Wastewater based environmental surveillance of toxigenic Vibrio cholerae in Pakistan.

BACKGROUND: Pakistan has been experiencing intervals of sporadic cases and localized outbreaks in the last two decades. No proper study has been carried out in order to find out the environmental burden of toxigenic V. cholerae as well as how temporal and environmental factors associated in driving cholera across the country. METHODS: We tested waste water samples from designated national environment surveillance sites in Pakistan with RT-PCR assay. Multistage sampling technique were utilized for samples collection and for effective sample processing Bag-Mediated Filtration system, were employed. Results were analysed by district and month wise to understand the geographic distribution and identify the seasonal pattern of V. cholera detection in Pakistan. RESULTS: Between May 2019, and February 2020, we obtained and screened 160 samples in 12 districts across Pakistan. Out of 16 sentinel environmental surveillance sites, 15 sites showed positive results against cholera toxigenic gene with mostly lower CT value (mean, 34±2) and have significant difference (p < 0.05). The highest number of positive samples were collected from Sindh in month of November, then in June it is circulating in different districts of Pakistan including four Provinces respectively. CONCLUSION: V. cholera detection do not follow a clear seasonal pattern. However, the poor sanitation problems or temperature and rainfall may potentially influence the frequency and duration of cholera across the country. Occurrence of toxigenic V. cholerae in the environment samples showed that cholera is endemic, which is an alarming for a potential future cholera outbreaks in the country.

Improving Immunoassay Performance with Cleavable Blocking of Microarrays.

Among the key issues that are commonly associated with the development of microarray-based assays are nonspecific binding and diffusion constraints. Here we present a novel strategy addressing both of these challenges simultaneously. The essence of the method consists in blocking the microarray surface with a blocking agent containing a perfluoroalkyl chain and a disulfide linker. The resulting surface is hydrophobic, and no immiscible liquid layer remains on it upon cyclically draining and replenishing the sample solution, ensuring an efficient mass transfer of an analyte onto a microarray. Prior to the signal detection procedure, disulfide bonds are chemically cleaved, and the perfluoroalkyl chains are removed from the microarray surface along with nonspecifically adsorbed proteins, resulting in extremely low background. Using conventional fluorescent detection, we show a 30-fold increase in signal/background ratio compared to a common epoxy-modified glass substrate. The combination of this technique with magnetic beads detection results in a simple and ultrasensitive cholera toxin (CT) immunoassay. The limit of detection (LOD) is 1 fM, which is achieved with an analyte binding time of 1 h. Efficient mass transfer provides highly sensitive detection of whole virus particles despite their low diffusion coefficient. The achieved LOD for vaccinia virus is 104 particles in 1 mL of sample. Finally, we have performed for the first time the simultaneous detection of whole virus and CT protein biomarker in a single assay. The developed technique can be used for multiplex detection of trace amounts of pathogens of various natures.

Janus Micromotors Coated with 2D Nanomaterials as Dynamic Interfaces for (Bio)-Sensing.

In this work, we study the interaction of graphdiyne oxide (GDYO)-, graphene oxide (GO)-, or black phosphorous (BP)-wrapped Janus micromotors using a model system relying on a fluorescence-labeled affinity peptide, which is released upon specific interaction with a target Cholera Toxin B. Such ON-OFF-ON system allows mimicking similar processes occurring at (bio)-interfaces and to study the related sorption and desorption kinetics. The distinct surface properties of each nanomaterial play a critical role in the loading/release capacity of the peptide, greatly influencing the release profiles. Sorption obeys a second-order kinetic model using the two-dimensional (2D) nanomaterials in connection with micromotors, indicating a strong influence of chemisorption process for BP micromotors. Yet, release kinetics are faster for GDYO and GO nanomaterials, indicating a contribution of π and hydrophobic interactions in the probe sorption (Cholera Toxin B affinity peptide) and target probe release (in the presence of Cholera Toxin B). Micromotor movement also plays a critical role in such processes, allowing for efficient operation in low raw sample volumes, where the high protein content can diminish probe loading/release, affecting the overall performance. The loading/release capacity and feasibility of the (bio)-sensing protocol are illustrated in Vibrio cholerae and Vibrio parahaemolyticus bacteria cultures as realistic domains. The new concept described here holds considerable promise to understand the interaction of micromotor with biological counterparts in a myriad of biomedical and other practical applications, including the design of novel micromotor-based sensors.

TiO2 Nanomaterials in Photoelectrochemical and Electrochemiluminescent Biosensing.

Titanium dioxide (TiO2) is increasingly being used in biosensing applications. Herein, we review the most recent developments in photoelectrochemical (PEC) and electrochemiluminescent (ECL) biosensing based on TiO2 nanomaterials, as well as the mechanisms that lead to the improved performance of biosensors that incorporate these nanomaterials. The merits of TiO2-based ECL and PEC biosensing strategies are summarized by highlighting some illustrative examples that have been reported within the last 5 years. The future prospects for and challenges in this field are also discussed.

An extended core nanocoax pillar architecture for enhanced molecular detection.

Biosensors that incorporate nanomaterials and nanofabrication techniques enable molecular detection of chemical and biological macromolecules with a high degree of specificity and ultrasensitivity. Here, we present a novel fabrication process that yields a nanostructure capable of detecting biological macromolecules. The extended core nanocoax (ECC) structure builds on a previously reported nanocoaxial-based sensor. The fabrication of the device incorporates an extended inner pillar, with controllable extension above the annulus and into the surrounding solution. This new design eliminates structural constraints inherent in the original nanocoax architecture. We also provide results demonstrating improvement in biosensing capability. Specifically, we show the capability of the new architecture to detect the B subunit of the Vibrio cholerae toxin at improved sensitivity (100 pg/ml) in comparison to optical enzyme-linked immunosorbant assay (1 ng/ml) and previously reported coaxial nanostructures (2 ng/ml).

On-Chip Electrochemical Detection of Cholera Using a Polypyrrole-Functionalized Dendritic Gold Sensor.

Rapid diagnosis of an infectious disease outbreak in the field is critical for limiting the escalation of an outbreak into an epidemic. Devices suited to point-of-care (POC) diagnosis of cholera must not only demonstrate clinical laboratory levels of sensitivity and specificity but do so in a portable and low-cost manner, with a simplistic readout. We report work toward an on-chip electrochemical immunosensor for the detection of cholera toxin subunit B (CTX), based on a dendritic gold architecture biofunctionalized via poly(2-cyanoethyl)pyrrole (PCEPy). The dendritic electrode has an ∼18× greater surface area than a planar gold counterpart, per electrochemical measurements, allowing for a higher level of detection sensitivity. A layer of PCEPy polymer generated on the dendritic surface facilitated the performance of an electrochemical enzyme-linked immunosorbant assay (ELISA) for CTX on-chip, which demonstrated a detection limit of 1 ng mL-1, per a signal-to-noise ratio of 2.6. This was more sensitive than detection using a simple planar gold electrode (100 ng mL-1) and also matched the diagnostic standard optical ELISA, but on a miniaturized platform with electrical readout. The ability to meet POC demands makes biofunctionalized gold dendrites a promising architecture for on-chip detection of cholera.

Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin.

Aptamers are envisioned to serve as powerful synthetic substitutes to antibodies in a variety of bioanalytical assay formats. However, lateral flow assays (LFAs) remain dominated by antibody-based strategies. In this study, a LFA for the detection of cholera toxin as a model analyte is developed and optimized using a synthetic aptamer and a naturally occurring receptor as biorecognition elements and directly compared with solely aptamer and aptamer and antibody-based alternative approaches. The aptamer (CT916) recently selected by our group, GM1 receptors and an anti-cholera toxin antibody were evaluated. Relying solely on molecules that can easily be synthesized while aiming for high sensitivity, we applied a novel combination of capture aptamer and GM1 cell receptor-labeled liposomes for cholera toxin detection, achieving a limit of detection (LOD) of 2 ng ml-1 (3σ)/10 ng ml-1 (visual) in ∼15 min. To put our novel aptasensor into perspective, we developed a competitive lateral flow assay, exploiting the competition of cholera toxin in solution with immobilized cholera toxin for binding of aptamer-coated gold nanoparticles (AuNPs) (LOD = 51 ng ml-1 (3σ)/100 ng ml-1 (visual), assay time ∼10 min). As dual simultaneously binding aptamers were not available, we designed aptamer antibody pair-based lateral flow assays using aptamer-coated AuNPs which yielded a LOD of 5 ng ml-1 (by the 3σ rule)/10 ng ml-1 (visual) in a 10 min assay and an even better LOD of 0.6 ng ml-1 (3σ)/1 ng ml-1 (visual), with an ∼20 min total assay time. All set-ups are highly specific and provide an excellent alternative for cholera toxin detection in places where professional knowledge and sophisticated equipment are not readily available and cost efficient, simple, and rapid tests are needed, while the combination of GM1 cell receptor-labeled liposomes and aptamers is clearly the most promising.

Serum zonulin in patients with inflammatory bowel disease: a pilot study.

BACKGROUND: In recent years it has been supposed that impaired intestinal permeability represents an early event preceding the onset of inflammatory bowel disease (IBD). Since zonulin has been proposed as a biomarker of intestinal permeability, we investigated its role in patients with IBD and the correlation between serum and fecal zonulin. METHODS: A total of 118 IBD patients (86 Crohn's disease [CD] and 32 ulcerative colitis [UC]) and 23 healthy controls (HC) were prospectively enrolled. A serum sample was collected for all the subjects included in the study. A stool specimen collected in the same day of blood drawing was available for a subgroup of 33 IBD patients. Serum and fecal zonulin were tested by ELISA. Non-parametric statistical tests were used for data analysis. RESULTS: Serum zonulin concentration was higher in IBD patients compared to HC (34.5 [26.5-43.9] ng/mL vs. 8.6 [6.5-12.0] ng/mL, P<0.001) showing an area under the curve of 0.98 for their discrimination. No difference in serum zonulin concentration was observed between patients with CD and those with UC (P=0.074). An inverse correlation was observed between serum zonulin concentration and disease duration (rs=-0.30, P=0.001); no correlation was observed between serum and fecal zonulin (rs=0.15, P=0.394). CONCLUSIONS: Serum zonulin is highly sensitive for the evaluation of intestinal permeability in IBD patients. There is no correlation between zonulin values in serum and feces.

On-chip biosynthesis of GM1 pentasaccharide-related complex glycans.

A functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and to apply glycan-involved applications simultaneously. GM3 trisaccharide, GM2 tetrasaccharide, and GM1 pentasaccharide were successfully directly biosynthesized on lactose-immobilized surfaces through three consecutive glycosyltransferase reactions along with small amounts of enzymes and donors, without any additional processes. Biosynthesized GM1 pentasaccharide-related complex glycans were demonstrated to provide information on the substrate specificity of whole cholera toxin. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan-involved applications such as glycan-protein interaction analysis and glycan biomarker-based diagnosis.

Microbial patterns in patients with histamine intolerance.

Histamine intolerance represents a controversially discussed disorder. Besides an impaired degradation of orally supplied histamine due to diamine oxidase (DAO) deficiency, a deranged gut flora may also contribute to elevated histamine levels. Our aim was to determine the intestinal bacterial composition in patients with proven histamine intolerance in comparison to other food intolerances and healthy controls. A total of 64 participants were included in the study, encompassing 8 patients with histamine intolerance (HIT), 25 with food hypersensitivity (FH), 21 with food allergy and 10 healthy controls (HC). All participants underwent blood testing for total and food-specific immunoglobulin E, plasma histamine and DAO serum activity. Stool samples were used to analyze stool histamine and zonulin levels and bacterial composition by 16s rRNA sequencing. No significant differences in stool histamine levels were observed, but HIT patients showed elevated levels of stool zonulin. Microbiota analysis revealed increased levels of Proteobacteria (5.4%) and a significantly reduced alpha-diversity in the HIT group (P = 0.019). On family level, HC showed a significantly higher abundance of Bifidobacteriaceae compared to other study groups (P = 0.005), with lowest levels in the HIT group (P = 0.036). Also significantly reduced abundances of the genera Butyricimonas (P = 0.026) and Hespellia (P = 0.025) were observed in the HIT patients, whereas Roseburia were significantly elevated (P = 0.021). We concluded that the altered occurrence of Proteobacteria and Bifidobacteriaceae, reduced alpha-diversity as well as elevated stool zonulin levels suggest a dysbiosis and intestinal barrier dysfunction in histamine intolerant patients, which in turn may play an important role in driving disease pathogenesis.

Ultrasensitive Detection of Bacterial Protein Toxins on Patterned Microarray via Surface Plasmon Resonance Imaging with Signal Amplification by Conjugate Nanoparticle Clusters.

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20 nm AuNP via streptavidin bridge, followed by attachment of 5 nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently sized conjugated nanoparticles effectively increases their packing density on surface and significantly improves SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 × 10-16 to 2.51 × 10-13 mol/cm2 with the limit of detection of 5.01 × 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.

Intestinal Integrity Biomarkers in Early Antiretroviral-Treated Perinatally HIV-1-Infected Infants.

Intestinal fatty acid binding protein (iFABP) levels did not differ between human immunodeficiency virus type 1 (HIV-1)- infected infants and uninfected infants exposed to HIV-1, but those who breastfed had substantially lower levels. Zonulin levels increased from 3 to 5.3 months of age with perinatal acquisition of HIV-1 despite early antiretroviral treatment. Biomarkers of intestinal integrity (ie, iFABP and zonulin) were compared in 56 HIV-1-positive African infants who received early antiretroviral treatment and 53 HIV-1-exposed but uninfected (HEU) controls. Despite heightened inflammation and immune activation in HIV-positive infants, iFABP and zonulin levels at 3 months of age were not different from those in HEU infants and largely were not correlated with inflammatory and immune activation biomarkers. However, zonulin levels increased and became significantly higher in HIV-positive infants as compared to HEU infants by 5 months of age, despite viral suppression due to antiretroviral treatment. These findings have implications for intestinal integrity biomarker profiling in perinatal HIV-1 infection.

[Mass spectrometry virulence marker Vibrio cholerae.]

Identification mass-spectrometry of marker proteins of toxicity of V. cholerae for development the express of diagnostics of the causative agent of cholera on the basis of the computer analysis in the MALDI-ToF format of electronic profiles became the purpose of our research. Subjected to the computer analysis mass and spectrometer electronic passports 140 of strains of V. cholerae with obviously known characteristic of strains which were divided into 2 basic groups in the parameter of existence or lack of a gene of cholera toxin (ctx-and ctx+). We for the first time revealed a taxon - the specific marker protein ceous peak having a molecular mass of 3202 Da characteristic of strains of V.cholerae ctx + and unrepresentative for V.cholerae ctx-.

Plasmonic Sensing with 3D Printed Optics.

Three-dimensional (3D) printing has undergone an exponential growth in popularity due to its revolutionary and near limitless manufacturing capabilities. Recent trends have seen this technology utilized across a variety of scientific disciplines, including the measurement sciences, but precise fabrication of optical components for high-performance biosensing has not yet been demonstrated. We report here 3D printing of high-quality, custom prisms by stereolithography that enable Kretschmann-configured plasmonic sensing of bacterial toxins. Simple benchtop polishing procedures render a smooth surface that supports propagation of surface plasmon polaritons with a deposited gold layer, which exhibit high bulk refractive index sensitivities and are capable of discriminating trace levels of cholera toxin on a supported lipid membrane interface. Further evidence of the flexibility of this manufacturing technique is demonstrated with printed prisms of varied geometries and in situ monitoring of nanoparticle growth by total internal reflection spectroscopy. This work represents the first example of 3D printed light-guiding sensing platforms and demonstrates the versatility and broad perspective of 3D printing in optical detection.

Detection of Cholera Toxin by an Immunochromatographic Test Strip.

As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139 infection, detection of CT is an important biomarker for diagnosis of the disease. The procedure for pathogenicity analysis of V. cholerae isolates must be carefully developed for the reason that the amount of CT produced by V. cholerae varies according to the medium used and culture conditions (i.e. temperature and aeration status) applied. Here we describe a reproducible rapid method for analysis of CT production by toxigenic V. cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of purified recombinant CT.

Oral Supplementation with Bovine Colostrum Decreases Intestinal Permeability and Stool Concentrations of Zonulin in Athletes.

Increased intestinal permeability has been implicated in various pathologies, has various causes, and can develop during vigorous athletic training. Colostrum bovinum is a natural supplement with a wide range of supposed positive health effects, including reduction of intestine permeability. We assessed influence of colostrum supplementation on intestinal permeability related parameters in a group of 16 athletes during peak training for competition. This double-blind placebo-controlled study compared supplementation for 20 days with 500 mg of colostrum bovinum or placebo (whey). Gut permeability status was assayed by differential absorption of lactulose and mannitol (L/M test) and stool zonulin concentration. Baseline L/M tests found that six of the participants (75%) in the colostrum group had increased intestinal permeability. After supplementation, the test values were within the normal range and were significantly lower than at baseline. The colostrum group Δ values produced by comparing the post-intervention and baseline results were also significantly lower than the placebo group Δ values. The differences in stool zonulin concentration were smaller than those in the L/M test, but were significant when the Δ values due to intervention were compared between the colostrum group and the placebo group. Colostrum bovinum supplementation was safe and effective in decreasing of intestinal permeability in this series of athletes at increased risk of its elevation.

Increased intestinal permeability, measured by serum zonulin, is associated with metabolic risk markers in overweight pregnant women.

BACKGROUND: Increased intestinal permeability with subsequent metabolic endotoxemia, i.e., elevated circulating levels of bacterial lipopolysaccharide, LPS, has been introduced as a novel initiator of obesity related metabolic disturbances in non-pregnant individuals. The objective was to investigate the extent to which intestinal permeability, measured by serum zonulin concentration, is related to metabolic endotoxemia and metabolic risk markers in overweight pregnant women. METHODS: This was a cross-sectional study including 100 pregnant overweight women in early pregnancy. Serum zonulin was analyzed using ELISA, and markers for metabolic endotoxemia (LPS), inflammation (high-sensitive C-reactive protein and glycoprotein acetylation GlyA), glucose metabolism (fasting glucose and insulin), and lipid metabolism were measured. RESULTS: Higher serum zonulin concentration associated positively with LPS (P=0.02), inflammatory markers (P<0.001), insulin (P<0.001), insulin resistance (P<0.001), and triglycerides (P=0.001), and negatively with insulin sensitivity (P=0.001) (ANOVA with Tukey's corrections or Kruskal-Wallis nonparametric test with Bonferroni correction for zonulin quartiles). All the observed associations were confirmed (P<0.015) in a linear regression model adjusted with potential confounding factors. Both LPS and GlycA showed positive relationship with insulin resistance, serum insulin, triglycerides, total and LDL-cholesterol and negative relationship with insulin sensitivity (P≤0.03) in the univariate linear regression. Positive relationship was also found between LPS and HDL-cholesterol (P=0.03). CONCLUSIONS: Our findings suggest that increased serum zonulin concentration, i.e., increased intestinal permeability, contributes to metabolic endotoxemia, systemic inflammation, and insulin resistance in overweight pregnant women. By reinforcing intestinal barrier, it may be possible to manipulate maternal metabolism during pregnancy with subsequent health benefits.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin.

Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.