BackgroundMalaria is highly prevalent in many parts of India and is mostly caused by the parasite species Plasmodium vivax followed by Plasmodium falciparum. Chloroquine (CQ) is the first-line treatment for blood stage P. vivax parasites, but cases of drug resistance to CQ have been reported from India. One of the surveillance strategies which is used to monitor CQ drug resistance, is the analysis of single nucleotide polymorphisms (SNPs) of the associated gene markers. Susceptibility to CQ can also be determined by copy number assessment of multidrug resistant gene (mdr-1). The current study has examined the prevalence of SNPs in P. vivax orthologs of P. falciparum chloroquine resistant and multi-drug resistant genes (pvcrt-o and pvmdr-1, respectively) and pvmdr-1 copy number variations in isolates from the highly endemic Mangaluru city near the South Western Coastal region of India.MethodsA total of 140 blood samples were collected from P. vivax infected patients attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of these 140 samples, sequencing was carried out for 54 (38.5%) and 85 (60.7%) isolates for pvcrt-o and pvmdr-1, respectively. Single nucleotide polymorphisms (SNPs) in the pvcrt-o and pvmdr-1 genes were analysed by direct sequencing method, while copy number variations of 60 isolates (42. 8%) were determined by real time PCR.ResultsOut of 54 clinical isolates analysed for pvcrt-o, three (5.6%) showed K10 insertion and the rest had wild type sequence. This is the first report to show K10 insertion in P. vivax isolates from India. Further, out of 85 clinical isolates of P. vivax analysed for mutations in pvmdr-1 gene, only one isolate had wild type sequence (~ 1%) while the remaining (99%) carried mutant alleles. Seven non-synonymous mutations with two novel mutations (I946V and Y1028C) were observed. Of all the observed mutations in pvmdr-1 gene, T958M was most highly prevalent (present in 90% of samples) followed by F1076L (76%), and Y976F (7%). Amplification of pvmdr-1 gene was observed in 31.6% of the isolates, out of 60 amplified.ConclusionThe observed variations both in pvmdr-1 and pvcrt-o genes indicate a trend towards parasite acquiring CQ resistance in this endemic area.

The precise role of autophagy in P. falciparum remains largely unknown. Although a limited number of autophagy genes have been identified in this apicomplexan, only PfAtg8 has been characterized to a certain extent. On the basis of the expression levels of PfAtg8 and the putative PfAtg5, we report that the basal autophagy in this parasite is quite robust and mediates not only the intraerythrocytic development but also fresh invasion of red blood cells (RBCs) in the subsequent cycles. We demonstrate that the basal autophagy responds to both inducers and inhibitors of autophagy. In addition, the parasite survival upon starvation is temporally governed by the autophagy status. Brief periods of starvation, which induces autophagy, help survival while prolonged starvation decreases autophagy leading to stalled parasite growth and reduced invasion. Thus, starvation-induced autophagy is context dependent. Importantly, we report characterization of another autophagy marker in this parasite, the putative PfAtg5 (Pf3D7_1430400). PfAtg5 is expressed in all the intraerythrocytic stages and partially colocalizes with ER, mitochondria, apicoplast and PfAtg8. It is also present on the double membrane bound vesicles. Altogether, these studies pave way for the detailed dissection of P. falciparum autophagy machinery and insights into molecular and functional characterization of its players for developing new therapeutics as antimalarials.

BackgroundGenes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) are the targets of sulfadoxine–pyrimethamine (SP) present in artemisinin based combination therapy (ACT; artesunate + sulfadoxine pyrimethamine) for Plasmodium falciparum. Although SP is generally not used to treat vivax infection, mutations in dhfr and dhps that confer antifolate resistance in Plasmodium vivax are common; which may be attributed to its sympatric existence with P. falciparum. Current study was aimed to determine the pattern of mutations in dhfr and dhps in P. vivax isolates from Mangaluru region.MethodsA total of 140 blood samples were collected from P. vivax-infected people attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of 140 isolates, 25 (18%) and 50 (36%) isolates were selected randomly for sequence analysis of pvdhfr and pvdhps genes respectively. Fragment of pvdhps and full length pvdhfr were amplified, sequenced and analysed for single nucleotide polymorphisms. dhps was analysed by PCR–RFLP also, to detect the two specific mutations (A383G and A553G).ResultsAnalysis of pvdhps sequences from 50 isolates revealed single and double mutants at 38 and 46% respectively. Three non-synonymous mutations (K55R, S58R and S117N) were identified for pvdhfr. Among these, K55R was detected for the first time.ConclusionsThe current study indicates that P. vivax dhps and dhfr mutant alleles are prevalent in this area, suggesting significant SP pressure.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2316-3) contains supplementary material, which is available to authorized users.

: The present study was planned to investigate and compare the protein profile viz. total proteins, albumin, globulin, albumin/globulin ratio, uric acid, urea and creatinine in follicular fluid and serum in buffaloes. The electrophoretic pattern of proteins in follicular fluid of different sizes of ovarian follicles and serum were also studied. Buffalo ovaries, at random stage of oestrous cycle and with unknown reproductive status were obtained from Deonar Abattoir, Chembur, Mumbai, during their e-visceration. Pairs of ovary from each buffalo were collected in separate sterile plastic bags. They were carried to the laboratory in thermos flask containing ice packs. In the laboratory, the ovaries were cut open at the hylus using a pair of sterile scissors. These were washed with tap water, distilled water and finally with deionised water. The follicles visible on its surface were classified based on their diameter as small (<5 mm), medium (5-10 mm) and large (>10 mm) follicles using digital vernier caliper. The fluids from these follicles were aspirated using 26 gauge-2 ml syringe. Twenty four samples each from small, medium and large sized follicles along with blood samples of buffaloes belonging to respective category were collected. Besides, blood samples from 12 buffaloes during mid-oestrous cycle from a private farm (Imom Son's Dairy Farm, Thane-400 604) were collected. Blood samples were allowed to clot at room temperature. Clear serum was separated by centrifugation at 1500 rpm for 30 min. The ovarian follicular fluid samples were centrifuged at 2000 rpm for 15 minutes in order to remove the cell debris. The follicular fluid and the blood samples were analysed for total proteins, albumin, globulin, albumin/globulin ratio, urea, creatinine and uric acid using STAT FAX 2000 Autoanalyser and kits. A random sample from the three classes of follicles each and a serum sample were used for protein fractionation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of the present study indicated that the protein components in the follicular fluid and serum exhibited increase / decrease in accordance with follicle size. The electrophoretic pattern of follicular fluid and serum showed significant difference between two fluid compartments. The small reservoir of fluid of follicles reflects the biochemical activity of the follicle. It is, therefore, suggested to carry out further studies to elucidate the precise role of these biochemical components and separated proteins which will help in understanding of the basic changes ongoing during follicular development, so that the optimal environment could be established for the maturation of viable oocytes.