellent. Extensive monitoring of parasite clearance dynamics after treatment is needed to determine whether responsiveness to artemisinin-based combination therapies (ACT) is changing in Africa. In this study, Kenyan children with uncomplicated falciparum malaria were randomly assigned to pyronaridine-artesunate (PA) or artemether-lumefantrine (AL) treatment. Parasite clearance was evaluated over 7 days following the start of treatment by quantitative polymerase chain reaction (qPCR) and direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA), a simplified molecular malaria diagnostic. Residual parasitemia at day 7 was detected by qPCR in 37.1% (26/70) of AL-treated children and in 46.1% (35/76) of PA-treated participants (P = 0.275). Direct-on-blood PCR nucleic acid lateral flow immunoassay detected residual parasites at day 7 in 33.3% (23/69) and 30.3% (23/76) of AL and PA-treated participants, respectively (P = 0.692). qPCR-determined parasitemia at day 7 was associated with increased prevalence and density of gametocytes at baseline (P = 0.014 and P = 0.003, for prevalence and density, respectively) and during follow-up (P = 0.007 and P = 0.011, respectively, at day 7). A positive db-PCR-NALFIA outcome at day 7 was associated with treatment failure (odds ratio [OR]: 3.410, 95% confidence interval [CI]: 1.513-7.689, P = 0.003), but this association was not found for qPCR (OR: 0.701, 95% CI: 0.312-1.578, P = 0.391). Both qPCR and db-PCR-NALFIA detected substantial residual submicroscopic parasitemia after microscopically successful PA and AL treatment and can be useful tools to monitor parasite clearance. To predict treatment outcome, db-PCR-NALFIA may be more suitable than qPCR.

In vivo study of Plasmodium falciparum chloroquine susceptibility in three departments of Haiti

BackgroundMalaria is considered a public health priority in Haiti, with a goal to eliminate by year 2020. Chloroquine is the first-line treatment recommended by the Ministry of Public Health and Population. In order to verify the suitability of chloroquine for uncomplicated malaria treatment, an in vivo study of susceptibility of Plasmodium falciparum to chloroquine was conducted from January 2013 to March 2015 in six localities in the south of Haiti.ResultsSixty-one patients who presented with confirmed P. falciparum malaria were included in the study and followed until day 28 after having taken 25 mg/kg of chloroquine orally over 3 days. The sample included 28 children under the age of 10, 9 adolescents aged 10–19 years, and 24 adults aged 20 years and over. Among them, 30 were monitored on day 3 (49%) and 33 on day 28 (59%). Clinical and parasitological monitoring was carried out on day 7 on 28 subjects, on day 14 on 13 subjects and on day 21 on 18 subjects. Residual parasitaemia with presence of trophozoites was found in 7 of 30 subjects on day 3 (23%), and in 6 of 28 subjects on day 7 (21%) who had a temperature less than 37.5 °C. These patients can be considered as late parasitological failures. All monitoring performed on day 28 was negative. Gametocytes were found in 3 patients (9%) despite the use of primaquine. The continuing low parasitaemia on day 3 and 7 in more than one fifth of cases raises the question of the efficacy of chloroquine in southern Haiti.ConclusionsResults suggest a decrease of chloroquine susceptibility for treatment of P. falciparum malaria cases in southern Haiti. Consequently, there is a need to strengthen malaria treatment surveillance and to study the effectiveness of chloroquine in Haiti by monitoring patients after treatment.

Plasmodium Falciparum Clearance in Malaria Treated Children at Lake Alau Settlements, North Eastern Nigeria: Effects of Body Weight

The influence of body weights on Plasmodium falcifarum clearance in malaria positive children was conducted using AT+SP and AQ+SP combination therapies, at the malaria holo - endemic and risk settlements of lake - alau, North Eastern Nigeria. A total of 313 children (6 - 59 months) were admitted into the study, to assess the efficacies of the combination therapies in children (6 - 59 months) using the effects of Body weights (kg) on parasite densities. Each child's weight was determined on days 0, 1, 2, 3, 4, 7, 14 and 28 during the follow up periods. The drugs were administered between 0-3 days. Finger prick and venipuncture (syringe) techniques were employed and blood was sampled by pricking using sterilized needle on days 0, 1, 2, 3, 4, 7, 14 and 28 for the assessment of parasite densities count (per µl) while blood smear was prepared using Giemsa stained slides making thick and thin blood smears. The result shows body weight exerted negative effects on Plasmodium falciparum between the range of 71.63 to 96.0% for AT+SP and 87.79 to 96.38% for AQ+SP in the first three days of treatment. Furthermore, results showed for each kg gain in body weight there was a synchronous parasite clearance by 325.79 versus 1199.91∕µ, 16.621 versus 157.06∕µ, 17.445 versus 153.5∕µ for AT+SP versus AQ+SP on days 1, 2, and 3, respectively. The terminal resultsindicated a higher residual parasitaemia at the onset of late phase in children treated with AQ+SP (195.17 to 113.92∕µ) tcompared to AT+SP (182.48 to 25.531∕µ). The respective parasite densities clearance per kg body weight for days 7, 14 and 28 for AT+SP versus AQ+SP was 33.82 versus 35.887∕µ, 10.076 versus 29.578∕µ and 2.3548 versus 26.368∕µ respectively. Thus, on each of the late follow-up days, the rate of parasite depletion was higher in AQ+SP than AT+SP.*Corresponding Author: muskokoss@gmail.com

Aptamer-based detection of disease biomarkers in mouse models for chagas drug discovery.

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo.

Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence.

Background. Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown.Methods. We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequence–based amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment.Results. Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.6–39.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.17–3.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.61–5.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.08–31.01; P < .001).Conclusions. Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia.

Extended malaria parasite clearance time in African children following artemisinin-combination therapy enhances transmission to Anopheles mosquitoes

Artemisinin resistance was recently shown to have spread or emerged on the Thailand/Myanmar border. Evidence is accumulating that the parasite clearance time after artemisinin-based combination therapy (ACT) is increasing in settings in Asia and Africa. It is currently unknown if an extended parasite clearance time after ACTs has consequences for the individual patient or confers a higher malaria transmission potential. 298 children in Mbita, Western Kenya, with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL, n = 153) ordihydroartemisinin-piperaquine (DP, n = 145). Parasite carriage post-treatment was determined by microscopy and qPCR, gametocyte carriage by quantitative nucleic acid sequence based amplication. Infectiousness to mosquitoes was determined by mosquito membrane feeding assays. Both drugs were efficacious as judged by standard trial outcomes. Sub-patent residual parasitaemia on day 3 was detected by qPCR in 36.11% (95% CI 25.11 - 48.29) of children treated with AL, and in 30.16% (95% CI 19.23 - 43.02) of children treated with DP. After adjustment for age, treatment arm and enrolment parasite density, children with an extended parasite clearance time were significantly more likely to have microscopically detected recurrent parasitaemia during follow-up (Odds Ratio: 19.51, 95% CI 5.24 - 72.71, p < 0.001). Children with an extended parasite clearance time were also more likely to be infectious to mosquitoes (Odds Ratio 2.76; 95% CI 1.14 - 6.67, p = 0.02) and gave rise to a higher oocyst load in mosquitoes (Incidence Rate Ratio 2.80, 95% CI 1.49 - 5.24, p = 0.001). Our findings indicate that an extended parasite clearance time after ACTs has consequences for the individual patient and for the population at large due to higher transmission potential. The high prevalence of residual subpatent parasitaemia after treatment may be due to novel parasite genotypes with reduced drug sensitivity, inadequate population-level immunity, or the higher sensitivity of qPCR for detection of persisting parasites.

Declining Responsiveness of Plasmodium falciparum Infections to Artemisinin-Based Combination Treatments on the Kenyan Coast

BackgroundThe emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies.MethodsOn the Kenyan coast we studied the treatment responses in 474 children 6–59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995)ResultsThe proportion of patients with residual parasitemia on day 1 rose from 55% in 2005–2006 to 87% in 2007–2008 (odds ratio, 5.4, 95%CI, 2.7–11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7–9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005–2006 and 2007–2008 (OR body temperature >37.5°C, 2.8, 1.9–4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof.ConclusionsThe significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates.Trial RegistrationControlled-Trials.com ISRCTN88705995

Is parasite clearance clinically important after malaria treatment in a high transmission area? A 3-month follow-up of home-based management with herbal medicine or ACT

Argemone mexicana (AM), a validated herbal medicine for uncomplicated malaria, seems to prevent severe malaria without completely clearing parasites in most patients. This study, in a high transmission area of South Mali, explores whether residual parasitaemia at day 28 was associated with subsequent malaria episodes and/or anaemia. Three hundred and one patients were randomly assigned to AM or artesunate/amodiaquine as first line treatment, of whom 294 were followed up beyond the standard 28 days, to 84 days. From day 29 to day 84, there were no significant differences between treatment groups in new clinical episodes of uncomplicated malaria (0.33 vs 0.31 episodes/patient), severe malaria (<6% per month of patients aged ≤5 years) or moderate anaemia (hematocrit <24%: 1.1% in both groups at day 84). Total parasite clearance at day 28 was not correlated with incidence of uncomplicated or severe malaria or of moderate anaemia over the subsequent two months. Total parasite clearance at day 28 was not clinically important in the context of high transmission. If this finding can be confirmed, some antimalarials which are clinically effective but do not completely clear parasites could nevertheless be appropriate in high transmission areas. Such a policy could be tested as a way to delay resistance to artemisinin combination therapies.

Rapid clearance of Plasmodium falciparum hyperparasitaemia after oral amodiaquine treatment in patients with uncomplicated malaria

Amodiaquine is one of the possible alternative therapeutic options to treat chloroquine-resistant Plasmodium falciparum infections in Africa. In this study, its efficacy to treat patients with acute uncomplicated falciparum malaria and high parasitaemia (≥5% of infected erythrocytes in the peripheral blood) was assessed by using the standard protocol developed by the World Health Organization. The mean pre-treatment parasitaemia was 10.8% (range, 5.0–35%). All 32 patients who completed the study (four lost to follow-up) had an adequate clinical and parasitological response on day 14. As compared with the pre-treatment parasitaemia, the mean parasitaemia decreased by 97.8% on day 2 (two patients with negative smears) and 99.97% on day 3 (22 patients with negative smears; the other ten patients had low residual parasitaemia ranging from 24 to 400 asexual parasites/μl). Our results suggest that, in areas of stable transmission in Africa, hyperparasitaemic patients with uncomplicated malaria can be safely treated with oral amodiaquine provided that their clinical and parasitological responses are closely monitored for the first 3 days and controlled on day 7 and 14.

Towards an Understanding of the Mechanism of Pyrimethamine-Sulfadoxine Resistance in Plasmodium falciparum : Genotyping of Dihydrofolate Reductase and Dihydropteroate Synthase of Kenyan Parasites

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were "pure" triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.

Effect of two successive annual treatments with single doses of ivermectin on microfilaraemia due to Wuchereria bancrofti var. pacifica

Between 1986 and 1988 a single-blind, dose-ranging study was carried out in French Polynesia to determine the efficacy and tolerability of single 50, 100, 150 and 200 μg/kg doses of ivermectin in Wuchereria bancrofti carriers. Forty male microfilariae (mf) carriers between 18 and 50 years of age, in whom mf density was ⩾20 mf/ml, were treated twice at a one-year interval. Twelve months after the second treatment, in carriers who were given a dose ⩾100 ng/kg, mean mf density was 4–7% of the initial pretreatment mf density. Therefore, several successive annual treatments with single doses ⩾100 μg/kg of ivermectin should result in reducing mf densities to a very low level. Nevertheless, at 9 months after the second treatment, residual parasitaemia ranged from 1 to 2182 mf/ml (median 85) in 30 patients. Finally, in patients with pretreatment mf counts ⩽150 mf/ml, mean mf density was 2·8 and 8·9 mf/ml, respectively, during the 2 six-month periods following treatment, while in patients with pretreatment mf densities >150 mf/ml (median 1500) it was 92·3 and 334·1 mf/ ml during the same periods. These results suggest that, when implementing filariasis control programmes, the best strategy might be administration of several treatments with a single dose of ivermectin every 6 months to the entire population, at least in French Polynesia. Afterwards, when mf densities had been reduced to a relatively low level (100–150 mf/ ml), annual treatments could be considered.

Trypanosoma cruzi variants with reduced virulence obtained by mutagenesis.

Previous reports have indicated that by mutagen treatment of mouse tumour cells in vitro it is possible to obtain at high frequency stable tumour cell variants that fail to form tumours in syngeneic mice because of increased immunogenicity. By analogy with these tumour cell variants, we examined whether variants with reduced virulence could be obtained by mutagen treatment of trypomastigotes derived from a Trypanosoma cruzi strain that was adapted to culture and produced lethal infections in DBA/2 mice at a dose of 5 X 10(4) parasites. A very large frequency of T. cruzi clones were obtained that failed to provoke an acute lethal infection after injection of 5 X 10(5) parasites. Most of these variants with reduced virulence (vir-) multiplied actively in normal mice until day 8 after injection. After that time the parasitaemia decreased gradually. For most variants a low level of residual parasitaemia persisted for more than 100 days. Unlike the situation encountered with mouse tumour cell variants it was not possible to demonstrate the presence of new antigens on the T. cruzi vir- variants. However, these variants seemed to have acquired an increased immunogenicity since they provoked the rejection of virulent parasites injected concomitantly. Mice that had been immunized with living vir- clones were protected against a challenge with a virulent clone derived from the original parasite population.