Imported Plasmodium falciparum malaria in HIV-infected patients: a report of two cases

As HIV becomes a chronic infection, an increasing number of HIV-infected patients are travelling to malaria-endemic areas. Association of malaria with HIV/AIDS can be clinically severe. Severe falciparum malaria is a medical emergency that is associated with a high mortality, even when treated in an Intensive Care Unit. This article describes two cases of HIV-positive patients, who returned from malaria-endemic areas and presented a parasitaemia > 5% of erythrocytes and clinical signs of severe falciparum malaria, both with > 350 CD4 cell count/μl, absence of chemoprophylaxis and successful response. Factors like drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections

Plasmodium vivax dhfr and dhps mutations in isolates from Madagascar and therapeutic response to sulphadoxine-pyrimethamine

<p>Abstract</p> <p>Background</p> <p>Four of five <it>Plasmodium </it>species infecting humans are present in Madagascar. <it>Plasmodium vivax </it>remains the second most prevalent species, but is understudied. No data is available on its susceptibility to sulphadoxine-pyrimethamine, the drug recommended for intermittent preventive treatment during pregnancy. In this study, the prevalence of <it>P. vivax </it>infection and the polymorphisms in the <it>pvdhfr </it>and <it>pvdhps </it>genes were investigated. The correlation between these polymorphisms and clinical and parasitological responses was also investigated in <it>P. vivax</it>-infected patients.</p> <p>Methods</p> <p><it>Plasmodium vivax </it>clinical isolates were collected in eight sentinel sites from the four major epidemiological areas for malaria across Madagascar in 2006/2007. <it>Pvdhfr </it>and <it>pvdhps </it>genes were sequenced for polymorphism analysis. The therapeutic efficacy of SP in <it>P. vivax </it>infections was assessed in Tsiroanomandidy, in the foothill of the central highlands. An intention-to-treat analysis of treatment outcome was carried out.</p> <p>Results</p> <p>A total of 159 <it>P. vivax </it>samples were sequenced in the <it>pvdhfr/pvdhps </it>genes. Mutant-types in <it>pvdhfr </it>gene were found in 71% of samples, and in <it>pvdhps </it>gene in 16% of samples. Six non-synonymous mutations were identified in <it>pvdhfr</it>, including two novel mutations at codons 21 and 130. For <it>pvdhps</it>, beside the known mutation at codon 383, a new one was found at codon 422. For the two genes, different combinations were ranged from wild-type to quadruple mutant-type. Among the 16 patients enrolled in the sulphadoxine-pyrimethamine clinical trial (28 days of follow-up) and after adjustment by genotyping, 3 (19%, 95% CI: 5%–43%) of them were classified as treatment failure and were <it>pvdhfr </it>58R/117N double mutant carriers with or without the <it>pvdhps </it>383G mutation.</p> <p>Conclusion</p> <p>This study highlights (i) that genotyping in the <it>pvdhfr </it>and <it>pvdhps </it>genes remains a useful tool to monitor the emergence and the spread of <it>P. vivax </it>sulphadoxine-pyrimethamine resistant in order to improve the national antimalarial drug policy, (ii) the issue of using sulphadoxine-pyrimethamine as a monotherapy for intermittent preventive treatment of pregnant women or children.</p

Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

BACKGROUND: In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. METHODS: The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. RESULTS: 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. CONCLUSION: No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers

Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP) drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ) exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. <it>Plasmodium falciparum in vitro </it>resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the <it>cytochrome b </it>gene. ATQ -resistant <it>Plasmodium yoelii </it>and <it>Plasmodium berghei </it>lines have been obtained and resistant lines have amino acid mutations in their CYT <it>b </it>protein sequences. <it>Plasmodium chabaudi </it>model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the <it>P. chabaudi </it>clones, to select a resistant parasite line and to perform genotypic characterization of the <it>cytb </it>gene of these clones.</p> <p>Methods</p> <p>To select for ATQ resistance, <it>Plasmodium. chabaudi chabaudi </it>clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. <it>Plasmodium chabaudi cytb </it>gene was amplified and sequenced.</p> <p>Results</p> <p>ATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i) multiple blood passages in the absence of the drug, (ii) freeze/thawing of parasites in liquid nitrogen and (iii) transmission through a mosquito host, <it>Anopheles stephensi</it>. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six-fold increase in MCD to ATQ relative to AS-3CQ.</p> <p>Conclusions</p> <p>A mutation was found on the <it>P. chabaudi cytb </it>gene from the AS-ATQ sample a substitution at the residue Tyr268 for an Asn, this mutation is homologous to the one found in <it>P. falciparum </it>isolates resistant to ATQ.</p

Detection of high levels of mutations involved in anti-malarial drug resistance in Plasmodium falciparum and Plasmodium vivax at a rural hospital in southern Ethiopia

<p>Abstract</p> <p>Background</p> <p>In Ethiopia, malaria is caused by <it>Plasmodium falciparum </it>and <it>Plasmodium vivax</it>, and anti-malarial drug resistance is the most pressing problem confronting control of the disease. Since co-infection by both species of parasite is common and sulphadoxine-pyrimethamine (SP) has been intensively used, resistance to these drugs has appeared in both <it>P. falciparum </it>and <it>P. vivax </it>populations. This study was conducted to assess the prevalence of anti-malarial drug resistance in <it>P. falciparum </it>and <it>P. vivax </it>isolates collected at a rural hospital in southern Ethiopia.</p> <p>Methods</p> <p>A total of 1,147 patients with suspected malaria were studied in different months across the period 2007-2009. <it>Plasmodium falciparum dhfr </it>and <it>dhps </it>mutations and <it>P. vivax dhfr </it>polymorphisms associated with resistance to SP, as well as <it>P. falciparum pfcrt </it>and <it>pfmdr1 </it>mutations conferring chloroquine resistance, were assessed.</p> <p>Results</p> <p>PCR-based diagnosis showed that 125 of the 1147 patients had malaria. Of these, 52.8% and 37.6% of cases were due to <it>P. falciparum </it>and <it>P. vivax </it>respectively. A total of 10 cases (8%) showed co-infection by both species and two cases (1.6%) were infected by <it>Plasmodium ovale</it>. <it>Pfdhfr </it>triple mutation and <it>pfdhfr/pfdhps </it>quintuple mutation occurred in 90.8% (95% confidence interval [CI]: 82.2%-95.5%) and 82.9% (95% CI: 72.9%-89.7%) of <it>P. falciparum </it>isolates, respectively. <it>Pfcrt </it>T76 was observed in all cases and <it>pfmdr1 </it>Y86 and <it>pfmdr1 </it>Y1246 in 32.9% (95% CI: 23.4%-44.15%) and 17.1% (95% CI: 10.3-27.1%), respectively. The <it>P. vivax dhfr </it>core mutations, N117 and R58, were present in 98.2% (95% CI: 89.4-99.9%) and 91.2% (95% CI: 80.0-96.7%), respectively.</p> <p>Conclusion</p> <p>Current molecular data show an extraordinarily high frequency of drug-resistance mutations in both <it>P. falciparum </it>and <it>P. vivax </it>in southern Ethiopia. Urgent surveillance of the emergence and spread of resistance is thus called for. The level of resistance indicates the need for implementation of entire population access to the new first-line treatment with artemether-lumefantrine, accompanied by government monitoring to prevent the emergence of resistance to this treatment.</p

Active case detection, treatment of falciparum malaria with combined chloroquine and sulphadoxine/pyrimethamine and vivax malaria with chloroquine and molecular markers of anti-malarial resistance in the Republic of Vanuatu

<p>Abstract</p> <p>Background</p> <p>Chloroquine-resistant <it>Plasmodium falciparum </it>was first described in the Republic of Vanuatu in the early 1980s. In 1991, the Vanuatu Ministry of Health instituted new treatment guidelines for uncomplicated <it>P. falciparum </it>infection consisting of chloroquine/sulphadoxine-pyrimethamine combination therapy. Chloroquine remains the recommended treatment for <it>Plasmodium vivax</it>.</p> <p>Methods</p> <p>In 2005, cross-sectional blood surveys at 45 sites on Malo Island were conducted and 4,060 adults and children screened for malaria. Of those screened, 203 volunteer study subjects without malaria at the time of screening were followed for 13 weeks to observe peak seasonal incidence of infection. Another 54 subjects with malaria were followed over a 28-day period to determine efficacy of anti-malarial therapy; chloroquine alone for <it>P. vivax </it>and chloroquine/sulphadoxine-pyrimethamine for <it>P. falciparum </it>infections.</p> <p>Results</p> <p>The overall prevalence of parasitaemia by mass blood screening was 6%, equally divided between <it>P. falciparum </it>and <it>P. vivax</it>. Twenty percent and 23% of participants with patent <it>P. vivax </it>and <it>P. falciparum </it>parasitaemia, respectively, were febrile at the time of screening. In the incidence study cohort, after 2,303 person-weeks of follow-up, the incidence density of malaria was 1.3 cases per person-year with <it>P. vivax </it>predominating. Among individuals participating in the clinical trial, the 28-day chloroquine <it>P. vivax </it>cure rate was 100%. The 28-day chloroquine/sulphadoxine-pyrimethamine <it>P. falciparum </it>cure rate was 97%. The single treatment failure, confirmed by <it>merozoite surface protein-2 </it>genotyping, was classified as a day 28 late parasitological treatment failure. All <it>P. falciparum </it>isolates carried the Thr-76 <it>pfcrt </it>mutant allele and the double Asn-108 + Arg-59 <it>dhfr </it>mutant alleles. <it>Dhps </it>mutant alleles were not detected in the study sample.</p> <p>Conclusion</p> <p>Peak seasonal malaria prevalence on Malo Island reached hypoendemic levels during the study observation period. The only <it>in vivo </it>malaria drug efficacy trial thus far published from the Republic of Vanuatu showed chloroquine/sulphadoxine-pyrimethamine combination therapy for <it>P. falciparum </it>and chloroquine alone for <it>P. vivax </it>to be highly efficacious. Although the chloroquine-resistant <it>pfcrt </it>allele was present in all <it>P. falciparum </it>isolates, mutant alleles in the <it>dhfr </it>and <it>dhps </it>genes do not yet occur to the extent required to confer sulphadoxine-pyrimethamine resistance in this population.</p

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BACKGROUND: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. METHODOLOGY/PRINCIPAL FINDINGS: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance