Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most... more
Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon ru...
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Background: Available DNA isolation methods for Plasmodium involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of Plasmodium... more
Background: Available DNA isolation methods for Plasmodium involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction of Plasmodium DNA from dried filter paper spot (DBS), appropriate for resource-limited setups. Methods: The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed in terms of DNA quantity, PCR detection sensitivity, time requirement, cost effectiveness, labor intensiveness and degree of shearing. The reliability of this method was confirmed by comparing it with other in use methods for Plasmodium DNA isolation. Results: Pure DNA was obtained with this method, as exemplified by the absorbance ratio (260nm /280nm) of 1.2. The protocol produced digestible, PCR-grade genomic DNA, also found to be suitable for sequencing. DNA isolated remained stable and retained its integrity after...
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Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian and Latin American region. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often... more
Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian and Latin American region. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs- failure of detection in low transmission settings, and deletion of one of the target proteins- Histidine rich protein (HRP) are evident from multiple reports; thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides- C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5,458 cyclic constrained peptides (in duplicate) against P. falciparum infected sera. Peptides (C6, A8, B7- cyclic constrai...
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Background: Combined in-silico and in-vitro approaches were adopted to investigate the antiplasmodial activity of Catharanthus roseus and Tylophora indica plant extracts as well as their isolated components (vinblastine, vincristine and... more
Background: Combined in-silico and in-vitro approaches were adopted to investigate the antiplasmodial activity of Catharanthus roseus and Tylophora indica plant extracts as well as their isolated components (vinblastine, vincristine and tylophorine). </P><P> Methods: We employed molecular docking to prioritize phytochemicals from a library of 26 compounds against Plasmodium falciparum multidrug-resistance protein 1 (PfMDR1). Furthermore, Molecular Dynamics (MD) simulations were performed for a duration of 10 ns to estimate the dynamical structural integrity of ligand-receptor complexes. </P><P> Results: The retrieved bioactive compounds viz. tylophorine, vinblastin and vincristine were found to exhibit significant interacting behaviour; as validated by in-vitro studies on chloroquine sensitive (3D7) as well as chloroquine resistant (RKL9) strain. Moreover, they also displayed stable trajectory (RMSD, RMSF) and molecular properties with consistent interaction ...
A series of pyrazole-pyrazoline substituted with benzenesulfonamide were synthesized and evaluated for their antimalarial activity in vitro and in vivo. The compounds were active against both chloroquine (CQ) sensitive (3D7) and CQ... more
A series of pyrazole-pyrazoline substituted with benzenesulfonamide were synthesized and evaluated for their antimalarial activity in vitro and in vivo. The compounds were active against both chloroquine (CQ) sensitive (3D7) and CQ resistant (RKL-9) strains of Plasmodium falciparum. Seven compounds (7e, 7i, 7j, 7l, 7m, 7o and 7p) exhibiting ECless than 2 μM. A mechanistic study of compound 7o revealed that these compound act through the inhibition of β-hematin. The study indicated that these compounds can serve as lead compounds for further development of potent antimalarial drugs.
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Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism... more
Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.
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Antimalarial drug resistance is a serious challenge to malaria control worldwide. In vitro sensitivity assays provide an early indication of emerging drug resistance. In vitro susceptibility of field and culture adapted Plasmodium... more
Antimalarial drug resistance is a serious challenge to malaria control worldwide. In vitro sensitivity assays provide an early indication of emerging drug resistance. In vitro susceptibility of field and culture adapted Plasmodium falciparum isolates to different antimalarials was compared using two Methods: World Health Organization (WHO) micro-test (MARK III) and histidine rich protein II (HRP II) based enzyme- linked immunosorbent assay (ELISA). In total, 50 P. falciparum isolates were collected from five states, viz. Chhattisgarh, Meghalaya, Mizoram, Tripura and Odisha of India during December 2011-September 2014. The isolates were revived and evaluated for their susceptibility to chloroquine (CQ), monodesethylamodiaquine (AQ), mefloquine (MQ), quinine (QN) and artemisinin (ART) using the WHO micro-test (Mark III) and HRP II ELISA. The data were analyzed using non- linear regression analysis. The geometric mean (GM) IC50 values of different antimalarials for WHO Mark III assay w...
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A new series of oxazoline-pyrazoline hybrids (4a-p) were synthesized by condensation reaction of substituted oxazoline based chalcones (3a-m) and substituted hydrazines in methanol. Some of the compounds exhibited promising in vitro... more
A new series of oxazoline-pyrazoline hybrids (4a-p) were synthesized by condensation reaction of substituted oxazoline based chalcones (3a-m) and substituted hydrazines in methanol. Some of the compounds exhibited promising in vitro antimalarial activity for chloroquine sensitive CQ(S) (3D7) strain and chloroquine resistant CQ(R) (RKL9) strain. The most potent analogue 4i (IC50 0.322 μg/ml) exhibited significant in vivo antimalarial potential against Plasmodiumberghei mouse model. The stable complex of 4i with hematin (1:1 stoichiometry) suggests that heme may be one possible target for these hybrid compounds. The study has revealed potential of title compounds as lead for the development of antimalarial agents.
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Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous... more
Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds...