Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

In vitro anti-microbial activity of the Cuban medicinal plants Simarouba glauca DC, Melaleuca leucadendron L and Artemisia absinthium L

In the present study, an extensive in vitro antimicrobial profiling was performed for three medicinal plants grown in Cuba, namely Simarouba glauca, Melaleuca leucadendron and Artemisia absinthium. Ethanol extracts were tested for their antiprotozoal potential against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Antifungal activities were evaluated against Microsporum canis and Candida albicans whereas Escherichia coli and Staphylococcus aureus were used as test organisms for antibacterial activity. Cytotoxicity was assessed against human MRC-5 cells. Only M. leucadendron extract showed selective activity against microorganisms tested. Although S. glauca exhibited strong activity against all protozoa, it must be considered non-specific. The value of integrated evaluation of extracts with particular reference to selectivity is discussed.

Night and crepuscular mosquitoes and risk of vector-borne diseases in areas of piassaba extraction in the middle Negro River basin, state of Amazonas, Brazil

A study of crepuscular and night-biting mosquitoes was conducted at remote settlements along the Padauiri River, middle Negro River, state of Amazonas, Brazil. Collections were performed with human bait and a CDC-light trap on three consecutive days per month from June 2003-May 2004. In total, 1,203 h of collection were performed, of which 384 were outside and 819 were inside houses. At total of 11,612 specimens were captured, and Anophelinae (6.01%) were much less frequent than Culicinae (93.94%). Anopheles darlingi was the most frequent Anophelinae collected. Among the culicines, 2,666 Culex (Ae.) clastrieri Casal & Garcia, 2,394 Culex. (Mel.) vomerifer Komp, and 1,252 Culex (Mel.) eastor Dyar were the most frequent species collected. The diversity of insects found reveals the receptivity of the area towards a variety of diseases facilitated by the presence of vectors involved in the transmission of Plasmodium, arboviruses and other infectious agents.

Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives) isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae)

In the present study, in vitro techniques were used to investigate a range of biological activities of known natural quassinoids isobrucein B (1) and neosergeolide (2), known semi-synthetic derivative 1,12-diacetylisobrucein B (3), and a new semi-synthetic derivative, 12-acetylneosergeolide (4). These compounds were evaluated for general toxicity toward the brine shrimp species Artemia franciscana, cytotoxicity toward human tumour cells, larvicidal activity toward the dengue fever mosquito vector Aedes aegypti, haemolytic activity in mouse erythrocytes and antimalarial activity against the human malaria parasite Plasmodium falciparum. Compounds 1 and 2 exhibited the greatest cytotoxicity against all the tumor cells tested (IC50 = 5-27 µg/L) and against multidrug-resistant P. falciparum K1 strain (IC50 = 1.0-4.0 g/L) and 3 was only cytotoxic toward the leukaemia HL-60 strain (IC50 = 11.8 µg/L). Quassinoids 1 and 2 (LC50 = 3.2-4.4 mg/L) displayed greater lethality than derivative 4 (LC50 = 75.0 mg/L) toward A. aegypti larvae, while derivative 3 was inactive. These results suggest a novel application for these natural quassinoids as larvicides. The toxicity toward A. franciscana could be correlated with the activity in several biological models, a finding that is in agreement with the literature. Importantly, none of the studied compounds exhibited in vitro haemolytic activity, suggesting specificity of the observed cytotoxic effects. This study reveals the biological potential of quassinoids 1 and 2 and to a lesser extent their semi-synthetic derivatives for their in vitro antimalarial and cytotoxic activities.

Expansion of host range as a driving force in the evolution of Toxoplasma

The apicomplexan parasite Toxoplasma gondii is unusual in being able to infect almost any cell from almost any warm-blooded animal it encounters. This extraordinary host-range contrasts with its far more particular cousins such as the various species of the malaria parasite Plasmodium where each species of parasite has a single genus or even species of host that it can infect. Genetic and genomic studies have revealed a key role for a number of gene families in how Toxoplasma invades a host cell, modulates gene expression of that cell and successfully evades the resulting immune response. In this review, I will explore the hypothesis that a combination of sexual recombination and expansion of host range may be the major driving forces in the evolution of some of these gene families and the specific genes they encompass. These ideas stem from results and thoughts published by several labs in the last few years but especially recent papers on the role of different forms of rhoptry proteins in the relative virulence of F1 Toxoplasma progeny in a particular host species (mice).

In vitro antiplasmodial activity and toxicity assessment of plant extracts used in traditional malaria therapy in the Lake Victoria Region

As part of our program screening the flora of the Lake Victoria Region, a total of 54 organic extracts from seven plant families (8 species) were individually tested for antiplasmodial activity against chloroquine-sensitive [Sierra Leone (D-6)] and chloroquine-resistant [Vietnam (W-2)] strains. Only 22% of these extracts exhibited very high in vitro antiplasmodial activity. Six methanol (MeOH) extracts and one chloroform extract showed in vitro antiplasmodial activity against the D-6 Plasmodium falciparum strain, while only three MeOH extracts were active against the W-2 strain. All of the ethyl acetate extracts proved to be inactive against both strains of P. falciparum. A brine shrimp cytotoxicity assay was used to predict the potential toxicity of the extracts. The cytotoxicity to antiplasmodial ratios for the MeOH extracts were found to be greater than 100, which could indicate that the extracts are of low toxicity.

Malaria entomological inoculation rates in gold mining areas of Southern Venezuela

A longitudinal study of malaria vectors aiming to describe the intensity of transmission was carried out in five villages of Southern Venezuela between January 1999-April 2000. The man-biting, sporozoite and entomological inoculation rates (EIR) were calculated based on 121 all-night collections of anophelines landing on humans, CDC light traps and ultra violet up-draft traps. A total of 6,027 female mosquitoes representing seven species were collected. The most abundant species were Anopheles marajoara Galvão & Damasceno (56.7%) and Anopheles darlingi Root (33%), which together accounted for 89.7% of the total anophelines collected. The mean biting rate for An. marajoara was 1.27 (SD + 0.81); it was 0.74 (SD + 0.91) for An. darlingand 0.11 (SD + 0.10) for Anopheles neomaculipalpus Curry and the overall biting rate was 2.29 (SD + 1.06). A total of 5,886 mosquitoes collected by all three methods were assayed by ELISA and 28 pools, equivalent to 28 mosquitoes, yielded positive results for Plasmodium spp. CS protein. An. neomaculipalpus had the highest sporozoite rate 0.84% (3/356), followed by An. darlingi 0.82% (16/1,948) and An. marajoara 0.27% (9/3,332). The overall sporozoite rate was 0.48% (28/5,886). The rates of infection by Plasmodium species in mosquitoes were 0.37% (22/5,886) for Plasmodium vivax(Grassi & Feletti) and 0.10% (6/5,886) for Plasmodium falciparum (Welch). The estimated overall EIR for An. darling was 2.21 infective bites/person/year, 1.25 for An. marajoara and 0.34 for An. neomaculipalpus. The overall EIR was four infective bites/person/year. The biting rate, the sporozoite rate and the EIR are too low to be indicators of the efficacy of control campaigns in this area.

The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae)

The Anopheles (Nyssorhynchus) albitarsis complex includes six species: An. albitarsis, Anopheles oryzalimnetes Wilkerson and Motoki, n. sp., Anopheles marajoara, Anopheles deaneorum, Anopheles janconnae Wilkerson and Sallum, n. sp. and An. albitarsis F. Except for An. deaneorum, species of the complex are indistinguishable when only using morphology. The problematic distinction among species of the complex has made study of malaria transmission and ecology of An. albitarsis s.l. difficult. Consequently, involvement of species of the An. albitarsis complex in human Plasmodium transmission is not clear throughout its distribution range. With the aim of clarifying the taxonomy of the above species, with the exception of An. albitarsis F, we present comparative morphological and morphometric analyses, morphological redescriptions of three species and description of two new species using individuals from populations in Brazil, Paraguay, Argentina and Venezuela. The study included characters from adult females, males, fourth-instar larvae, pupae and male genitalia of An. albitarsis, An. marajoara, An. deaneorum and An. oryzalimnetes n. sp. For An. janconnae n. sp. only characters of the female, male and male genitalia were analyzed. Fourth-instar larvae, pupae and male genitalia characteristics of all five species are illustrated. Bionomics and distribution data are given based on published literature records.

Species composition and natural infectivity of anthropophilic Anopheles (Diptera: Culicidae) in the states of Córdoba and Antioquia, Northwestern Colombia

Malaria is a serious health problem in the states of Córdoba and Antioquia, Northwestern Colombia, where 64.4% of total Colombian cases were reported in 2007. Because little entomological information is available in this region, the aim of this work was to identify the Anopheles species composition and natural infectivity of mosquitoes distributed in seven localities with highest malaria transmission. A total of 1,768 Anopheles mosquitoes were collected using human landing catches from March 2007-July 2008. Ten species were identified; overall, Anopheles nuneztovari s.l. was the most widespread (62%) and showed the highest average human biting rates. There were six other species of the Nyssorhynchus subgenus: Anopheles albimanus (11.6%), Anopheles darlingi (9.8%), Anopheles braziliensis (6.6%), Anopheles triannulatus s.l. (3.5%), Anopheles albitarsis s.l. and Anopheles oswaldoi s.l. at < 1%; and three of the Anopheles subgenus: Anopheles punctimacula, Anopheles pseudopunctipennis s.l. and Anopheles neomaculipalpusat < 1% each. Two species from Córdoba, An. nuneztovari and An. darlingi, were found to be naturally infected by Plasmodium vivax VK247, as determined by ELISA and confirmed by nested PCR. All species were active indoors and outdoors. These results provide basic information for targeted vector control strategies in these localities.

Potential evidence of parasite avoidance in an avian malarial vector

Epidemiological studies of malaria or other vector-transmitted diseases often consider vectors as passive actors in the complex life cycle of the parasites, assuming that vector populations are homogeneous and vertebrate hosts are equally susceptible to being infected during their lifetime. However, some studies based on both human and rodent malaria systems found that mosquito vectors preferentially selected infected vertebrate hosts. This subject has been scarcely investigated in avian malaria models and even less in wild animals using natural host-parasite associations. We investigated whether the malaria infection status of wild great tits, Parus major, played a role in host selection by the mosquito vector Culex pipiens. Pairs of infected and uninfected birds were tested in a dual-choice olfactometer to assess their attractiveness to the mosquitoes. Plasmodium-infected birds attracted significantly fewer mosquitoes than the uninfected ones, which suggest that avian malaria parasites alter hosts' odours involved in vector orientation. Reaction time of the mosquitoes, that is, the time taken to select a host, and activation of mosquitoes, defined as the proportion of individuals flying towards one of the hosts, were not affected by the bird's infection status. The importance of these behavioural responses for the vector is discussed in light of recent advances in related or similar model systems.

Malaria seroprevalence in blood bank donors from endemic and non-endemic areas of Venezuela

In Venezuela, a total of 363,466 malaria cases were reported between 1999-2009. Several states are experiencing malaria epidemics, increasing the risk of vector and possibly transfusion transmission. We investigated the risk of transfusion transmission in blood banks from endemic and non-endemic areas of Venezuela by examining blood donations for evidence of malaria infection. For this, commercial kits were used to detect both malaria-specific antibodies (all species) and malaria antigen (Plasmodium falciparum only) in samples from Venezuelan blood donors (n = 762). All samples were further studied by microscopy and polymerase chain reaction (PCR). The antibody results showed that P. falciparum-infected patients had a lower sample/cut-off ratio than Plasmodium vivax-infected patients. Conversely, a higher ratio for antigen was observed among all P. falciparum-infected individuals. Sensitivity and specificity were higher for malarial antigens (100 and 99.8%) than for antibodies (82.2 and 97.4%). Antibody-positive donors were observed in Caracas, Ciudad Bolívar, Puerto Ayacucho and Cumaná, with prevalences of 1.02, 1.60, 3.23 and 3.63%, respectively. No PCR-positive samples were observed among the donors. However, our results show significant levels of seropositivity in blood donors, suggesting that more effective measures are required to ensure that transfusion transmission does not occur.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates.

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Thrombocytopenia in malaria: who cares?

Despite not being a criterion for severe malaria, thrombocytopenia is one of the most common complications of both Plasmodium vivax and Plasmodium falciparum malaria. In a systematic review of the literature, platelet counts under 150,000/mm³ ranged from 24-94% in patients with acute malaria and this frequency was not different between the two major species that affected humans. Minor bleeding is mentioned in case reports of patients with P. vivax infection and may be explained by medullary compensation with the release of mega platelets in the peripheral circulation by megakaryocytes, thus maintaining a good primary haemostasis. The speculated mechanisms leading to thrombocytopenia are: coagulation disturbances, splenomegaly, bone marrow alterations, antibody-mediated platelet destruction, oxidative stress and the role of platelets as cofactors in triggering severe malaria. Data from experimental models are presented and, despite not being rare, there is no clear recommendation on the adequate management of this haematological complication. In most cases, a conservative approach is adopted and platelet counts usually revert to normal ranges a few days after efficacious antimalarial treatment. More studies are needed to specifically clarify if thrombocytopenia is the cause or consequence of the clinical disease spectrum.