Pre-Pandemic Cross-Reactive Immunity against SARS-CoV-2 among Central and West African Populations.

For more than two years after the emergence of COVID-19 (Coronavirus Disease-2019), significant regional differences in morbidity persist. These differences clearly show lower incidence rates in several regions of the African and Asian continents. The work reported here aimed to test the hypothesis of a pre-pandemic natural immunity acquired by some human populations in central and western Africa, which would, therefore, pose the hypothesis of an original antigenic sin with a virus antigenically close to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To identify such pre-existing immunity, sera samples collected before the emergence of COVID-19 were tested to detect the presence of IgG reacting antibodies against SARS-CoV-2 proteins of major significance. Sera samples from French blood donors collected before the pandemic served as a control. The results showed a statistically significant difference of antibodies prevalence between the collected samples in Africa and the control samples collected in France. Given the novelty of our results, our next step consists in highlighting neutralizing antibodies to evaluate their potential for pre-pandemic protective acquired immunity against SARS-CoV-2. In conclusion, our results suggest that, in the investigated African sub-regions, the tested populations could have been potentially and partially pre-exposed, before the COVID-19 pandemic, to the antigens of a yet non-identified Coronaviruses.

Antimalarial Activity of Human Group IIA Secreted Phospholipase A2 in Relation to Enzymatic Hydrolysis of Oxidized Lipoproteins.

The level of human group IIA secreted phospholipase A2 (hGIIA sPLA2) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA2s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA2 was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA2s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA2 is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA2s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA2 Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA2, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Design and synthesis of simplified speciophylline analogues and ß-carbolines as active molecules against Plasmodium falciparum.

A structure-activity relationship study of active molecules against chloroquine-resistant Plasmodium falciparum K1 strain is reported. Structurally simplified analogues of antiplasmodial active alkaloids presented similar levels of activity as their corresponding natural products extracted from Guiera senegalensis and Mitragyna inermis with IC50 values on chloroquine-resistant P. falciparum K1 strain of up to 10.6 µM for spirooxindoles and 13.8 µM for ß-carbolines. The identification of such simpler and cheaper structural analogues is crucial to efficiently study these natural products' action mode as well as developing new cures against malaria.

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in the Democratic Republic of the Congo: emergence of highly resistant pfdhfr/pfdhps alleles.

Background: In 2005, the Democratic Republic of the Congo (DRC) switched to artesunate/amodiaquine as the first-line antimalarial in response to increasing sulfadoxine/pyrimethamine resistance and adopted intermittent preventive treatment using sulfadoxine/pyrimethamine in pregnancy. Objectives: To determine the prevalence of molecular markers of sulfadoxine/pyrimethamine resistance in southwestern DRC 10 years after the new policy was instituted. Methods: From March 2014 to December 2015, blood samples were collected from symptomatic patients presenting to outpatient centres in urban and rural areas. A total of 2030 confirmed Plasmodium falciparum isolates were genotyped at codons associated with sulfadoxine/pyrimethamine resistance. Results: The prevalence of pfdhfr-N51I, C59R and S108N and pfdhps-A437G mutations was consistently high; the prevalence of the pfdhps-K540E mutation was low but increased since its first report in 2008 in the same region, reaching 17.6% by 2015. The pfdhps-A581G mutation increased from ∼4.5% in 2014 to ∼14.0% in 2015 at urban sites while in rural areas it remained low (∼4.0%). The mutations pfdhfr-I164L and pfdhps-A613S were detected for the first time in DRC. Also, 11 (0.8%) isolates revealed the presence of the newly described pfdhps-I431V mutation. Combining pfdhfr and pfdhps alleles, quintuple and sextuple mutations were observed, with the emergence of septuple (IRNI/IAGEGA)- and octuple (IRNI/VAGKGS)-mutant genotypes. Conclusions: Intermittent preventive treatment using sulfadoxine/pyrimethamine during pregnancy remains warranted in southwestern DRC. However, the expansion of pfdhps-K540E mutation and emergence of mutants that cause higher levels of sulfadoxine/pyrimethamine resistance is concerning and may present a challenge for future preventive interventions in the country.

Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis.

In vitro antiplasmodial activity of cepharanthine.

BACKGROUND: New classes of anti-malarial drugs are needed to control the alarming Plasmodium falciparum resistance toward current anti-malarial therapy. The ethnopharmacological approach allows the discovery of original chemical structures from the vegetable biodiversity. Previous studies led to the selection of a bisbenzylisoquinoline, called cepharanthine and isolated from a Cambodian plant: Stephania rotunda. Cepharanthine could exert a mechanism of action different from commonly used drugs. Potential plasmodial targets are reported here. METHODS: To study the mechanism of action of cepharanthine, a combined approach using phenotypic and transcriptomic techniques was undertaken. RESULTS: Cepharanthine blocked P. falciparum development in ring stage. On a culture of synchronized ring stage, the comparisons of expression profiles showed that the samples treated with 5 µM of cepharanthine (IC90) were significantly closer to the initial controls than to the final ones. After a two-way ANOVA (p-value < 0.05) on the microarray results, 1,141 probes among 9,722 presented a significant differential expression.A gene ontology analysis showed that the Maurer's clefts seem particularly down-regulated by cepharanthine. The analysis of metabolic pathways showed an impact on cell-cell interactions (cytoadherence and rosetting), glycolysis and isoprenoid pathways. Organellar functions, more particularly constituted by apicoplast and mitochondrion, are targeted too. CONCLUSION: The blockage at the ring stage by cepharanthine is described for the first time. Transcriptomic approach confirmed that cepharanthine might have a potential innovative antiplasmodial mechanism of action. Thus, cepharanthine might play an ongoing role in the progress on anti-malarial drug discovery efforts.

Diversity of Anopheles mosquitoes in Binh Phuoc and Dak Nong Provinces of Vietnam and their relation to disease.

BACKGROUND: Human malaria is still a burden in Dak Nong and Binh Phuoc Provinces in south-central Vietnam that border Cambodia. Several Anopheles species that transmit human malarial Plasmodium may also transmit Wuchereria bancrofti, the nematode that causes Bancroftian lymphatic filariasis. The objective of this study was to investigate the role of Anopheles species in the transmission of these two pathogens in the two highly malaria endemic provinces of Vietnam. METHODS: Anopheles mosquitoes were collected in Dak Nong and Binh Phuoc Provinces in November and December of 2010 and 2011. Human landing catches, paired collections on human and buffalo, and resting captures were made with mouth aspirators. Collections were also made with light traps. Morphological and PCR-based methods were used to identify the species. Real-time PCR was used to detect Plasmodium species and W. bancrofti in individual mosquitoes. RESULTS: Twenty-four Anopheles species were identified among 797 captured mosquitoes. Anopheles dirus was found in both provinces and was the predominant species in Binh Phuoc Province; An. maculatus was the most prevalent species in Dak Nong Province. Anopheles minimus was collected only in Binh Phuoc Province. Some specimens of An. minimus and An. pampanai were misidentified based on morphology. Four specimens of An. scanloni were identified, and this is the first report of this species of the Dirus Complex in Vietnam. Two females, one An. dirus and one An. pampanai, collected in Binh Phuoc Province were infected with P. vivax, for an overall infection rate of 0.41% (2/486): 0.28% for An. dirus (1/361) and 20% for An. pampanai (1/5). No mosquitoes were found to be infected with P. falciparum, P. knowlesi or W. bancrofti in either province. CONCLUSION: A diversity of Anopheles species occurs in Dak Nong and Binh Phuoc Provinces of Vietnam, several of which are considered to be actual and potential vectors of malarial protozoa and microfilariae. It is highly likely that two of the species, An. dirus and An. pampanai, are active in malaria transmission based on the detection of P. vivax in females of these species. This is the first report of An. scanloni in Vietnam.

Ethnobotany, phytochemistry and pharmacology of Stephania rotunda Lour.

ETHNOPHARMACOLOGICAL RELEVANCE: Stephania rotunda Lour. (Menispermaceae) is an important traditional medicinal plant that is grown in Southeast Asia. The stems, leaves, and tubers have been used in the Cambodian, Lao, Indian and Vietnamese folk medicine systems for years to treat a wide range of ailments, including asthma, headache, fever, and diarrhoea. AIM OF THE REVIEW: To provide an up-to-date, comprehensive overview and analysis of the ethnobotany, phytochemistry, and pharmacology of Stephania rotunda for its potential benefits in human health, as well as to assess the scientific evidence of traditional use and provide a basis for future research directions. MATERIAL AND METHODS: Peer-reviewed articles on Stephania rotunda were acquired via an electronic search of the major scientific databases (Pubmed, Google Scholar, and ScienceDirect). Data were collected from scientific journals, theses, and books. RESULTS: The traditional uses of Stephania rotunda were recorded in countries throughout Southeast Asia (Cambodia, Vietnam, Laos, and India). Different parts of Stephania rotunda were used in traditional medicine to treat about twenty health disorders. Phytochemical analyses identified forty alkaloids. The roots primarily contain l-tetrahydropalmatine (l-THP), whereas the tubers contain cepharanthine and xylopinine. Furthermore, the chemical composition differs from one region to another and according to the harvest period. The alkaloids exhibited approximately ten different pharmacological activities. The main pharmacological activities of Stephania rotunda alkaloids are antiplasmodial, anticancer, and immunomodulatory effects. Sinomenine, cepharanthine, and l-stepholidine are the most promising components and have been tested in humans. The pharmacokinetic parameters have been studied for seven compounds, including the three most promising compounds. The toxicity has been evaluated for liriodenine, roemerine, cycleanine, l-tetrahydropalmatine, and oxostephanine. CONCLUSION: Stephania rotunda is traditionally used for the treatment of a wide range of ailments. Pharmacological investigations have validated different uses of Stephania rotunda in folk medicine. The present review highlights the three most promising compounds of Stephania rotunda, which could constitute potential leads in various medicinal fields, including malaria and cancer.

Quantitative analysis of cepharanthine in plasma based on semiautomatic microextraction by packed sorbent combined with liquid chromatography.

The spread of Plasmodium falciparum resistance toward most of the used drugs requires new antimalarial compounds. Taking advantage of the biodiversity, the ethnopharmacological approach opens the way for the discovery and the characterization of potent original molecules. Previous works led to the selection of a bisbenzylisoquinoline, cepharanthine, extracted from Stephania rotunda, which is mainly present in Cambodia. A sensitive and selective liquid chromatography method has been developed for the determination of cepharanthine in mouse plasma. The method involved a semiautomated microextraction by packed sorbent (MEPS) using 4 mg of solid phase silica-C8 sorbent. LC separation was performed on a Kinetex XB-C18 column (2.6 µm) with a mobile phase of acetonitrile containing formic acid and 10 mM ammonium formate buffer pH 3.5. Data were acquired at 282 nm with a diode array detector. The drug/internal standard peak area ratios were linked via linear relationships to plasma concentrations (75-2,000 ng/mL). Precision was below 5% and accuracy was 99.0-102%. Extraction recovery of cepharanthine was 56-58%. The method was successfully used to determine the pharmacokinetic profile of cepharanthine in healthy and Plasmodium berghei infected mice. The infection did not impact pharmacokinetic parameters of cepharanthine.

In vitro and in vivo combination of cepharanthine with anti-malarial drugs.

BACKGROUND: Stephania rotunda is used by traditional health practitioners in Southeast Asia to treat a wide range of diseases and particularly symptoms related to malaria. Cepharanthine (CEP) is an alkaloid isolated from this plant with potential innovative antiplasmodial activity. The analysis of interactions between antiplasmodial drugs is necessary to develop new drugs combinations to prevent de novo emergence of resistance. The objective of this study was to evaluate the anti-malarial activity of CEP in combination with usual anti-malarial compounds, both in vitro and in vivo. METHODS: A fixed ratio method using the isotopic micro test was performed on the chloroquine-resistant plasmodial strain W2 to build isobolograms from eight CEP-based combinations with standard anti-malarial drugs. The efficacy of two combinations was then evaluated in the BALB/c mouse infected with Plasmodium berghei ANKA strain. RESULTS: In vitro, efficiency gains were observed when CEP was combined with chloroquine (CQ), lumefantrine (LUM), atovaquone (ATO), piperaquine (PPQ) and particularly monodesethylamodiaquine (MdAQ), whereas an antagonistic interaction was observed with dihydroartemisinin (DHA) and mefloquine (MQ). In vivo, the combination of CEP with CQ or amodiaquine (AQ) improved significantly the survival of mice and extended the delay for parasitic recrudescence. CONCLUSION: All these observations suggest that CEP could be an interesting lead compound in the development of a combination therapy against malaria.

Usefulness of a rapid immuno-migration test for the detection of canine monocytic ehrlichiosis in Africa.

A rapid immuno-migration test for the serological detection of canine monocytic ehrlichiosis, Witness® Ehrlichia (WE) (Zoetis, France), was evaluated in 528 serum samples from dogs living in endemic areas of West and East Africa: Senegal (N=208), Ivory Coast (N=7), Sudan (N=27), and Djibouti (N=286). Of these dogs, 200 were French military working dogs (MWD) temporarily residing in Africa. The WE test results were compared with those obtained by indirect immunofluorescence (IFA). The sensitivity of WE was 97% [94.2, 98.7] with a specificity of 100% [98.6, 100]. In MWD, the seroprevalence (IFA) was 7%; in native dogs, it reached 77.1%. This significant difference can be explained by prophylactic measures from which MWD benefit. The WE test represents a simple, fast and reliable test for the detection of anti-Ehrlichia canis antibodies. Its implementation for the diagnosis of clinical cases has been validated in the field, and its use allows easy detection of asymptomatic dogs that may be carriers of E. canis.

In vitro piperaquine susceptibility is not associated with the Plasmodium falciparum chloroquine resistance transporter gene.

BACKGROUND: Dihydroartemisinin-piperaquine is a new ACT that is administered as single daily dose for three days and has been demonstrated to be tolerated and highly effective for the treatment of uncomplicated Plasmodium falciparum malaria. Piperaquine was used alone to replace chloroquine as the first-line treatment for uncomplicated malaria in China in response to increasing chloroquine resistance in the 1970s. However, the rapid emergence of piperaquine-resistant strains that resulted in the cessation of its use in China in the 1980s, suggests that there is cross-resistance between piperaquine and chloroquine. Very few data are available on cross-resistance between piperaquine and chloroquine, and the data that do exist are often contradictory. METHODS: In total, 280 P. falciparum isolates, collected between April 2008 and June 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country, were assessed ex vivo for piperaquine and chloroquine susceptibilities by using the standard 42-hour 3H-hypoxanthine uptake inhibition method. The chloroquine resistance-associated mutation K76T in pfcrt was also investigated for the 280 isolates. RESULTS: The IC50 for piperaquine ranged from 9.8 nM to 217.3 nM (mean = 81.3 nM. The IC50 for chloroquine ranged from 5.0 nM to 1,918 nM (mean = 83.6 nM. A significant but low correlation was observed between the Log IC50 values for piperaquine and chloroquine (r = 0.145, p < 0.001). However, the coefficient of determination of 0.021 indicates that only 2.1% of the variation in the response to piperaquine is explained by the variation in the response to chloroquine. The mean value for piperaquine was 74.0 nM in the Pfcrt K76 wild-type group (no = 125) and 87.7 nM in the 76 T mutant group (no = 155). This difference was not significant (p = 0.875, Mann Whitney U test). CONCLUSIONS: The present work demonstrates that there was no cross-resistance between piperaquine and chloroquine among 280 P. falciparum isolates and that piperaquine susceptibility is not associated with pfcrt, the gene involved in chloroquine resistance. These results confirm the efficacy of piperaquine in association with dihydroartemisinin and support its use in areas in which parasites are resistant to chloroquine.

A new, rapid and sensitive bioluminescence assay for drug screening on Leishmania.

We validated a new method, based on luciferine/luciferase bioluminescence, for drug screening on promastigotes of different Leishmania species. Results obtained with this new, rapid, reproducible, and reliable method are in good accordance with results obtained by the conventional MTT assay. This bioluminescence assay has a lower detection limit.

Taxonomic assessment of Anopheles crawfordi and An. dangi of the Hyrcanus Group of subgenus Anopheles in Vietnam.

Anopheles dangi, introduced as a new species of the Hyrcanus Group of subgenus Anopheles in an illustrated dichotomous key for the identification of the Anopheles mosquitoes of Vietnam published in 1987, was distinguished from Anopheles crawfordi based on the presence of a humeral pale spot on the base of the costal vein of the wing. However, this character has been known to occur occasionally in An. crawfordi. To determine whether An. dangi is distinct from An. crawfordi, we analyzed nucleotide sequences of the COI, COII and Cyt-b genes of mtDNA and the D3 gene of rDNA obtained from specimens collected in south-central Vietnam that were identified as An. dangi and An. crawfordi based on the presence or absence, respectively, of a humeral pale spot. Maximum Likelihood and Bayesian analyses of the sequences showed a low mean genetic distance of 0.004 for specimens identified as An. crawfordi and 0.008 for those identified as An. dangi. The mean genetic distance between the two nominal species was 0.006, compared with 0.077 for any group versus the outgroup taxa Anopheles dirus and Anopheles minimus, and the specimens of the two forms clustered in a single strongly supported clade. Consequently, An. dangi is merely a morphological variant of An. crawfordi and is deemed to be a synonym of that nominal species.

A twenty-year follow-up of canine leishmaniosis in three military kennels in southeastern France.

BACKGROUND: Canine leishmaniosis (CanL) is enzootic in southeastern France, and military working dogs (MWD) posted in this area are highly exposed. To assess the efficiency of prevention, we performed a serological and clinical follow-up of 80 MWD in the Var and Corsica regions during the 20-year period from 1993 to 2012. The systematic and specific prevention of CanL using a deltamethrin-impregnated collar (DMC) was implemented in 2002. FINDINGS: Out of 80 dogs tested annually, the cumulative serological and clinical incidence was 42.5% (34/80) and 21.2% (17/80) respectively, during the first period, and these numbers decreased to 6.2% (5/80) and 2.5% (2/80) during the second period (p < 0.00001). Considering the incidence of serology since 2002, the CanL risk has been reduced by 85.2% and the level of protection of MWD reached 93.8%. Dogs without collars had a 10.4-fold greater chance of becoming infected than protected dogs. Although other ecological factors might have influenced the epidemiology of CanL, DMC usage was the main factor affecting dog exposure to CanL. CONCLUSIONS: The prevention strategy based on DMC proved highly efficient in our population of MWD, as it effectively controlled the disease. This result is also of interest to public health, as dogs are reservoirs for Leishmania infantum.

Real-time PCR assay for discrimination of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in the Ivory Coast and in the Comoros Islands.

BACKGROUND: Plasmodium ovale is one of the five malaria species infecting humans. Recent data have shown that the name of this neglected species masks two distinct genotypes also called curtisi and wallikeri. Some authors show that these species could be sympatric. These two subspecies are not differentiated by microscopy techniques and malaria rapid diagnostic tests. This diagnostic defect is the result of low parasitaemia, antigenic polymorphism and absence of antibodies performance and requires the use of sequencing techniques. An accurate and easy discrimination detection method is necessary. METHODS: A new molecular assay was developed to easily identify the two genotypes of P. ovale. This tool allowed the study of 90 blood samples containing P. ovale, confirmed by molecular biology techniques, which were obtained from patients with imported malaria. RESULTS: The new marker was validated on well genotyped samples. The genotype of 90 P. ovale samples mainly imported from the Ivory Coast and the Comoros Islands was easily and quickly realized. The distribution of the two subspecies was described with a significant number of samples and showed that the two genotypes were present in the studied countries. CONCLUSION: This work confirms the presence of the two species in the same country for the first time, in the Ivory Coast and the Comoros Islands. A better genotyping of P. ovale types may improve a better characterization of the clinical pathophysiology for each.

Enhanced basophil reactivities during severe malaria and their relationship with the Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein.

Recent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derived Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruited P. falciparum-infected patients in Senegal with mild malaria (MM; n = 19) or severe malaria (SM; n = 9) symptoms and healthy controls (HC; n = 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measured ex vivo using fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.

Differential association of Plasmodium falciparum Na+/H+ exchanger polymorphism and quinine responses in field- and culture-adapted isolates of Plasmodium falciparum.

Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na(+)/H(+) exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC(50)) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.

Polymorphism of Plasmodium falciparum Na(+)/H(+) exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam.

BACKGROUND: The Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam. METHOD: Ninety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay. RESULTS: Significant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC(50) of 682 nM versus median IC(50) of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC(50) of 704 nM versus median IC(50) of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC(50) showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7. CONCLUSIONS: The haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.