Performance of OptiMAL(R) in the diagnosis of Plasmodium vivax and Plasmodium falciparum infections in a malaria referral center in Colombia

Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL(R), is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL(R) in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL(R) for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL(R) in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.     

A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax

A multiplex PCR was designed for the differential diagnosis of the two parasite species by targeting the 18S rRNA gene with a set of primer combinations, amplifying DNA fragments of 1451-bp and 833-bp for P. falciparum and P. vivax, respectively. The sensitivity of this PCR test was high, as minimal as 0.1 parasite per one microliter of blood sample and a minimum of four copies of the target gene could be detected. For the diagnosis of mixed infection of two Plasmodium spp., there were no apparent competition or cross-reaction between the majority and minority Plasmodium species. The multiplex PCR was evaluated on 210 clinical samples and 60 normal controls. The PCR test yielded highly concordant results with microscopic examination, with the only one exception of a mixed (P. falciparum plus P. vivax) infection case, which was diagnosed as a single infection of P. falciparum by microscopy. We propose that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting P. falciparum and P. vivax.     

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.     

Purine-less death in Plasmodium falciparum induced by immucillin-H, a transition state analogue of purine nucleoside phosphorylase.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.     

Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya

We previously reported isolation of DNA probe which specifically recognizes Plasmodium falciparum and developed a simple method for its use. The sensitivity and specificity of this DNA probe method have now been extensively field tested in comparison with those of conventional microscopic examination of blood films in two separate studies in Malindi, Kenya, involving a total of 1179 patients. In the second study, which used improved techniques, sensitivity of the DNA probe was 89% when compared to microscopy. We conclude that the DNA probe method compares favorably with conventional microscopy in detecting parasite densities as low as 25 parasites per microliter of blood. A significant advantage of the DNA probe method is that it utilizes a standardized procedure which can simultaneously and reproducibly analyze a large number of samples without opportunity for significant reader bias.     

Plasmodium falciparum: analysis of B epitopes of a polypeptide antigen expressed in Escherichia coli, using monoclonal antibodies

Monoclonal antibodies were raised against a recombinant molecule corresponding to the polypeptide 72 kDa, previously described as possibly related to protection in Plasmodium falciparum infection. Selection of hybridoma cell lines was done by immunofluorescence to guarantee the reactivity of the monoclonal antibodies both against the recombinant and the native molecule of the parasite. Monoclonal antibodies were characterized by serological and immunochemical techniques. Competitive binding assays between monoclonal antibodies defined four different B epitopes. One epitope is specific for P. falciparum, a second is also present in P. vivax, while the two others seem to be ubiquitous and are also present in the rodent parasite P. chabaudi. The ubiquitous epitope 72.C is apparently the only one recognized by squirrel monkey sera presenting protective antibodies against the asexual blood infection by P. falciparum.     

Imported Malaria in the United Kingdom

Over 2,000 cases of imported malaria have been confirmed by blood examination. Ninety percent. of cases from tropical Africa were infected with P. falciparum. Most of the patients were Caucasians and had primary infections. All developed fever within a month after arrival and most of them within two weeks of arrival. In some patients malaria parasites were seen in routine blood films.Developing forms of P. falciparum were always present in the peripheral blood of patients suffering from a primary attack which was not diagnosed or treated until a week or more after the onset of fever.All deaths investigated were caused by P. falciparum and were primary infections.In not one of the P. falciparum infections did the victim continue taking prophylactic drugs for more than a few days after leaving the endemic area. Had drugs been continued for one month probably not a single overt case of P. falciparum would have occurred.A primary attack of P. falciparum malaria is seldom, if ever, classical in that the fever is never tertian and may resemble clinically many other diseases.Children in boarding-schools returning from the tropics should be supplied with prophylactic tablets and instructions to the matron. If there is an epidemic of a fever any students who have recently returned from the tropics should have a blood film examined for malaria.The risk of contracting malaria among drug addicts is considerable, especially with P. falciparum.     

Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR)

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.     

Molecular epidemiology of drug resistance markers of Plasmodium falciparum in Yunnan Province, China

ABSTRACT: BACKGROUND: The mutations in Plasmodium falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes were associated with anti-malaria drug resistance. The aim of this study was to investigate the prevalence of polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps and pfatp6 in Yunnan Province. Finger-prick blood samples were collected from malaria-positive patients from Yunnan Province in 2009-2010. Single-nucleotide polymorphisms (SNPs) in the resistance-related genes were analysed by various PCR-based methods. RESULTS: A total of 108 blood samples were collected. Although chloroquine has not been used to treat falciparum malaria for nearly 30 years, 95.3% of the parasites still carried the pfcrt K76T mutation, whereas the majority of isolates displayed the wild-type pfmdr1 N86 and D1246 sequences. The molecular level of sulphadoxine-pyrimethamine resistance in P. falciparum was high. The most prevalent mutation was pfdhfr C59R (95.9%), whereas the frequencies of the quadruple, triple and double mutants were 22.7% (N51I/C59R/S108N/I164L), 51.5% (N51I/C59R/S108N, N51I/C59R/I164L and C59R/S108N/ I164L) and 21.6% (N51I/ C59R, C59R/S108N and C59R/I164L), respectively. A437G (n=77) and K540E (n=71) were the most prevalent mutations in pfdhps, and 52.7% of the samples were double mutants, among which A437G/K540E was the most common double mutation (37/49). Quadruple mutants were found in 28.0% (26/93) of samples. A total of 8.6% of isolates (8/93) carried the S436A/A437G/A581G triple mutation. No mutations were found in pfatp6 codons 623 or 769, but another two mutations (N683K and R756K) were found in 4.6% (3/97) and 9.2% (6/97) of parasite isolates, respectively. CONCLUSIONS: This study identified a high frequency of mutations in pfcrt, pfdhfr and pfdhps associated with CQ and SP resistance in P. falciparum and no mutations linked to artemisinin resistance (pfatp6). Molecular epidemiology should be included in routine surveillance protocols and used to provide complementary information to assess the appropriateness of the current national anti-malarial drug policy.     

New prospects for the epidemiologic and immunologic study of malaria by the Western blotting technic

A new modification in western blotting technique now permits the analysis of micro samples of blood collected from inhabitants of malaria endemic areas using several different antigens of Plasmodium falciparum. Compared to Immuno Fluorescent Assay and to Immuno Enzymology, ELISA (using somatic antigens and exoantigens of P. falciparum) the western blotting method gives a more detailed analysis. It seems to open new prospects for seroepidemiological studies of human malaria and for the selection of antigenic fractions that may allow the preparation of a vaccine.     

Thermal properties of red blood cells infected by malaria parasites

Three membrane thermotropic transitions at 8, 20, and 40 degrees C have been detected in human red blood cells (RBC) by using spin-labeled stearic acids. Red blood cells infected in vitro by Plasmodium falciparum showed the disappearance of the 8 degrees C transition and a lowering of the 40 degrees C transition to 32 degrees C. The disappearance of the 8 degrees C transition was observed in synchronized cultures of P. falciparum trophozoites as well as in mouse RBC infected in vivo by an asynchronous population of P. berghei. Furthermore, erythrocytes infected by P. falciparum showed an increase in the phosphorylation of protein 4.1. This protein was shown previously to be involved in the 8 degrees C transition, (T. Forte, T. L. Leto, M. Minetti, and V. T. Marchesi, Biochemistry 24, 7876-7880 (1985). Our results suggest that the malaria parasite invasion produces a disorganization of the protein 4.1-membrane interaction.     

Plasmodium falciparum: generation of a cDNA library enriched in sporozoite-specific transcripts by directional tag subtractive hybridization

We have adapted the "directional tag subtractive hybridization" technique as a means of investigating stage-specific gene expression in Plasmodium falciparum. This technique utilizes unidirectional cDNA libraries cloned into separate lambda vectors and involves hydroxyapatite chromatographic separation of target antisense cDNA and driver sense strand cRNA followed by PCR amplification of cDNA sequences specific to the target stage. This technique enabled efficient subtraction of asexual blood stage sequences from a P. falciparum sporozoite cDNA library and led to identification of novel sporozoite sequences. This technique can be applied to study gene expression in parasite stages that are difficult to obtain routinely.     

A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii-infected mice

Efficacy of vaccine candidates against toxoplasmosis may be expressed in terms of reduction in cyst number in brains of animals vaccinated and then challenged with a cyst-forming strain of Toxoplasma gondii, compared to non-vaccinated animals. Cyst number generally has been determined by microscopic examination of brain homogenate samples, a technique which has a low sensitivity and is time-consuming. Here we describe a quantitative competitive PCR method, which allows quantifying T. gondii DNA in brain samples. The method uses a primer pair, which allows the amplification of a 301 bp fragment of the 35-fold repeated T. gondii B1 gene and an internal standard (non-homologous competitor) derived from phage lambda, which can be amplified using the same primers and whose size and G/C content are similar to that of the B1 target sequence. The method is sensitive (as few as 10 parasites can be quantified), reproducible, and is not affected by the presence of DNA extracted from mouse brain by means of a simple and rapid technique. It is suitable to quantify the parasite load in the brain of infected mice and to evaluate efficacy of toxoplasmosis vaccine candidates.     

Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh

A combination of chloroquine (CQ) and primaquine (PQ) had been used as the first-line treatment of uncomplicated Plasmodium falciparum malaria in Rangamati, Bangladesh until the end of 2004. Doctors or medical staffs had felt that CQ plus PQ had become less effective against uncomplicated falciparum malaria patients, but that it was more effective against the minority-indigenous patients than the Bengali patients. The efficacy of CQ plus PQ and the mutation status of the CQ resistance transporter (pfcrt) gene of infecting P. falciparum were, thus, investigated for 45 uncomplicated falciparum malaria patients in Rangamati in 2004. The total failure rate was 57.8%. One or two pfcrt sequences (CIETH and SMNTH at positions 72, 74-76, and 97, mutation underlined) with K76T mutation known to be related to CQ-resistant phenotype were detected in 38 patients' blood samples. Of the 38 patients, in total 15 patients (14/25 minority-indigenous and 1/13 Bengali patients) resulted in adequate clinical and parasitological response (ACPR). There was a statistically significant difference in ACPR rate between the minority-indigenous patients and the Bengali patients. P. falciparum with mutant or resistant pfcrt (pfcrt-resistant) was detected by PCR in blood samples on day 28 for 10 ACPR minority-indigenous patients but not for the only one Bengali ACPR patient, who all were infected with pfcrt-resistant P. falciparum on day 0. The minority-indigenous patients, but not Bengalis, are suggested to be often cured by CQ plus PQ, leaving a very few parasites detectable only by PCR, even when they are infected with pfcrt-resistant P. falciparum.     

Changing patterns of the IL-2/IL-2R pathway of lymphocyte activation following exposure to Plasmodium falciparum products: a study with squirrel monkey peripheral blood mononuclear cells

Abstract Squirrel monkeys are experimental hosts useful for studies on human malaria. In the present work, in vitro lymphocyte reactivity was measured by proliferation in the presence of Plasmodium falciparum -derived products, and found to depend on the previous malarial status of the animals and upon the source and/or the nature of the P. falciparum -derived material. Special attention was given to the IL-2/IL-2R pathway of lymphocyte activation. Culture supernatant from P. falciparum -parasitized erythrocytes exerted an inhibitory effect towards T lymphocytes obtained from P. falciparum -non-immune squirrel monkeys, when activated, for instance, by PHA. These lymphocytes did not incorporate tritiated thymidine (neither they did proliferate) although they expressed IL-2 α and β binding chains and secreted IL-2 (or at least TCGF). This inhibitory effect could not be rescued by the addition of rhIL-2, although the assayed lymphocytes could retain the ability to continue their cell cycle progression and divide after removal of the P. falciparum -derived inhibitory product(s). The incidence of anti-mitogenic molecules which impair the IL-2/IL-2R pathway of lymphocyte activation in malaria related processes is discussed.     

Filtration and agar embedding applied to fine needle aspirates for tumor diagnosis by electron microscopy. A combined technique.

OBJECTIVE: To develop a novel method for processing of fine needle aspirates subjected to electron microscopic (EM) study. STUDY DESIGN: Included 70 cases of poorly differentiated malignant tumors in which a definitive diagnosis was not possible on light microscopic (LM) examination and that thus required application of an ancillary technique such as FNA/EM, for diagnosis. We have established a novel method of processing, a technique of filtration through nylon mesh filters to eliminate red blood cells (RBCs) and necrotic debris, followed by agar well embedding to avoid loss of diagnostic material during processing without centrifugation at later steps after agar embedding, thus minimizing the time required for processing. It was successfully carried out in 70 cases. RESULTS: The combined technique was extremely effective in eliminating RBCs and necrotic debris. It also avoided further loss of valuable diagnostic material. An accurate diagnosis was rendered in 70 cases; that was not possible by LM alone. The whole procedure saves two to three hours of processing as centrifugation is not required after the agar embedding step. CONCLUSION: This technique was found to be cost- and time-effective, particularly suitable for developing countries, where financial resources are limited.     

Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.