In Silico Analysis Reveals High Levels of Genetic Diversity of Plasmodium knowlesi Cell Traversal Protein for Ookinetes and Sporozoites (PkCelTOS) in Clinical Samples.

The cell-traversal protein for ookinetes and sporozoites (CelTOS), expressed on the surface of ookinetes and sporozoitesin Plasmodium species, is a promising malaria vaccine candidate. CelTOS is essential for parasite invasion into mosquito midgut and human hepatocytes, thereby contributing to malaria transmission and disease pathogenesis. This study explores the genetic diversity, polymorphisms, haplotypes, natural selection, phylogenetic analysis, and epitope prediction in the full-length Plasmodium knowlesi CelTOS gene in clinical samples from Sarawak, Malaysian Borneo, and long-term laboratory strains from Peninsular Malaysia and the Philippines. Our analysis revealed a high level of genetic variation in the PkCelTOS gene, with a nucleotide diversity of π ~ 0.021, which was skewed towards the 3' end of the gene. This level of diversity is double that observed in PfCelTOS and 20 times that observed in PvCelTOS from worldwide clinical samples. Tests of natural selection revealed evidence for positive selection within clinical samples. Phylogenetic analysis of the amino acid sequence of PkCelTOS revealed the presence of two distinct groups, although no geographical clustering was observed. Epitope prediction analysis identified two potential epitopes (96AQLKATA102 and 124TIKPPRIKED133) using the IEDB server and one epitope (125IKPPRIKED133) by Bcepred server on the C' terminal region of PkCelTOS protein. Both the servers predicted a common epitope region of nine amino acid length (IKPPRIKED) peptide, which can be studied in the future as a potential candidate for vaccine development. These findings shed light on the genetic diversity, polymorphism, haplotypes, and natural selection within PkCelTOS in clinical samples and provide insights about its future prospects as a potential candidate for P. knowlesi malaria vaccine development.

Design and development of novel N-(4-aminobenzoyl)- l-glutamic acid conjugated 1,3,5-triazine derivatives as Pf-DHFR inhibitor: An in-silico and in-vitro study.

In the present work, a library of 120 compounds was prepared using various aliphatic and aromatic amines. Finally, 10 compounds were selected through in silico screening carrying 4-aminobenzoyl-l-glutamic acid and 1,3,5-triazine moiety. The docking results of compounds 4d16 and 4d38 revealed higher binding interaction with amino acids Asp54 (-537.96 kcal/mol) and Asp54, Phe116 (-618.22 kcal/mol) against wild (1J3I) and quadruple mutant (1J3K) type of Pf-DHFR inhibitors and were comparable to standard WR99210. These compounds were developed by facile and microwave-assisted synthesis via nucleophilic substitution reaction and characterized by different spectroscopic methods. In vitro antimalarial assay results also suggested that these two compounds were having higher antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strain out of the ten synthesized compounds with IC50 13.25 µM and 14.72 µM, respectively. These hybrid scaffolds might be useful in the lead discovery of a new class of Pf-DHFR inhibitors.

Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development.

BACKGROUND: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen. METHODS: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique. RESULTS: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation. CONCLUSIONS: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

Microwave-assisted synthesis of hybrid PABA-1,3,5-triazine derivatives as an antimalarial agent.

The present manuscript deals with the development of novel p-aminobenzoic acid (PABA) associated 1,3,5-triazine derivatives as antimalarial agents. The molecules were developed via microwave-assisted synthesis and structures of compounds were ascertained via numerous analytical and spectroscopic techniques. The synthesized compounds were also subjected to ADMET analysis. In a docking analysis, the title compounds showed high and diverse binding affinities towards wild (-162.45 to -369.38 kcal/mol) and quadruple mutant (-165.36 to -209.47 kcal/mol) Pf-DHFR-TS via interacting with Phe58, Arg59, Ser111, Ile112, Phe116. The in vitro antimalarial activity suggested that compounds 4e, 4b, and 4h showed IC50 ranging from 4.18 to 8.66 µg/ml against the chloroquine-sensitive (3D7) strain of Plasmodium falciparum. Moreover, compounds 4g, 4b, 4e, and 4c showed IC50 ranging from 8.12 to 12.09 µg/ml against chloroquine-resistant (Dd2) strain. In conclusion, our study demonstrated the development of hybrid PABA substituted 1,3,5-triazines as a novel class of Pf-DHFR inhibitor for antimalarial drug discovery.

Sputum Proteomics Reveals a Shift in Vitamin D-binding Protein and Antimicrobial Protein Axis in Tuberculosis Patients.

Existing understanding of molecular composition of sputum and its role in tuberculosis patients is variously limited to its diagnostic potential. We sought to identify infection induced sputum proteome alteration in active/non tuberculosis patients (A/NTB) and their role in altered lung patho-physiology. Out of the study population (n = 118), sputum proteins isolated from discovery set samples (n = 20) was used for an 8-plex isobaric tag for relative and absolute concentration analysis. A minimum set of protein with at least log2(ATB/NTB) >±1.0 in ATB was selected as biosignature and validated in 32 samples. Predictive accuracy was calculated from area under the receiver operating characteristic curve (AUC of ROC) using a confirmatory set (n = 50) by Western blot analysis. Mass spectrometry analysis identified a set of 192 sputum proteins, out of which a signature of ß-integrin, vitamin D binding protein:DBP, uteroglobin, profilin and cathelicidin antimicrobial peptide was sufficient to differentiate ATB from NTB. AUC of ROC of the biosignature was calculated to 0.75. A shift in DBP-antimicrobial peptide (AMP) axis in the lungs of tuberculosis patients is observed. The identified sputum protein signature is a promising panel to differentiate ATB from NTB groups and suggest a deregulated DBP-AMP axis in lungs of tuberculosis patients.

Estimation of biofilm, proteinase & phospholipase production of the Candida species isolated from the oropharyngeal samples in HIV-infected patients.

BACKGROUND & OBJECTIVES: Candida, the most common opportunistic infection in acquired immunodeficiency syndrome (AIDS), attributes its pathogenicity to its virulence factors, mainly the biofilms, the proteinases and the phospholipases. There is a significant interplay of these factors during the HIV infection. This study was aimed to estimate the biofilm, proteinase and phospholipase production in Candida species isolated from the oropharyngeal samples in the HIV-infected patients. METHODS: A total of 126 consecutive HIV-positive patients were screened for Candida growth using oropharyngeal swabs. Identification was done by Gram staining, germ tube test, chlamydospore identification, chromagar and biochemical tests on Vitek 2. Biofilm production was observed on Sabouraud's dextrose broth with glucose, phospholipase production in egg yolk agar medium and proteinase production in bovine serum albumin agar medium. RESULTS: Of a total of 126 patients, 53 (42.06%) showed Candida growth: Candida albicans (n=46, 86.8%) was most common followed by the non-albicans Candida (NAC) (n=7, 13.93%). Of a total 33 (62.3%) biofilm positive isolates, significant production was observed in the NAC species (P <0.05). C. albicans reported the highest phospholipase (n=37/41, 90.24%) and proteinase (n=37/43, 86%) activities in a total of 41 (77%) phospholipase positive and 43 (81.1%) proteinase positive isolates. INTERPRETATION & CONCLUSIONS: Although C. albicans was the most common Candida species identified in HIV positive patients, the emergence of NAC was of special concern. Virulence factors such as biofilms, proteinases and phospholipases were noted in both these groups. Further research is required for better understanding of the pathogenic role of Candida species so as to aid in therapeutic interventions.

Bacterial Pathogens Associated with Community-acquired Pneumonia in Children Aged Below Five Years.

OBJECTIVE: To determine the spectrum of bacterial pathogens causing community-acquired pneumonia in children below 5 years of age. METHODS: Children aged below 5 years satisfying the WHO criteria for pneumonia, severe pneumonia or very severe pneumonia, and with the presence of lung infiltrates on chest X-ray were enrolled. Two respiratory samples, one for culture and the other for PCR analysis, and a blood sample for culture were collected from every child. RESULTS: Of the 180 samples processed, bacterial pathogens were detected in 64.4%. Streptococcus pneumoniae and Hemophilus influenzae were most frequently detected. The performance of PCR analysis and culture were identical for the typical bacterial pathogens; atypical pathogens were detected by PCR analysis only. CONCLUSION: S. pneumoniae and H. influenza were the most commonly detected organisms from respiratory secretions of children with community acquired pneumonia.