Clinostomum complanatum: Anthelmintic potential of curcumin on the infective progenetic metacercarial stage.

The emerging resistance against commonly used antiparasitic drugs has driven investigators to explore alternative approaches using plant-derived active ingredients. These compounds have been tested for antiviral, antibacterial, and anthelmintic properties, particularly against adult worms. However, their effects on larval forms have been neglected. Curcumin is a polyphenol that is a significant constituent of the rhizome of Curcuma longa and possesses various biological activities, including antioxidant, anti-inflammatory, anti-infectious, and anti-carcinogenic. In the present study, the anthelmintic potential of curcumin was tested in vitro for its efficacy against the zoonotically important larval form, the progenetic metacercariae of Clinostomum complanatum, which were procured from the forage fish, Trichogaster fasciatus. Curcumin produced time and concentration-dependent inhibition in the motility of treated metacercarial worms, with the maximum inhibition of motility reported at 60 µM along with a significant increase of (36-92%) in ROS and (57-112%) in GSH levels at the end of a period of 6 h. In contrast, curcumin at the highest concentration significantly inhibited the activities of the antioxidant and detoxification enzymes SOD (36%) and GST (16%), respectively, in addition to altering the polypeptide profile and inhibiting cysteine proteases. The tegumental surface appeared to be highly disrupted in curcumin-treated worms, exhibiting severe blebbing, shearing of the tegument, and spine erosion. Such changes would affect the tegumental functions and survival of worms in the hostile microenvironment. This would render worms more susceptible to host-mediated rejection responses. Based on the results of the present study, it is inferred that C. complanatum could serve as an excellent model for screening novel anthelmintic drugs against larval trematodes of great economic significance. Furthermore, we conclude that curcumin could be exploited as an excellent phytotherapeutic agent against the virulent larval form under investigation.

Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum.

Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55 kDa. The optimal pH and temperature are found to be 8.0 and 37 °C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15 min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50 °C and 60 °C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.

Conserved Antigenic Structure of Contemporary Wild Poliovirus Type 1 Strains Endemic in Pakistan.

BACKGROUND: Elimination of poliovirus in Pakistan and Afghanistan is challenged by notions against the role of oral poliovirus vaccine (OPV) in eradicating contemporary wild poliovirus (WPV) strains. METHODS: A total of 1055 WPV type 1 (WPV1) strains isolated between 2013 and 2018 were categorized into 68 antigenic groups and tested for neutralization by OPV-derived antibodies. Molecular docking was conducted to determine neutralization efficiency of antibodies against WPV. The clinical significance of WPV1 variants was assessed to ascertain their role in patient outcomes. RESULTS: We found that 88% of WPV1 strains isolated from paralytic children belonged to a single antigenic lineage identical to the WPV1 strain detected in 1993. WPV1 antigenic variants were effectively neutralized by OPV-derived antibodies, with geometric mean titers comparable to the neutralization titers found for 3 strains in OPV (OPV1-3, 7.96-9.149 [95% confidence interval, 6.864-10.171]; WPV1 strains, 7.542-8.786 [6.493-9.869]). Docking examination underscored a strong antigen-antibody interaction despite variations within the viral protein 1 epitopes. There was no significant association (P = .78) with clinical prognosis among patients infected with antigenically diverse WPV1 strains and patient outcomes, including death. CONCLUSIONS: Our findings substantiate the robustness of OPV for neutralizing the contemporary WPV1 strains endemic in Pakistan and Afghanistan. Vaccination coverage must be augmented to achieve early eradication.

Genetic Epidemiology Reveals 3 Chronic Reservoir Areas With Recurrent Population Mobility Challenging Poliovirus Eradication in Pakistan.

BACKGROUND: Pakistan is among 3 countries endemic for wild poliovirus type 1 (WPV1) circulation that are still struggling for eradication of poliomyelitis. Active clinical and environmental surveillance with meticulous laboratory investigations provide insights into poliovirus transmission patterns and genomic diversity to inform decisions for strategic operations required to achieve eradication. METHODS: We analyzed epidemiological and virological data to comprehend the current epidemiological status of WPV1 in Pakistan during 2015-2017. Stool specimens of patients with acute flaccid paralysis (AFP) and sewage samples collected from 60 environmental sites were tested. Viral culturing, intratypic differentiation by real-time polymerase chain reaction, and nucleic acid sequencing of the VP1 region of the poliovirus genome to determine genetic relatedness among WPV1 strains were applied. RESULTS: Poliovirus isolates were grouped into 11 distinct clusters, which had ≥95% nucleotide homology in the VP1 coding region. Most of the poliovirus burden was shared by 3 major reservoirs: Karachi, Peshawar, and Quetta block (64.2% in 2015, 75.4% in 2016, and 76.7% in 2017). CONCLUSIONS: Environmental surveillance reveals importations and pockets of unimmunized children that dictate intensive target mop-up campaigns to contain poliovirus transmission. A decrease in the number of orphan isolates reflects effective combination of AFP and environmental surveillance in Pakistan. The genetic data reflect sustained transmission within reservoir areas, further expanded by periodic importations to areas of high immunity reflected by immediate termination of imported viruses. Improved immunization coverage with high-quality surveillance is vital for global certification of polio eradication.

Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing.

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

In vitro anthelmintic effect of biologically synthesized silver nanoparticles on liver amphistome, Gigantocotyle explanatum.

In order to ensure global food security a rationale approach is required to control all those factors which directly or indirectly affect the food productivity. The neglected helminthic diseases alone are responsible for huge economic losses to the agrarian stakeholders. The problem is further compounded by the emerging drug resistance in flukes against the commonly used anthelmintics like triclabendazole. Therefore, the search for alternatives including the nano-based approaches has become a necessity to develop future control strategies. In the present study the effect of biologically synthesized silver nanoparticles (AgNPs) was investigated on an economically important amphistome parasite, Gigantocotyle explanatum, obtained from the infected liver of the Indian water buffaloes, Bubalus bubalis. In vitro treatment of the adult worms with different doses of AgNPs severely affected the worm motility and caused ROS mediated damages in the treated flukes. The antioxidant system and the detoxification ability of the worms appeared to be disrupted along with pronounced DNA damage in the treated worms as compared to the controls. Following the treatment of worms with different concentrations of AgNPs there was a significant (p < 0.05) increase in lipid peroxidation and protein carbonylation levels which are the key oxidative stress markers. The tegumental surface which is metabolically active, was severely damaged as evident from the loss of papillae, severe blebbing, shearing and erosion of the surface structures. Such topographical disruptions would facilitate the penetration of the nanoparticles deep within the tissues that might greatly reduce the invasive potential of the flukes as evident from the decreased motility. Taken together our findings suggest that the AgNPs posses great anthelmintic potential and could be further exploited for the development of anthelmintic formulations which may be tested in vivo.

Low virulence potential and in vivo transformation ability in the honey bee venom treated Clinostomum complanatum.

The helminth parasites possess great capabilities to adapt themselves within their hosts and also develop strategies to render the commonly used anthelmintics ineffective leading to the development of resistance against these drugs. Besides using anthelmintics the natural products have also been tested for their anti-parasitic effects. Therapeutic efficacy of honey bee venom (HBV) has been tested in various ailments including some protozoal infections but very little is known about its anthelmintic properties. To investigate the anthelmintic effect of HBV the excysted progenetic metacercariae of Clinostomum complanatum, a heamophagic, digenetic trematode with zoonotic potential, infecting a wide variety of hosts, were obtained from Trichogaster fasciatus, a forage fish, which serves as the intermediate host. The metacercarial worms were in vitro incubated in RPMI-1640 medium containing HBV along with the controls which were devoid of HBV for the analysis of worm motility, enzyme activity, polypeptide profile and surface topographical changes. The motility of the worms was significantly reduced in a time dependent manner with an increase in the concentration of HBV. Following incubation of worms the release of cysteine proteases was inhibited in the presence of HBV as revealed by gelatine substrate gel zymography. As well as the polypeptide profile was also significantly influenced, particularly intensity/expression of Mr 19.4 kDa, 24 kDa and 34 kDa was significantly reduced upon HBV treatment. The HBV treatment also inhibited antioxidant enzyme, superoxide dismutase (SOD) and Glutathione-S-transferase (GST) significantly (p < 0.05) in the worms. The scanning electron microscopy of the HBV treated worms revealed tegumental disruptions and erosion of papillae as well as spines showing vacuolation in the tegument. The HBV treated worms also showed a marked decline in the transformation rate when introduced into an experimental host which further reflect the anthelmintic potential of HBV.

Genomic Surveillance Elucidates Persistent Wild Poliovirus Transmission During 2013-2015 in Major Reservoir Areas of Pakistan.

BACKGROUND: Despite tremendous efforts in the fight against polio, Pakistan bears the highest proportion of poliomyelitis cases among the 3 endemic countries including Afghanistan and Nigeria. Apart from insecurity and inaccessibility challenges, the substantial shift of unimmunized children from North Waziristan due to recent military operations was presumed to favor the widespread poliovirus infection in Pakistan. METHODS: To better understand the current epidemiological situation, we analyzed the virologic data of wild poliovirus type 1 (WPV1) strains detected in Pakistan during 2013-2015. RESULTS: Five genetic clusters (A-E) were identified with at least 5% nucleotide divergence in the viral protein 1 (VP1) coding region. Peshawar, Quetta, and Karachi were found to be the major endemic foci where multiple discrete genetic lineages of WPV1 were detected. Phylogenetic analysis suggests that wild poliovirus strains from endemic regions were genetically distant (with 5%-15% VP1 nucleotide divergence) from those detected in North Waziristan cases, excluding the possibility of a recent progenitor of WPV1 instigating single-source transmission across the country. Orphan lineages detected in Rawalpindi, Lahore, Hyderabad, Sukkur, and Jacobabad revealed silent transmission and the need for vigilant surveillance. Sustenance of analogous genetic lineages over a period of 3 years highlights multiple unimmunized foci present to maintain viral genetic diversity. CONCLUSIONS: Our findings are inconsistent with the hypothesis that impoverished populations from North Waziristan serve as a possible determinant of widespread poliomyelitis infection in Pakistan and further emphasize the need to scale-up clinical and environmental surveillance as well as immunization activities.

Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction.

An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.

Detection of multiple cocirculating wild poliovirus type 1 lineages through environmental surveillance: impact and progress during 2011-2013 in Pakistan.

BACKGROUND: The environmental surveillance has proven to be a useful tool to identify poliovirus circulation in different countries and was started in Pakistan during July 2009 to support the acute flaccid paralysis (AFP) surveillance system. METHODS: Sewage samples were collected from 27 environmental sampling (ENV) sites and processed for poliovirus isolation through 2-phase separation method. Poliovirus isolates were identified as Sabin-like or wild type through real-time polymerase chain reaction (PCR). Wild-type strains were subjected to VP1 gene sequencing and phylogenetic analysis performed using MEGA 5.0. RESULTS: During 2011-2013, a total of 668 samples were collected from 4 provinces that resulted in 40% of samples positive for wild poliovirus type-1 (WPV-1). None of the samples were positive for WPV-3. The areas with high frequency of WPV-1 detection were Karachi-Gadap (69%), Peshawar (82%), and Rawalpindi (65%), whereas the samples from Quetta and Sukkur remained negative for WPV during 2013. Phylogenetic analysis revealed 3 major clusters with multiple poliovirus lineages circulating across different country areas as well as in bordering areas of Afghanistan. CONCLUSIONS: Environmental surveillance in Pakistan has been proven to be a powerful tool to detect WPV circulation in the absence of poliomyelitis cases in many communities. Our findings emphasize the need to continue and expand such surveillance activities to other high-risk areas in the country.

Thermodynamics and kinetic analysis of membrane: Challenges and perspectives.

The ultimate structure of the membrane is determined using two important effects: (i) thermodynamic effect and (ii) kinetic effect. Controlling the mechanism of kinetic and thermodynamic processes in phase separation is essential for enhancing membrane performance. However, the relationship between system parameters and the ultimate membrane morphology is still largely empirical. This review focuses on the fundamental ideas behind thermally induced phase separation (TIPS) and nonsolvent-induced phase separation (NIPS) methods, including both kinetic and thermodynamic elements. The thermodynamic approach to understanding phase separation and the effect of different interaction parameters on membrane morphology has been discussed in detail. Furthermore, this review explores the capabilities and limitations of different macroscopic transport models used for the last four decades to explore the phase inversion process. The application of molecular simulations and phase field to understand phase separation has also been briefly examined. Finally, it discusses the thermodynamic approach to understanding phase separation and the consequence of different interaction parameters on membrane morphology, as well as possible directions for artificial intelligence to fill the gaps in the literature. This review aims to provide comprehensive knowledge and motivation for future modeling work for membrane fabrication via new techniques such as nonsolvent-TIPS, complex-TIPS, non-solvent assisted TIPS, combined NIPS-TIPS method, and mixed solvent phase separation.

Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola gigantica.

Fasciola gigantica, responsible for the zoonotic disease fasciolosis, pose a great threat to the livestock and human health worldwide. The triclabendazole (TCBZ) has been used for decades as a broad spectrum anthelmintic to control this perilous disease but the emergence of resistance in flukes against TCBZ has prompted researchers across the world to explore for new drugs and antigenic targets. World Health Organization has strongly recommended the utilization of neurobiologically significant biomolecules as new drug/antigenic targets because of their significant role in the physiology of parasites. Monoamine Oxidase (MAO) is an important neurobiological enzyme which catabolizes aminergic neurotransmitters thus preventing prolonged excitation of neurons and in non-neuronal cells it prevents cellular toxicity due to accumulation of toxic monoamines. Owing to the important role of MAO in the survival and perpetuation of parasites, multipronged approaches were undertaken for the characterization of MAO-A in F. gigantica. The activity of MAO was found to be 1.5 times higher in the mitochondrial samples than the whole homogenate samples. The adult worms of the F. gigantica appeared to possess both the isoforms of MAO i.e., MAO-A and MAO-B. The zymographic studies revealed strong enzyme activity in its native state as assessed through prominent dark bands at 250KDa in the zymogram. The enzyme was also found to be highly immunogenic as revealed by high antibody titer at 1:6400 dilution. The immunogenicity of MAO-A enzyme was further established in the Western Blots in which a strong band of 50KDa was distinctly evident. Despite ubiquitous presence of MAO in F. gigantica some regions like tegumental surface and intestinal caecae displayed strong immunofluorescence as compared to other regions. The detection of MAO-A in the F. gigantica samples in Dot-Blot assay indicate a great potential of this molecule for the immunodiagnostics of fasciolosis, particularly in the field conditions. The enzyme activity was sensitive to the specific inhibitor clorgyline in a concentration dependant manner, particularly in the late incubation period. The zymographic results also exhibited similar trend. The strong intensity of spots in Dot-blots indicate high immunogenicity of the MAO protein. The intensity of bands/spots in the samples of worms treated with clorgyline also declined, clearly indicating that the tropical liver fluke possesses prominent MAO-A activity.

Artificial intelligence and structural design of inorganic hollow fiber membranes: Materials chemistry.

A key challenge is to produce the uniform morphology and regular pore design of inorganic hollow fiber membranes (HFMs) due to involvement of multiple parameters including, fabrication process and materials chemistry. Inorganic HFMs required technical innovations via novel structural design and artificial intelligence (AI) to produce the uniform structure and regular pore design. Therefore, this review aims at critical analysis on the most recent and relevant approaches to tackle the issues related to tune the morphology and pore design of inorganic HFMs. Structural design and evaluation of routes towards the dope suspension, spinning, and sintering of inorganic HFMs are critically analysed. AI, driving forces and challenges involved for harnessing of materials are revealed in this review. AI programs used for the prediction of pore design and performance of HFMs have also been explained in this review. Overall, this review will provide the understanding to build the equilibrium in spinning and sintering processes to control the design of micro-channels, and structural properties of inorganic HFMs. This review has great significance to control the new design of membranes via AI programs. This review also explain the inorganic membrane efficiency as algal-bioreactor.

Conjugated microporous polymer membranes for light-gated ion transport.

Inspired by the light-gated ion channels in cell membranes that play important roles in many biological activities, herein, we developed an artificial light-gated ion channel membrane out of conjugated microporous polymers. Through bottom-up design of the monomer molecular structure and by the electropolymerization method, the membrane pore size and thickness were precisely controlled on the molecular level. The obtained membrane exhibited uniform pore size and highly sensitive light-switchable response. The photoisomerization of the polymer chain resulted in a reversible "on and off" light control over the pore size and subsequently led to light-gated ion transport across the membrane for a series of ions including hydrogen, potassium, sodium, lithium, calcium, magnesium, and aluminum ions.

Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Environmental Surveillance Reveals Complex Enterovirus Circulation Patterns in Human Populations.

BACKGROUND: Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples. METHODS: We used a next-generation sequencing approach analyzing reverse transcriptase polymerase chain reaction products synthesized directly from sewage concentrates. RESULTS: We determined whole-capsid genome sequences of multiple enterovirus strains from all 4 A to D species present in environmental samples from the United Kingdom, Senegal, and Pakistan. CONCLUSIONS: Our results indicate complex enterovirus circulation patterns in human populations with differences in serotype composition between samples and evidence of sustained and widespread circulation of many enterovirus serotypes. Our analyses revealed known and divergent enterovirus strains, some of public health relevance and genetically linked to clinical isolates. Enteroviruses identified in sewage included vaccine-derived poliovirus and enterovirus D-68 stains, new enterovirus A71 and coxsackievirus A16 genogroups indigenous to Pakistan, and many strains from rarely reported serotypes. We show how this approach can be used for the early detection of emerging pathogens and to improve our understanding of enterovirus circulation in humans.

Evaluation of the bag-mediated filtration system as a novel tool for poliovirus environmental surveillance: Results from a comparative field study in Pakistan.

Poliovirus (PV) environmental surveillance (ES) plays an important role in the global eradication program and is crucial for monitoring silent PV circulation especially as clinical cases decrease. This study compared ES results using the novel bag-mediated filtration system (BMFS) with the current two-phase separation method. From February to November 2016, BMFS and two-phase samples were collected concurrently from twelve sites in Pakistan (n = 117). Detection was higher in BMFS than two-phase samples for each Sabin-like (SL) PV serotype (p<0.001) and wild PV type 1 (WPV1) (p = 0.065). Seventeen sampling events were positive for WPV1, with eight discordant in favor of BMFS and two in favor of two-phase. A vaccine-derived PV type 2 was detected in one BMFS sample but not the matched two-phase. After the removal of SL PV type 2 (SL2) from the oral polio vaccine in April 2016, BMFS samples detected SL2 more frequently than two-phase (p = 0.016), with the last detection by either method occurring June 12, 2016. More frequent PV detection in BMFS compared to two-phase samples is likely due to the greater effective volume assayed (1620 mL vs. 150 mL). This study demonstrated that the BMFS achieves enhanced ES for all PV serotypes in an endemic country.

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica.

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out.