Detection of Wuchereria bancrofti DNA in wild caught vector and non-vector mosquitoes: implications for elimination of lymphatic filariasis.

BACKGROUND: Transmission Assessment Survey (TAS) is the WHO recommended method used for decision-making to stop or continue the MDA in lymphatic filariasis (LF) elimination programme. The WHO has also recommended Molecular Xenomonitoring (MX) of LF infection in vectors as an adjunct tool in settings under post-MDA or validation period. Screening of non-vectors by MX in post-MDA / validation settings could be useful to prevent a resurgence of LF infection, as there might be low abundance of vectors, especially in some seasons. In this study, we investigated the presence of LF infection in non-vectors in an area endemic for LF and has undergone many rounds of annual MDA with two drugs (Diethylcarbamazine and Albendazole, DA) and two rounds of triple drug regimens (Ivermectin + DA). METHODS AND RESULTS: Mosquitoes were collected from selected villages of Yadgir district in Karnataka state, India, during 2019. A total of 680 female mosquitoes were collected, identified morphologically by species and separated as pools. The female mosquitoes belonging to 3 species viz., Anopheles subpictus, Culex gelidus and Culex quinquefaciatus were separated, pooled, and the DNA extracted using less expensive method and followed by LDR based real-time PCR assay for detecting Wuchereria bancrofti infection in vector as well as non-vector mosquitoes. One pool out of 6 pools of An. subpictus, 2 pools out of 6 pools of Cx. gelidus, and 4 pools out of 8 pools of Cx. quinquefaciatus were found to be positive for W. bancrofti infection by RT-PCR. The infection rate in vectors and non-vectors was found to be 1.8% (95% CI: 0.5-4.2%) and 0.9% (95% CI: 0.2-2.3%), respectively. CONCLUSIONS: Our study showed that non-vectors also harbour W. bancrofti, thus opening an opportunity of using these mosquitoes as surrogate vectors for assessing risk of transmission to humans in LF endemic and post MDA areas.

In Vitro Antiviral Activity of Potential Medicinal Plant Extracts Against Dengue and Chikungunya Viruses.

Dengue and chikungunya are two important mosquito-borne infections which are known to occur extensively in tropical and subtropical areas. Presently, there is no treatment for these viral diseases. In vitro antiviral screening of 25 extracts prepared from the plants of Vitex negundo, Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, Anacardium occidentale, Cucurbita maxima, Simarouba glauca, and Embelia ribes using different solvents and four purified compounds (anacardic acid, chloroquinone, glaucarubinone, and methyl gallate) were carried out for their anti-dengue virus (DENV) and anti-chikungunya virus (CHIKV) activities. Maximum nontoxic concentrations of the chloroform, methanol, ethyl acetate, petroleum ether, dichloromethane, and hydroalcoholic extracts of eight plants were used. The antiviral activity was assessed by focus-forming unit assay, quantitative real-time RT-PCR, and immunofluorescence assays. Extracts from Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, and Cucurbita maxima showed both anti-DENV and CHIKV activity while extract from Vitex negundo showed only anti-DENV activity. Among the purified compounds, anacardic acid, chloroquinone and methyl gallate showed anti-dengue activity while only methyl gallate had anti-chikungunya activity. The present study had identified the plant extracts with anti-dengue and anti-chikungunya activities, and these extracts can be further characterized for finding effective phytopharmaceutical drugs against dengue and chikungunya.

Multi-centric evaluation of a stage-specific reverse transcriptase-polymerase chain reaction assay as a xenomonitoring tool for the detection of infective (L3) stage Wuchereria bancrofti in vectors.

Background & objectives: An infective stage specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay utilizing the abundant larval transcript-3 (Alt-3) gene of Wuchereria bancrofti was developed at ICMR-VCRC, Puducherry and found to be stage specific, and sensitive upon validation in the laboratory. This study was aimed at independently evaluating this assay for its utility as a monitoring/surveillance tool in the operational programme for elimination of lymphatic filariasis (LF) by four national research laboratories. Methods: Evaluation of the assay was carried out in a multi-centric mode in three phases. In phase I, a workshop was conducted to impart hands-on training to the scientists from the collaborating centres on the RT-PCR assay and in Phase II the assay was evaluated for specificity and sensitivity in detecting the infective (L3) stage larvae of W. bancrofti in its vector, Culex quinquefasciatus, using 50 coded pooled samples. Phase III evaluation was done on wild-caught mosquito vectors from selected endemic areas of Assam and Bhubaneswar States and Andaman Nicobar islands. Results: Phase I data indicated that the assay was able to detect all the pools of mosquito samples contaning L3 stage larvae of W. bancrofti as positive, even in the presence of other vector stages of the parasite indicating its stage specificity (100%). The assay was found highly sensitive (100%), detecting all the infected pools as positive and specific detecting all uninfected pools as negative. The results of phase II showed inter-laboratory variation. Phase III evaluation from all the centres suggested that the infectivity rate determined for pooled mosquitoes by the RT-PCR assay (0.5%) was comparable to that by dissection method (1.2%) (95% confidence interval overlaps). Interpretation & conclusions: Overall, the results from three of the four participating centres indicated that the assay is at least as sensitive and stage specific as the conventional mosquito dissection technique, and hence, may be useful as a xenomonitoring tool for Transmission Assessment Survey in Mass Drug Administration programmes for LF.

Piperlongumine induces ROS mediated cell death and synergizes paclitaxel in human intestinal cancer cells.

Piperlongumine (PL), a herbal drug extracted from long pepper (Piper longum L), is known for its anti-inflammatory and anti-cancer properties. Although, its anti-cancer potential has been evaluated in cancer models like breast, pancreatic, gastric, hepatocellular and lung carcinoma, there is no report on its bio-activity evaluation in intestinal cancers. Here, we report the anti-neoplastic potential of PL against human intestinal carcinoma in-vitro and its possible mechanisms of action. Cytotoxicity studies demonstrate that PL inhibits cell proliferation of INT-407 and HCT-116 cells in a concentration and time-dependent manner. Also, PL elevated the levels of intracellular reactive oxygen species, which may lead to lethal oxidative stress, mitochondrial dysfunction, and nuclear fragmentation. Remarkably, P53, P21, BAX, and SMAD4 were significantly upregulated after PL treatment whereas; BCL2 and SURVIVIN were down-regulated. Moreover, the combination study also shows the synergistic effect of PL with the current chemotherapeutic drug paclitaxel. These findings suggest that PL possesses anti-neoplastic properties in intestinal cancer cells.

Evaluation of loop-mediated isothermal amplification assay for detection of scrub typhus in patients with acute febrile illness presenting to a Tertiary Care Center in Puducherry, India.

PURPOSE: Scrub typhus an acute febrile illness has diverse clinical manifestations, which overlap with other febrile illnesses. Due to this reason, it is misdiagnosed, leading to inappropriate treatment, sometimes resulting in fatality. Thus, accurate diagnosis of scrub typhus is important for appropriate treatment. This study evaluated the loop-mediated isothermal amplification (LAMP) assay as a diagnostic test for scrub typhus among patients with fever. MATERIALS AND METHODS: A total of 50 cases of acute febrile illness clinically resembling scrub typhus, with or without an eschar, or cases of pyrexia of unknown origin were included in the study. Blood samples collected from these cases were subjected to detection of IgM antibodies to Orientia tsutsugamushi by ELISA, conventional groEL polymerase chain reaction (PCR), and the LAMP assay. RESULTS: Twelve cases had fever for less than a week, and two had fever for more than 3 weeks. IgM antibodies to O. tsutsugamushi were detected in 37 out of 50 samples (74%). LAMP assay was positive in 33 samples (66%). groEL gene-based PCR detected 35 (70%) samples as positive. Two samples negative by LAMP assay were positive by this PCR. Twenty samples collected from patients with dengue, typhoid, and malaria tested by the LAMP assay were negative, indicating its good specificity. LAMP assay and the conventional groEL-based PCR could detect 72.7% and 74.3% of the samples, respectively before the 10th day after onset of fever, whereas IgM ELISA could detect only 40.5% of the 37 samples. CONCLUSION: This study suggests that LAMP assay could be a useful diagnostic test for detecting scrub typhus in the acute phase of the illness and a cheaper alternative to other molecular methods in resource poor settings.

Improving the Diagnosis of Scrub Typhus by Combining groEL Based Polymerase Chain Reaction and IgM ELISA.

INTRODUCTION: Scrub typhus, an acute febrile illness, caused by Orientia tsutsugamushi, is an important cause of pyrexia of unknown origin in regions of endemicity. This disease is mostly underdiagnosed or misdiagnosed, the reasons for this being a combination of factors which include clinical manifestations that can mimic other infections, lack of easy and reliable diagnostic methods and variation among strains in endemic areas. Hence, easy and reliable methods of diagnosis will contribute to rapid identification and treatment of the infection. AIM: The aim of the study was to compare four different methods of detection of scrub typhus and to identify one single test or a combination of tests detecting maximum number of cases. MATERIALS AND METHODS: One hundred and forty-five suspected scrub typhus cases were included in this study. Duration of fever and presence of eschar in each patient was noted down. Enzyme-Linked Immunosorbent Assay (ELISA) to detect Immunoglobulin M (IgM) antibodies and Polymerase Chain Reaction (PCR) to detect three genes of Orientia, namely, 56 kDa, 16S rRNA, and groEL were done on these samples. The results of each test were analyzed to identify the test picking up maximum number of positive samples. Statistical analysis was performed using Chi-square test. The level of significance was set at p<0.05. RESULTS: These tests showed that IgM ELISA (93%) and PCR (68%) picked up maximum number of positives. Statistical analysis performed using Chi-square test between the diagnostic assays showed that the p - value <0.001 was significant for IgM ELISA. Among the molecular markers, p-value was significant (<0.001) for groEL PCR. Further analysis of eschar positivity and duration of fever also showed that groEL PCR could detect DNA of the bacterium even in cases with 10 days of fever and this PCR was the best among the molecular markers used to detect the infection. CONCLUSION: This study suggests that IgM detection by ELISA and conventional groEL PCR, either in combination or alone, depending on the duration of fever, would enhance the diagnosis of scrub typhus.

Glaucarubinone sensitizes KB cells to paclitaxel by inhibiting ABC transporters via ROS-dependent and p53-mediated activation of apoptotic signaling pathways.

Multidrug resistance (MDR) is considered to be the major contributor to failure of chemotherapy in oral squamous cell carcinoma (SCC). This study was aimed to explore the effects and mechanisms of glaucarubinone (GLU), one of the major quassinoids from Simarouba glauca DC, in potentiating cytotoxicity of paclitaxel (PTX), an anticancer drug in KB cells. Our data showed that the administration of GLU pre-treatment significantly enhanced PTX anti-proliferative effect in ABCB1 over-expressing KB cells. The Rh 123 drug efflux studies revealed that there was a significant transport function inhibition by GLU-PTX treatment. Interestingly, it was also found that this enhanced anticancer efficacy of GLU was associated with PTX-induced cell arrest in the G2/M phase of cell cycle. Further, the combined treatment of GLU-PTX had significant decrease in the expression levels of P-gp, MRPs, and BCRP in resistant KB cells at both mRNA and protein levels. Furthermore, the combination treatments showed significant reactive oxygen species (ROS) production, chromatin condensation and reduced mitochondrial membrane potential in resistant KB cells. The results from DNA fragmentation analysis also demonstrated the GLU induced apoptosis in KB cells and its synergy with PTX. Importantly, GLU and/or PTX triggered apoptosis through the activation of pro-apoptotic proteins such as p53, Bax, and caspase-9. Our findings demonstrated for the first time that GLU causes cell death in human oral cancer cells via the ROS-dependent suppression of MDR transporters and p53-mediated activation of the intrinsic mitochondrial pathway of apoptosis. Additionally, the present study also focussed on investigation of the protective effect of GLU and combination drugs in human normal blood lymphocytes. Normal blood lymphocytes assay indicated that GLU is able to induce selective toxicity in cancer cells and in silico molecular docking studies support the choice of GLU as ABC inhibitor to enhance PTX efficacy. Thus, GLU has the potential to enhance the activity of PTX and hence can be a good alternate treatment strategy for the reversal of PTX resistance.

Resveratrol loaded gelatin nanoparticles synergistically inhibits cell cycle progression and constitutive NF-kappaB activation, and induces apoptosis in non-small cell lung cancer cells.

PURPOSE: Previously, we reported that the prepared resveratrol (RSV) loaded gelatin nanoparticles (GNPs) possessed enhanced anticancer effect than free RSV in non-small cell lung carcinoma cells and Swiss albino mice. The present study aims to explore the relevant mechanism of cell death induced by the combination of RSV-GNPs in NCI-H460 cells. METHODS AND RESULTS: To increase its bioavailability and anticancer efficacy, we have encapsulated RSV-GNPs by Coacervation method. The detailed methods of preparation and characterization of RSV-GNPs were reported in our earlier publication. RSV-GNPs treated cells showed a further increased level of lipid peroxidative markers, i.e. TBARS and LHP in NCI-H460 cells. Activities of antioxidant enzymes SOD, CAT, GPx and GSH levels were decreased upon the treatment with RSV-GNPs in NCI-H460 cells. The nuclear fragmentation was evaluated by DAPI staining and data showed condensed apoptotic bodies upon treatment with the combination of RSV-GNPs compared to RSV alone treatment group. In addition, cell death induced by RSV-GNPs was mainly due to apoptosis which was characterized by a nuclear DNA fragmentation in a ladder-pattern was obtained from the genomic DNA analysis. Moreover, Western blotting analysis showed that apoptosis induced by RSV-GNPs is associated with the increased Bax, p53, p21, caspase-3 protein levels, and decreased Bcl-2 and NF-κB proteins expression, which indicates the involvement of mitochondria-dependent apoptosis in the anticancer efficacy of RSV-GNPs in NCI-H460 cells. It was also found that this enhanced anticancer efficacy of RSV-GNPs induced cell arrest in the G0/G1 phase of cell cycle. CONCLUSIONS: Taken together, the results of our study clearly suggested that the cell death induced by the combination of RSV-GNPs would involve alteration in expression of p53, p21, caspase-3, Bax, Bcl-2 and NF-κB, indicating oxidative mechanism in NCI-H460 cells. Based on these results, it is concluded that GNPs is an ideal way to deliver RSV because of its high loading efficiency and superior efficacy in NCI-H460 cells.

Development of Fourth Generation ABC Inhibitors from Natural Products: A Novel Approach to Overcome Cancer Multidrug Resistance.

Multidrug resistance (MDR) in cancer caused due to overexpression of ABC drug transporters is a major problem in modern chemotherapy. Molecular investigations on MDR have revealed that the resistance is due to various transport proteins of the ABC superfamily which include Phosphoglycoprotein (P-gp/MDR1/ ABCB1), multidrug resistance-associated protein-1 (MRP1), and the breast cancer resistance protein (BCRP). They have been characterized functionally and are considered as major players in the development of MDR in cancer cells. These ATP-dependent transporter proteins cause MDR either by decreased uptake of the drug or increased efflux of the drug from the target organelles. Several MDR-reversing agents are being developed and are in various stages of clinical trials. The first three generations of ABC modulators such as quinine, verapamil, cyclosporine-A, tariquitor, PSC 833, LY335979, and GF120918 required to be administered in high doses to reverse MDR and were associated with adverse effects. Additionally, these modulators non-selectively inhibit ABC and adversely accumulate chemotherapeutic drugs in brain and kidney. Currently, research has stepped up towards reversing MDR by using natural products which exhibitted potential as chemosensitizers. Globally, there is a rich biodiversity of natural products which can be sourced for developing drugs. These products may provide more lead compounds with superior activity, foremost to the development of more effective therapies for MDR cancer cells. Here, we briefly review the status of natural products for reversing MDR modulators, and discuss the long term goal of MDR strategies in current clinical settings.

Functional analysis of a highly conserved abundant larval transcript-2 (alt-2) intron 2 repeat region of lymphatic filarial parasites.

The filarial-specific protein abundant larval transcript-2 (ALT-2) is expressed exclusively in the infective larval stage (L3) and is a crucial protein for establishing immunopathogenesis in human hosts. The alt-2 gene has a conserved minisatellite repeat (29 or 27bp) in intron 2 (IR2) whose significance within lymphatic filarial species is unknown. Here, we report the role of IR2 in the regulation of alt-2 gene expression using an in vitro model. Using electrophoretic mobility shift assays, we identified the presence of a putative nuclear protein binding region within IR2. Subsequent transient expression experiments in eukaryotic cell lines demonstrated that the IR2 downregulated the expression of a downstream luciferase reporter gene, which was further validated with RT-PCR. We therefore identify IR2 as a suppressor element that regulates L3 stage-specific expression of alt-2.

Functional analysis of genetic polymorphism in Wuchereria bancrofti glutathione S-transferase antioxidant gene: impact on protein structure and enzyme catalysis.

Wuchereria bancrofti glutathione S-transferase (Wb-GST) is referred as a promising chemotherapeutic target for lymphatic filariasis. GST represents the major class of detoxifying enzymes of the tissue dwelling parasitic helminths. Though many inhibition studies were carried out for Wb-GST, understanding its genetic distribution in parasite population is necessary to develop ideal inhibitor. Our genetic polymorphic studies exposed the existence of three variant Wb-GST alleles in the four endemic regions of India. Moreover, it also revealed the variability in the distribution of Wb-GST alleles in the studied population. Therefore we cloned, expressed and purified the recombinant variant Wb-GST proteins to study the mutation impact on its structure and hence on its catalysis. Among the studied mutations, the I60F/G78S substitutions in the N-terminal domain and loop region connecting the two domains of Wb-GST lowered the affinity for glutathione and its analog, S-hexyl glutathione. Moreover, molecular modeling and docking studies revealed that the I60F/G78S mutations affected the proximity of Trp38 and Arg95 in glutathione binding site resulting in weaker interaction with S-hexyl glutathione. Besides, the variants also had lower affinity (Ki) and higher IC50 values for well-known GST inhibitors. Interestingly, the Wb-GST variant proteins showed enhanced catalytic efficiency for lipid peroxidation products which are produced due to oxidative stress. Thus, our study provides evidence for the functional impact of mutations on Wb-GST protein and also spotlights the mechanisms of parasite survival against the host oxidative stress environment.

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection.

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Lymphatic filarial species differentiation using evolutionarily modified tandem repeats: generation of new genetic markers.

Polymerase chain reaction based methods are promising tools for the monitoring and evaluation of the Global Program for the Elimination of Lymphatic Filariasis. The currently available PCR methods do not differentiate the DNA of Wuchereria bancrofti or Brugia malayi by a single PCR and hence are cumbersome. Therefore, we designed a single step PCR strategy for differentiating Bancroftian infection from Brugian infection based on a newly identified gene from the W. bancrofti genome, abundant larval transcript-2 (alt-2), which is abundantly expressed. The difference in PCR product sizes generated from the presence or absence of evolutionarily altered tandem repeats in alt-2 intron-3 differentiated W. bancrofti from B. malayi. The analysis was performed on the genomic DNA of microfilariae from a number of patient blood samples or microfilariae positive slides from different Indian geographical regions. The assay gave consistent results, differentiating the two filarial parasite species accurately. This alt-2 intron-3 based PCR assay can be a potential tool for the diagnosis and differentiation of co-infections by lymphatic filarial parasites.