Entomological assessment in 'hotspots' of four lymphatic filariasis endemic districts, Central Nepal during post-MDA surveillance.

BACKGROUND OBJECTIVES: Annual mass drug administration (MDA) is the main strategy to interrupt the transmission of lymphatic filariasis (LF) in the community. The main aim of monitoring the MDA program, for its effectiveness and interruption of LF is the post-MDA surveillance using antigen survey in children born after MDA. The latest technique of new research suggests that xenomonitoring is an effective tool for monitoring LF intervention. The objective of this study was to assess the W. bancrofti infection/or infectivity in vector mosquitoes by xenomonitoring during post-MDA surveillance. METHODS: A descriptive cross-sectional study was conducted in the hotspots of selected four districts of Central Nepal. A gravid trap technique was used for sampling mosquitoes. Infection/or infectivity was determined via the dissection of vector mosquitoes. Anopheles, Aedes, Armigerus and Culex species were collected from hotspots of four endemic districts, two from the hilly region (Lalitpur and Dhading) and two from Terai region (Bara and Mahottari) of Central Nepal. RESULTS: A total of 4450 mosquitoes belonging to four genera, Anopheles, Culex, Armigeres, and Aedes were collected from four hotspots. The distribution of Culex quinquefasciatus was found to be the highest, 88.9% (n=3955/4450) followed by Cx. vishnui (4.5%), Armigeres sp (5.8%), An. culicifascies (0.2%), Aedes spp (0.8%). The proportion of female mosquitoes trapped is significantly higher. A total of 3344 parous Cx. quinquefasciatus mosquitoes were dissected for any larval stage of W. bancrofti. We could not find any filarial infection in dissected mosquito samples. INTERPRETATION CONCLUSION: We conclude that the gravid trap is an efficient tool for the collection of gravid Cx. quinquefasciatus mosquitoes for xenomonitoring studies of filariasis endemic regions. Vector composition indicated a maximum number of vector mosquitoes of lymphatic filariasis were trapped compared with the other three species. Distribution and density of Cx. quinquefasciatus was found highest in four hotspots of endemic districts. None of the Cx. quinquefasciatus dissected were found to be infected by larval forms of filaria. Since the low levels of infection persistence in the human population in these hot spots, vector infection and infectivity can't be ignored. Microscopic xenomonitoring at a low level of infection persistent is less likely to be efficient so molecular xenomonitoring along with a large sample should be required in each of the hot spots of the districts. Additionally, area is receptive so further vector control intervention should be required to reduce the risk of resurgence of infection.

Assessment of microfilaremia in 'hotspots' of four lymphatic filariasis endemic districts of Nepal during post-MDA surveillance.

BACKGROUND: The lymphatic filariasis (LF) elimination program in all sixty-three endemic districts of Nepal was based on annual mass drug administration (MDA) using a combination of diethylcarbamazine (DEC) and albendazole for at least 5 years. The MDA program was started in the Parsa district of the Terai region and at least six rounds of MDA were completed between 2003 and 2017 in all filariasis endemic districts of Central Nepal. Transmission Assessment Survey (TAS) report indicated that circulating filarial antigen (CFA) prevalence was below the critical value i.e., ≤ 2% in selected LF endemic districts of Central Nepal. Based on the TAS report, antigen-positive cases were found clustered in the foci of those districts which were considered as "hotspots". Hence the present study was designed to assess microfilaremia in hotspots of four endemic districts of Central Nepal after the MDA program. METHODOLOGY AND PRINCIPAL FINDINGS: The present study assessed microfilaremia in hotspots of four endemic districts i.e. Lalitpur and Dhading from the hilly region and Bara and Mahottari from the Terai region of Central Nepal. Night blood samples (n = 1722) were collected by finger prick method from the eligible sample population irrespective of age and sex. Community people's participation in the MDA program was ensured using a structured questionnaire and chronic clinical manifestation of LF was assessed using standard case definition. Two districts one each from the hilly region (Lalitpur district) and Terai region (Bara district) showed improved microfilaria (MF) prevalence i.e. below the critical level (<1%) while the other two districts are still over the critical level. There was a significantly high prevalence of MF in male (p = <0.05) and ≥41 years of age group (p = <0.05) community people in the hotspots of four endemic districts. People who participated in the previous rounds of the MDA program have significantly low MF prevalence. The upper confidence limit of MF prevalence in all hotspots of four districts was above the critical level (>1%). Chronic clinical manifestation of LF showed significant association with the older age group (≥41 years) but not with sex. CONCLUSIONS: The study revealed LF transmission improved in hotspots of two districts while continued in others but the risk of LF resurgence cannot be ignored since the upper confidence level of MF prevalence is over 1% in all the hotspots studied districts. High MF prevalence is well correlated with the number of MDA rounds but not with the MDA coverage. Community people involved in MDA drug uptake in any previous and last rounds have significantly less MF infection. Hence it is recommended that before deciding to stop the MDA rounds it is essential to conduct the MF survey at the hotspots of the sentinel sites.

Assessment of antigenemia among children in four hotspots of filarial endemic districts of Nepal during post-MDA surveillance.

BACKGROUND: Sixty-three out of 77 districts reported lymphatic filariasis (LF) endemic in Nepal. Mass drug administration (MDA) with diethylcarbamazine (DEC) and albendazole (ALB) treatment program was continued for 6 to 11 rounds in these districts. Nepal government has stopped the MDA program based on the transmission assessment survey (TAS) report of 2014 and 2018 indicating Wuchereria bancrofti antigenemia prevalence < 2%. But the persistence of low levels of the circulating filarial antigen (CFA) in some foci of four endemic districts of Central Nepal, i.e., 0.4% in Dhading, 0.7% in Mahottari, 0.21% in Lalitpur and 1.2% in Bara district could responsible for enhancing the risk of infection resurgence. Hence the present study was designed to assess antigenic prevalence using Filariasis Test Strip (Alere, Scarborough ME) in children born after MDA in hotspot areas of four endemic districts of Central Nepal. RESULTS: The present study covers 70% children of the eligible population. The result revealed significantly high CFA prevalence in hotspots of Mahottari district belonging to the Terai region and Dhading district belonging to the hilly region, i.e., 13% and 10%, respectively, compared to baseline prevalence and TAS report. While in Lalitpur district and Bara district CFA prevalence was still found to be less than 2%. A higher number of MDA rounds covered in hotspots were found significantly associated with the low antigenic prevalence of W. bancrofti. Whereas median treatment coverage and inter-quartile range (IQR) in study districts were not found significantly associated with CFA prevalence. Although the clinical manifestation of hydrocele (1%) was found in all four study districts, it was not due to the W. bancrofti infection. CONCLUSIONS: Two hotspot regions, one each from the Terai (Mahottari) and hilly (Dhading) districts were found highly prevalent with CFA and significantly associated with the number of MDA rounds but were not associated with treatment coverage and IQR. Higher CFA prevalence was observed in hotspots where baseline prevalence was high together indicating that rounds of MDA program need to be extended further in these hotspot regions of endemic districts.

Development of peptide-based ratiometric fluorescent probe for sensing heparan sulfate and heparin in aqueous solutions at physiological pH and quantitative detection of heparan sulfate in live cells.

Heparan sulfate (HS) plays a critical role in various biological processes as a vital component of the extracellular matrix. In this study, we synthesized three fluorescent probes (1-3) comprising Arg-rich peptides as HS receptors and a fluorophore capable of exhibiting red-shifted emissions upon aggregation. All three probes demonstrated ratiometric responses to HS and heparin in aqueous solutions. Remarkably, probe 3 exhibited a unique ratiometric response to HS in both aqueous solutions at physiological pH and HS proteoglycans on live cells. Probe 3 displayed exceptional sensing properties, including high biocompatibility, water solubility, visible light excitation, a large Stokes shift for ratiometric detection and remarkable selectivity and sensitivity for HS (with a low limit of detection: 720 pM). Binding mode studies unveiled the crucial role of charge interactions between probe 3 and negatively charged HS sugar units. Upon binding, the fluorophore segments of the probes overlapped, inducing green and red emission changes through restricted intramolecular rotation of the fluorophore moiety. Importantly, probe 3 was effectively employed to quantify the reduction of HS proteoglycan levels in live cells by inhibiting HS sulfation using siRNA and an inhibitor. It successfully detected decreased HS levels in cells treated with doxorubicin and irradiation, consistent with results obtained from western blot and immunofluorescence assays. This study presents the first ratiometric fluorescent probe capable of quantitatively detecting HS levels in aqueous solutions and live cells. The unique properties of peptide-based probe 3 make it a valuable tool for studying HS biology and potentially for diagnostic applications in various biological systems.

Ratiometric Fluorescence Sensing System for Lead Ions Based on Self-Assembly of Bioprobes Triggered by Specific Pb2+-Peptide Interactions.

Lead is one of the most toxic substances. However, there are few ratiometric fluorescent probes for sensing Pb2+ in aqueous solution as well as living cells because specific ligands for Pb2+ ions have not been well characterized. Considering the interactions between Pb2+ and peptides, we developed ratiometric fluorescent probes for Pb2+ based on the peptide receptor in two steps. First, we synthesized fluorescent probes (1-3) based on the tetrapeptide receptor (ECEE-NH2) containing hard and soft ligands by conjugation with diverse fluorophores that showed excimer emission when they aggregated. After investigation of fluorescent responses to metal ions, benzothiazolyl-cyanovinylene was evaluated as an appropriate fluorophore for ratiometric detection of Pb2+. Next, we modified the peptide receptor to decrease the number of hard ligands and/or to replace Cys with disulfide bond and methylated Cys for improving selectivity and cell permeability. From this process, we developed two fluorescent probes (3 and 8) among the probes (1-8) that exhibited remarkable ratiometric sensing properties for Pb2+ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb2+, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb2+-peptide interactions of the probes caused nanosized aggregates in which the fluorophores of the probes came close each other, exhibiting excimer emission. In particular, 8 based on tetrapeptide bearing a disulfide bond and two carboxyl groups with a good permeability successfully quantified intracellular uptake of Pb2+ in live cells through ratiometric fluorescent signals. The ratiometric sensing system based on specific metal-peptide interactions and excimer emission process could provide a valuable tool to quantify Pb2+ in live cells and pure aqueous solutions.

Development of ratiometric fluorescent probes based on peptides for sensing Pb2+ in aquatic environments and human serum.

The detection of Pb2+ ions in aquatic environments and biofluid samples is crucial for assessment of human health. Herein, we synthesized two fluorescent probes (1 and 2) consisting of the peptide receptor for Pb2+ and a benzothiazolyl-cyanovinylene fluorophore that exhibited excimer-like emission when it aggregated. The peptide-based probes sensitively detected Pb2+ in purely aqueous solution (1% DMF) through ratiometric fluorescent response with a decrease in monomer emission at 520 nm and an increase in excimer emission at 570 nm. Specially, probe 2 showed remarkable detection features such as high selectivity for Pb2+over 15 metal ions, high binding affinity (Kd = 5.83 × 10-7 M) for Pb2+, significant emission intensity changes, low detection limit (3.8 nM) of Pb2+, high water solubility, and visible light excitation (450 nm). Probe 2 was successfully used to quantify nanomolar concentration (0 âˆ¼ 800 nM) of Pb2+ in real water samples (ground water and tap water). Specially, 2 was successfully applied for the quantification of Pb2+ in human serum by combination of microwave-assisted human serum digestion and filtration of digested serum by anion exchange cartridge. We clearly investigated the binding mode of 2 with Pb2+ using 1H NMR, IR spectroscopy, pH titration, confocal microscopy, and size analysis. The peptide-based fluorescent probe might have great application potential for sensing Pb2+ in aquatic environments and biofluid samples.

Ratiometric fluorescent detection of lead ions in aquatic environment and living cells using a fluorescent peptide-based probe.

Ratiometric fluorescent detection using dual emission bands is highly necessary to quantify Pb(II) in aquatic environment and live cells. We synthesized a ratiometric fluorescent peptidyl probe (1) by conjugation of a peptide receptor for Pb(II) with an excimer-forming benzothiazolylcyanovinylene fluorophore. The peptidyl probe dissolved well in aqueous solution and displayed an emission band at 538 nm (λex = 460 nm). Upon addition of Pb(II) (0-20 µM), the emission maximum shifted from 538 nm to 575 nm and the emission intensity ratio (I575 /I538) increased significantly from 0.40 to 2.26. 1 exhibited a selective ratiometric response to Pb(II) over other metal ions. 1 with a low detection limit (1.2 ppb) of Pb(II) detected nanomolar concentrations (0-500 nM) of Pb(II) ions in groundwater and tap water. The cell-permeable probe detected intracellular Pb(II) by ratiometric fluorescent images. The binding mode study using NMR, IR and CD spectroscopy, and TEM revealed that the probe formed a 1:1 complex with Pb(II) and then formed red-emissive nanoparticles and fibrils. The probe exhibited desirable detection properties such as ratiometric detection, high solubility in water, visible light excitation, high selectivity and sensitivity for Pb(II), cell-permeability, and rapid response (< 6 min).

Ratiometric fluorescent detection of silver nanoparticles in aqueous samples using peptide-based fluorogenic probes with aggregation-induced emission characteristics.

The quantification of silver nanoparticles and Ag+ contamination in the aquatic ecosystem has attracted considerable interest. Benzoimidazolyl-cyanovinylene (1) was synthesized as an aggregation-induced emission fluorophore, and a fluorescent peptidyl probe (2 and 3) bearing this fluorophore was developed. The fluorescent peptidyl probes coordinated with Ag+ selectively among various metal ions, leading to a ratiometric response to Ag+ in pure aqueous solutions. Furthermore, an "in situ" protocol was developed to quantify AgNPs using 2 with H2O2 as an oxidizing reagent. The fluorescent detection method for Ag+ and AgNPs showed promising detection properties such as high selectivity, high sensitivity, fast response, visible light excitation, well-operations in pure aqueous solution, and large fluorescent signal change. The detection limits of Ag+ (0.64 ppb) and AgNPs (1.1 ppb) were significantly low. According to the binding mode study, Ag+ induced the formation of a 2:1 complex between 2 and Ag+ and the chirality of the peptide part of the probe was not critical for this process. The formation of aggregates of the probe triggered by Ag+ from AgNPs induced a significant change in fluorescence. Furthermore, the amounts of spiked AgNPs in groundwater and tap water were quantified using the fluorescent detection method with 2.

Fast and sensitive fluorescent detection of inorganic mercury species and methylmercury using a fluorescent probe based on the displacement reaction of arylboronic acid with the mercury species.

We present a reaction-based fluorescent probe (1) for Hg2+ and CH3Hg+, based on the displacement reaction of the arylboronic acid with the mercury species. 1 showed promising sensing properties for Hg2+ and CH3Hg+, such as high selectivity and sensitivity, turn-on response, fast response to Hg2+ (<2 min) and CH3Hg+ (<5 min), low detection limits and operation in purely aqueous solutions.

Selective red-emission detection for mercuric ions in aqueous solution and cells using a fluorescent probe based on an unnatural peptide receptor.

The selective ratiometric red-emission detection of Hg2+ ions in aqueous buffered solutions and live cells is still a significant challenge. In the present study, we synthesized a fluorescent probe (1) based on an unnatural peptide receptor containing sulfonamide groups with an aggregation-induced emission and twisted internal charge transfer (TICT)-active fluorophore, cyanostilbene. 1 exhibited a highly selective ratiometric response to Hg2+ among 14 metal ions tested by ratiometric red-emission at 600 nm, with a clear isoemissive point in purely aqueous solution containing 1% DMSO. The ratiometric response for Hg2+ ions was complete within 3 min and the ratiometric responses induced by Hg2+ ions did not suffer considerable interference from the other metal ions. The ratiometric response was complete for less than 7 µM Hg2+ and 1 had a potent binding affinity (7.42 × 10-6 M, R2 = 0.98) for Hg2+ and a nanomolar detection limit. 1 detected Hg2+ ions by ratiometric responses in aqueous buffered solutions over a wide range of pH (5.5-11.5). Binding mode studies using TEM, NMR, IR, and a mass spectrometer revealed that the sulfonamide groups of the unnatural peptide receptor played an important role in the complexation of Hg2+ and in the complexation-induced nano-sized aggregates, which resulted in a significant increase in emissions at 600 nm and a decrease in emissions at 535 nm. 1 quantified micro-molar concentrations (0-6 µM) of Hg2+ in tap water and groundwater by ratiometric detection. Furthermore, 1 passed through the lipid membranes of live cells and detected intracellular Hg2+ ions at 2 µM by a ratiometric red-emission change.

Ratiometric red-emission fluorescence detection of Al3+ in pure aqueous solution and live cells by a fluorescent peptidyl probe using aggregation-induced emission.

The development of a fluorescence method for the selective ratiometric detection of Al3+ ions in pure aqueous solutions and live cells is still a significant challenge. In the present study, we synthesized a new type of fluorescent probe using an Al3+-triggered self-assembly based on the dipeptide receptor and an aggregation-induced emission fluorophore. The fluorescent probe (1) bearing cyanostilbene with excitation by visible light detected Al3+ ions sensitively in pure aqueous buffered solution by ratiometric red-emission at 600 nm. 1 provided a highly selective ratiometric detection of Al3+ among 16 metal ions in aqueous solution. 1 exhibited sensitive ratiometric response to Al3+ in aqueous buffered solutions at pH ranging from 5 to 7.4. The detection limit (145 nM, R2 = 0.999) for Al3+ ions in pure aqueous solution was much lower than the maximum allowable level of Al3+ in drinking water demanded by the Environmental Protection Agency (EPA). The probe provided an efficient approach to detect low concentrations of Al3+ in ground water, tap water, and live cells by ratiometric red-emissions at 600 nm. The binding study using dynamic light scattering, NMR, IR, and TEM revealed that the complex between 1 and Al3+ self-assembled to form nanoparticles, resulting in the enhancement of the emission at 600 nm and a concomitant decrease in the emission at 535 nm.

Highly Sensitive Ratiometric Fluorescent Detection of Indium(III) Using Fluorescent Probe Based on Phosphoserine as a Receptor.

Indium is one of the most widely used scarce metals for manufacturing various electronic devices including notebooks, mobile phones, and PC monitors. Recent studies revealed that indium and its compound could cause several toxicities to human beings and animals. However, there is no report about ratiometric fluorescent detection of In(III) in aqueous solutions. We synthesized a fluorescent probe (1) for In(III) based on a phosphoserine as a receptor with a pyrene fluorophore using solid phase synthesis. 1 showed highly sensitive ratiometric response to In(III) in purely aqueous solutions by increasing excimer emission intensity at 476 nm with a concomitant decrease in monomer emission intensity at 395 nm. 1 showed sensitive ratiometric responses to In(III) over a wide range of pH (2 < pH < 8) and exhibited a highly selective ratiometric response to In(III) among 18 tested metal including Al(III) and Ga(III). Job's plot analysis indicated that 1 preferred to form a 2:1 complex with In(III) and the binding affinity for In(III) was measured to be 2.3 × 1012 M-2 ( R2 = 0.989). 1 showed linear ratiometric responses to nanomolar concentrations (0-750 nM) of In(III) and the detection limit was calculated to be 64 nM ( R2 = 0.992) in aqueous solution. The binding mode study using NMR, IR, and CD spectroscopies revealed that the phosphate and the amide groups of the receptor of 1 played an important role for the binding with In(III). Moreover, 1 was suitable for the ratiometric detection of In(III) in tap water and groundwater. 1 showed much better detection properties than those of the colorimetric methods using EDTA with Eriochrome black T (EBT) and 4-(2-pyridylazo) resorcinol (PAR) for the detection of In(III) in tap water and groundwater.

Microfilaria persistent foci during post MDA and the risk assessment of resurgence in India.

BACKGROUND: Pondicherry, a union territory in India, is an endemic district for bancroftian lymphatic filariasis transmitted by Culex quinquefasciatus where eight rounds of mass drug administration (MDA) were completed in 2011 (annually once from 2004 to 2011).The objectives of this study were to conduct a focal survey to assess microfilaria and antigen (Ag) prevalence among young adults and to assess vector infection and infectivity through a focal entomological survey. METHODS: Mosquitoes were collected using gravid traps in Sedurapet village of Pondicherry and dissected to enumerate W. bancrofti larvae stage first larval stage (L1), second larval stage (L2), and third larval stage (L3). Microfilarias (Mf) were detected using blood smears collected from inhabitants. RESULTS: A total of 360 individuals from 67 houses were enrolled in this study of which 290 (80.6%) were surveyed for the presence of Mf. Two Mf carriers were detected yielding an overall prevalence of 0.69% and two out of 85 (2.35%) were Mf antigen positive. Of the 2875 mosquitoes collected by gravid trap, Culex quinquefasciatus (93.9%) was the predominant species, followed by Anopheles subpictus (2.3%) and Culex vishnui (3.8%). The density of Cx. quinquefasciatus was 28.1 per trap-night. A total of 2429 Cx. quinquefasciatus were dissected and microscopically examined for abdominal conditions (gravid 85%, semi-gravid 9.4%, unfed 3.8%, and fully fed 1.9%) and filarial infection. One mosquito (infection rate equal to 0.04%) was found to harbor a second stage filarial larva, and none of the mosquitoes had infective stage larva. CONCLUSION: Our results show no reappearance of infection of lymphatic filariasis in Sedurapet village of Pondicherry after MDA, and thus, no further intervention is required in that area for possible resurgence of lymphatic filariasis. However, monitoring should be continued as part of post MDA activities until the endpoint of complete elimination is achieved. We demonstrated that xenomonitoring can be used to monitor the post MDA situation for possible risk of transmission to initiate control measures.

Dual Role of a Fluorescent Peptidyl Probe Based on Self-Assembly for the Detection of Heparin and for the Inhibition of the Heparin-Digestive Enzyme Reaction.

The detection of fluorescent probes for biomolecules and control of the function of a complex through a recognition process have not been investigated intensively. A fluorescent peptidyl probe (1) based on the self-assembly stimulated by heparin was synthesized. The fluorescent probe with an aggregation-induced emission fluorophore formed a self-assembling complex with heparin, resulting in a sensitive and selective turn-on response to heparin compared to its biological competitors. The detection limits for heparin were measured to be 138.0 pM (R2 = 0.976) in aqueous solution and 2.6 nM (R2 = 0.996) in aqueous solution containing human serum. Nanosized aggregates formed through the self-assembly of the complex showed potent resistance against the heparin-digestive enzyme. The dual role of the probe for the detection of heparin and the inhibition of heparinase-mediated digestion through the recognition process was used for the real-time monitoring of the enzyme activity of heparinase for the digestion of heparin. Furthermore, the dual role of the probe was applied for the detection of the oversulfated chondroitin sulfate contaminant in heparin.

Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R2=0.99) in aqueous solutions, 2.98nM (R2=0.98) in 1% serum samples, and 3.43nM (R2=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.