Analysis of treatment adherence and cost among patients with epilepsy: a four-year retrospective cohort study in Pakistan.

BACKGROUND: The adherence pattern of antiepileptic drugs (AEDs) among patients with epilepsy is relatively lower in the United States and different European countries. However, adherence and cost analysis of AEDs in Asian countries have not been thoroughly studied. Therefore, the present study aimed to analyze the cost and adherence of AEDs and its associated factors in patients followed in Pakistan. METHODS: Data from prescriptions collected from patients with epilepsy who have visited the Outpatient Department (OPD) of different tertiary care hospitals at the cosmopolitan city of Karachi, Pakistan from December 2015 to November 2019. The mean follow-up period for each participant was about 22 months. Pairwise comparisons from Cox regression/hazard ratios were used to assess the predictors of adherence. Direct costs of AEDs were calculated and presented as the annual cost of drugs. RESULTS: A total of 11,490 patients were included in this study, 51.2 % were male and 48.8 % were female with a mean age of 45.2 ± 15.8 y. Levetiracetam was found as the most prescribing AED in all study participants (32.9 %). Of them, 49.1 % of patients continued their initial recommended treatment. However, 31.3 % of patients have discontinued the therapy, while, 19.6 % were switched to other AED. Adherence with initial treatment was more profound in male (57.4 %) patients, compared to female with a mean age of 44.2 years. Lamotrigine users (60.6 %) showed a higher tendency to retain on initially prescribed drugs. The total cost of epilepsy treatment in the entire study cohort was 153280.5 PKR ($941.9). By applying the Cox regression analysis, it can be observed that the patients with increasing age (OR, 2.04), migraine (OR, 2.21), psychiatric disorders (OR, 4.28), other comorbidities (OR, 1.52) and users of other than top five prescribing AEDs (2.35) were at higher risk of treatment discontinuation. However, levetiracetam (OR, 0.69), valproic acid (OR, 0.52), carbamazepine (OR, 0.81), lamotrigine (OR, 0.80) or lacosamide (OR, 0.65) users have more chances to continue their initial therapy. CONCLUSIONS: Similar to western countries, the majority of patients with epilepsy exhibited low adherence with AEDs. Various associated factors for improving adherence were identified in this study.

Relationship of Obsessive-Compulsive Disorders with Religion and Psychosocial attitude among Local Medical College Students of Karachi: An epidemiological study.

OBJECTIVE: To evaluate the awareness, prevalence and attitude of medical students towards obsessive compulsive disorder. METHOD: The cross-sectional study was conducted at different medical institutions in Karachi from August to October 2018, and comprised medical students who were selected using random sampling. The participants were asked to fill demographic form as well as the obsessive compulsive disorder Yale-Brown scale-based questionnaire. Data was analysed using SPSS 23. RESULTS: Of the 1172 subjects, 602(51.4%) were aware of obsessive compulsive disorder, while 570(48.6%) were unaware. Washing, counting and arranging were higher among females than in males (p<0.004). Fear, shame and stigma were of major concerns (p<0.05). CONCLUSIONS: Awareness level of medical students regarding obsessive compulsive disorder was very low.

Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains.

A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40-63 µm) solid-phase extraction cartridges. AFs were separated using a LiChroCART-RP-18 (5 µm, 250 × 4.0 mm(2)) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min(-1) The fluorescence of each AF was detected at λex = 365 nm and λem = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB1, AFB2, AFG1 and AFG2), linearity (R(2) ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ -5.51), robustness (p < 0.0080), ruggedness (p < 0.0100) and average recoveries (89.2-97.8%). The limits of quantification of AFB1, AFB2, AFG1 and AFG2 were 0.080, 0.073, 0.062 and 0.066 ng g(-1), respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice (n = 20), wheat (n = 15) and maize (n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains.

Aflatoxins contamination in Pakistani brown rice: a comparison of TLC, HPLC, LC-MS/MS and ELISA techniques.

Advancement in the field of analytical food-chemistry has explored various experimental techniques for aflatoxins (AFs) quantification. The present study was aimed to compare four different techniques; thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the analysis of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in brown rice (n = 120) being collected from Karachi, Pakistan. All the four assays provide precised, accurate and comparable results. However, some differences were observed. For instance, TLC, HPLC and LC-MS/MS methodologies offered the advantage of the quantification of individual toxins in contrast to ELISA technique. The contamination ranges of AFB1/AFB2 as determined by TLC, HPLC and LC-MS/MS were 1.18-9.97/0.59-1.52, 0.16-10.54/0.26-1.35 and 0.11-10.88/0.38-1.48 µg/kg, respectively. However, AFG1 and AFG2 were not detected in any tested samples. Furthermore, owing to low-detection limit and sensitivity, HPLC and LC-MS/MS methodologies have identified greater number of contaminated samples in comparison to TLC and ELISA techniques. The overall average results of total AFs as provided by HPLC (3.79 µg/kg) and LC-MS/MS (3.89 µg/kg) were found higher in comparison to TLC (3.68 µg/kg) and ELISA (3.70 µg/kg). On the basis of achieved results, it was concluded that TLC, HPLC, LC-MS/MS and ELISA techniques are valuable tool for the quantification of AFs in cereals and grains. Furthermore, HPLC and LC-MS/MS techniques offer an added advantage for the detection of AFs in diminutive levels.

Natural co-occurrence of aflatoxins and ochratoxin A in liquorice roots.

The natural co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in liquorice roots obtained from different herbal outlets of Karachi, Pakistan, was evaluated. A total of 60 samples were obtained during 2019‒2021 and analysed using HPLC with fluorescence detection. The method was validated according to the European Union (EU) regulation. The incidence of AFs was 52 in all tested samples (87%), with a contamination range of 1.2‒6.4 µg kg-1 and an average of 2.1 ± 0.3 µg kg-1. In all samples, the AFs contamination was below the maximum limit (ML) of 10 µg kg-1 for total AFs as set by the EU. The OTA contamination in all samples ranged 1.5‒60.3 µg kg-1, with a mean of 18.9 ± 0.8 µg kg-1. In 36 samples (60%) the OTA contamination was below the ML of 20 µg kg-1 as set by the EU. These results led to the recommendation to test liquorice root on mycotoxins, as it comes to food quality standards.

Effectiveness of citrus fruit peel as a biosorbent for the mitigation of aflatoxins in vitro.

We assessed the effectiveness of novel and cost-effective citrus fruit peel (oranges, agro-waste material) for the removal of four aflatoxins B1, B2, G1, and G2 in vitro. The biosorbent was described using SEM, XRF, XRD, FITR spectroscopy, and point of zero charge. The adsorption performance was optimised in a batch experiment by altering the various parameters, such as biosorbent dose (1-15 mg/mL), the preliminary concentration of AFs (20-200 ng/mL), pH (1-9), the incubation period (10-60 min), and temperature (10-45 °C). Maximum removal (90%) was achieved when using biosorbent at 10 mg, each AF concentration 100 ng/mL, pH 3, and incubation time 45 min at 37 °C. The experimental data were well-described by the Langmuir isotherm model and the monolayer coverage (Qe) was calculated to be 78.5, 77.8, 79.2, and 75.6 ng/mg for aflatoxin B1, B2, G1, and G2, respectively. The thermodynamic and kinetic studies suggested that the adsorption performance was endothermic and obeyed the pseudo-second-order rate model. Studies at different pH also proved that the adsorption of toxins would be strong and sufficient under pH variation, as found in the gastrointestinal tract. Thus the biosorption of AFs by orange peel powder might be an efficient low price detoxification method in humans and animals.

Evaluating the pH of Various Commercially Available Beverages in Pakistan: Impact of Highly Acidic Beverages on the Surface Hardness and Weight Loss of Human Teeth.

The objectives of this study were to investigate the pH of common beverages and to evaluate the effects of common acidic beverages on the surface hardness and weight loss of human tooth specimens. A total of 106 beverages were conveniently purchased from supermarkets in Karachi, Pakistan. Prior to evaluation, beverages were refrigerated or stored at room temperature in accordance with the manufacturers' recommendations. Beverages were categorized into six groups: 'Sports and Energy drinks', 'Water', 'Fruit Juices and Drinks', 'Sodas', 'Milk and Flavored Milk' and 'Teas and Coffee'. Using a pH meter, the pH of each beverage was measured in triplicate at room temperature. In addition, the influence of five highly acidic beverages on the weight loss and surface hardness of human tooth specimens was evaluated using gravimetric analysis and the Vickers hardness tester, respectively. 'Sports and Energy drinks', 'Fruits Juices and Drinks' and 'Sodas' were the most acidic beverage categories, with a pH range of 3.00−5.00. A total of 33% of beverages tested in this study were highly acidic (pH less than 4.00), 29% of beverages were moderately acidic (pH 4.00−4.99) and 31% were mildly acidic (pH 5.00−6.99). Significant weight loss was observed in all immersed specimens compared to control counterparts (p < 0.05). Similarly, for surface hardness, five highly acidic beverages (Red Bull, Pepsi, Apple Cidra, Tang Mosambi and Tang Orange) significantly decreased the surface hardness of specimens (p < 0.05). The pH levels of commonly available beverages in Pakistan are highly acidic, which may encourage loss of minerals from teeth; hence, affecting their surface hardness.

Graphene oxide decorated with cellulose and copper nanoparticle as an efficient adsorbent for the removal of malachite green.

In this study, the graphene oxide surface was modified by grafting of nanocellulose and copper nanoparticles to promote the surface charge and adsorption efficiency for malachite green (MG). The structural and configurational properties of GO-CEL-Cu were verified by UV/Vis, SEM, TEM, EDX and FTIR spectroscopy and confirmed the electrostatic interaction and hydrogen bonding between GO, CEL and Cu-NPs. TEM images confirmed the deposition of Cu-NPs size between 24 and 37 nm on the GO surface. The uniform fine particles size makes strong interfacial interaction with GO sheets result in efficient load transfer from the matrix to the hybrid. The variable parameters such as adsorbent amount, MG concentration, pH, time and temperature were investigated to achieve optimum experimental condition. The experimental data was justified by Langmuir isotherm model with adsorption capacity for GO, GO-Cu, GO-CEL, GO-CEL-Cu as 127.3, 149.2, 156.8 and 207.1 mg/g, respectively. The spontaneity and endothermic nature of the process were confirmed by negative Gibbs free energy and followed the pseudo-second-order rate equation. Additionally, positive values of enthalpy and entropy suggesting endothermic process and increase randomness during process, respectively. In conclusion, nanocomposite is capable to adsorb the toxic dye due to its well economic, eco-friendly, well adsorption rate and regeneration ability.

Green Synthesis and Characterization of Carboxymethyl Cellulose Fabricated Silver-Based Nanocomposite for Various Therapeutic Applications.

PURPOSE: The current study proposed the simple, eco-friendly and cost-effective synthesis of carboxymethyl cellulose (CMC) structured silver-based nanocomposite (CMC-AgNPs) using Syzygium aromaticum buds extract. METHODS: The CMC-AgNPs were characterized by ultraviolet (UV) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transmission infra-red (FTIR), energy-dispersive X-ray (EDX), and dynamic light scattering (DLS) techniques. The synthesized nanocomposites were evaluated for their bactericidal kinetics, in-vivo anti-inflammatory, anti-leishmaniasis, antioxidant and cytotoxic activities using different in-vitro and in-vivo models. RESULTS: The spherical shape nanocomposite of CMC-AgNPs was synthesized with the mean size range of 20-30 nm, and the average pore diameter is 18.2 nm while the mean zeta potential of -31.6 ± 3.64 mV. The highly significant (P < 0.005) antibacterial activity was found against six bacterial strains with the ZIs of 24.6 to 27.9 mm. More drop counts were observed in Gram-negative strains after 10 min exposure with CMC-AgNPs. Significant damage in bacterial cell membrane was also observed in atomic force microscopy (AFM) after treated with CMC-AgNPs. Nanocomposite showed highly significant anti-inflammatory activity in cotton pellet induced granuloma model (Phase I) in rats with the mean inhibitions of 43.13% and 48.68% at the doses of 0.025 and 0.05 mg/kg, respectively, when compared to control. Reduction in rat paw edema (Phase II) was also highly significant (0.025 mg/kg; 42.39%; 0.05 mg/kg, 47.82%). At dose of 0.05 mg/kg, CMC-AgNPs caused highly significant decrease in leukocyte counts (922 ± 83), levels of CRP (8.4 ± 0.73 mg/mL), IL-1 (177.4 ± 21.3 pg/mL), IL-2 (83.7 ± 11.5 pg/mL), IL-6 (83.7 ± 11.5 pg/mL) and TNF-α (18.3 ± 5.3 pg/mL) as compared to control group. CMC-AgNPs produced highly effective anti-leishmaniasis activity with the viable Leishmania major counts decreased up to 36.7% within 24 h, and the IC50 was found to be 28.41 µg/mL. The potent DPPH radical scavenging potential was also observed for CMC-AgNPs with the IC50 value of 112 µg/mL. Furthermore, the cytotoxicity was assessed using HeLa cell lines with the LC50 of 108.2 µg/mL. CONCLUSION: The current findings demonstrate positive attributes of CMC fabricated AgNPs as a promising antibacterial, anti-inflammatory, anti-leishmaniasis, and antioxidant agent with low cytotoxic potential.

A mechanistic study on the inhibition of bacterial growth and inflammation by Nerium oleander extract with comprehensive in vivo safety profile.

BACKGROUND: Nerium oleander (L.) is well known traditionally used medicinal plant with several pharmacological activities. However, the anti-bacterial, anti-inflammatory activity and in vivo toxicity potential of floral parts of this plant are not reported. Therefore the present study was designed to investigate these activities of Nerium oleander ethanolic flower extract (NOEE) in different animal models. METHODS: Antimicrobial activity of plant extract was compared with five different antibiotics using the disk diffusion method. The time-killing kinetic assay and bacterial killing mechanism of NOEE were also performed. Anti-inflammatory activity was assessed using granuloma induced by cotton-pellet, rat paw edema induced by carrageenan and levels of different inflammatory biomarkers on healthy Wistar rats. The protein and mRNA expressions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were also measured. Acute (14 days) and sub-acute (28 days) oral toxicity studies were also performed on healthy Sprague Dawley rats. RESULTS: NOEE produced highly significant (P < 0.005) and significant (P < 0.05) zones of inhibition at 30 mg/mL and 20 mg/mL respectively against most of the tested bacterial strains. NOEE produced a more drop in viable counts of Gram-negative isolates within 20 min. After 12 h exposure with NOEE, the SEM images of MRSA showed the destruction of cell membrane. NOEE showed highly significant (P < 0.005) anti-inflammatory activity in cotton-pellet and carrageenan inflammatory models. In addition, treatment with NOEE also decreased the production of NO, PGE2, TNF-α and IL-1ß in the rat paw after treated with carrageenan. Similarly, NOEE also suppressed the inducible nitric oxide synthase (iNOS), TNF-α, IL-1ß, and cyclooxygenase-2 (COX-2) mRNA expressions. It is also showed highly significant reduction in total leukocyte count (73.09%) and C-reactive protein levels (54.60%). NOEE also inhibited COX-1, COX-2, 5-LO and 12-LO in a highly significant manner. Moreover, acute and sub-acute toxicity studies of NOEE in rats confirm the toxicity with hepatotoxicity at higher doses (2000 mg/kg) i.e. four times greater than the therapeutic dose. CONCLUSION: It is concluded that crude flower extract of N. oleander is a potent antimicrobial and anti-inflammatory agent with no toxicity potential at therapeutic doses.

Analysis of treatment cost and persistence among migraineurs: A two-year retrospective cohort study in Pakistan.

OBJECTIVES: The persistence pattern of anti-migraine drugs' use among migraineurs is very low in the United States and different European countries. However, the cost and persistence of antimigraine drugs in Asian countries have not been well-studied. Hence, the present study aimed to evaluate the treatment cost and persistence among migraineurs in Pakistan. METHODS: Data from prescriptions collected from migraineurs who visited the Outpatient Department (OPD) of different public and private sector tertiary-care hospitals of Karachi, Pakistan were used to conduct this retrospective cohort study from 2017 to 2019. The minimum follow up period for each migraineur was about 12 months for persistence analysis while dropped-out patients data were also included in survival analysis as right censored data. Pairwise comparisons from Cox regression/hazards ratio were used to assess the predictors of persistence with the reference category of non-binary variables i.e. hazard ratio = 1 for low frequency migraineurs and NSAIDs users. Persistence with anti-migraine drugs was estimated using the Kaplan-Meier curve along with the Log Rank test. RESULTS: A total of 1597 patients were included in this study, 729 (45.6%) were male and 868 (54.3%) were female. Non-steroidal anti-inflammatory drugs (NSAIDs) were the most prescribed class of drug initially for all classes of migraineurs (26.1%). Of them, 57.3% of migraineurs discontinued their treatment, 28.5% continued while 14.8% were switched to other treatment approaches. Persistence with initial treatment was more profound in female (58.8%) patients compared to males while the median age of continuers was 31 years. The total cost of migraine treatment in the entire study cohort was 297532.5 Pakistani Rupees ($1901.1). By estimating the hazard ratios (HR) using the Cox regression analysis, it can be observed that patients with high frequency (HR, 1.628; 95%CI, 1.221-2.179; p<0.0001) migraine, depression (HR, 1.268; 95%CI, 1.084-1.458; p<0.0001), increasing age (HR, 1.293; 95%CI, 1.092-1.458; p<0.0001), combination analgesics (HR, 1.817; 95%CI, 0.841-2.725; p = 0.0004) and prophylaxis drugs (HR, 1.314; 95%CI, 0.958-1.424; p<0.0001) users were at a higher risk of treatment discontinuation. However, patients with chronic migraine (HR, 0.881; 95%CI, 0.762-0.912; p = 0.0002), epileptic seizure (HR, 0.922; 95%CI, 0.654-1.206; p = 0.0002), other comorbidities (HR, 0.671; 95%CI, 0.352-1.011; p = 0.0003) and users of triptan(s) (HR, 0.701; 95%CI, 0.182-1.414; p = 0.0005) and triptan(s) with NSAIDs (HR, 0.758; 95%CI, 0.501-1.289; p<0.0001) had more chances to continue their initial therapy. CONCLUSION: Similar to western countries, the majority of migraineurs exhibited poor persistence to migraine treatments. Various factors of improved persistence were identified in this study.

Green synthesized and characterized copper nanoparticles using various new plants extracts aggravate microbial cell membrane damage after interaction with lipopolysaccharide.

In the present study, commercially available six plants leave extracts such as Eucalyptus camaldulensis, Azadirachta indica, Murraya koenigii, Avicennia marina, Rosa rubiginosa and Datura stramonium were utilized for the production of copper nanoparticles (CuNPs). The characterization of particles was performed by UV/Vis, TEM, SEM, EDX and FTIR spectroscopy. TEM images showed the creation of CuNPs having mean size ranged from 48 to 29 nm corresponding to different plant extracts. SEM analysis showed the formation of spherical form of NPs. FTIR spectroscopy verified the availability of phytochemical components as they serves the reducing, covering and stabilizing assistant of the CuNPs. Antimicrobial ability of NPs was performed against various clinical pathogenic strains by Oxford cup method. The synthesized NPs indicated potent antibacterial activity, with relatively low values of MIC between 15 and 60 µg/mL. The antibacterial effect of each CuNPs was observed in the resulting order A. indica > D. stramonium > M. koenigii > R. rubiginosa > A. marina > E. camaldulensis. After 12 h exposure with A. indica synthesized CuNPs, the SEM images of S. typhi showed destruction of cell membrane and cell lysis was clearly observed after interaction with lipopolysaccharide. In conclusion, these obtained CuNPs could be precisely applied in treatment protocols without any covering or core-shell procedures.

Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents.

In this study, a facile, ecological and economical green method is described for the fabrication of iron (Fe), copper (Cu) and silver (Ag) nanoparticles (NPs) from the extract of Syzygium cumini leaves. The obtained metal NPs were categorized using UV/Vis, SEM, TEM, FTIR and EDX-ray spectroscopy techniques. The Fe-, Cu- and Ag-NPs were crystalline, spherical and size ranged from 40-52, 28-35 and 11-19 nm, respectively. The Ag-NPs showed excellent antimicrobial activities against methicillin- and vancomycin-resistance Staphylococcus aureus bacterial strains and Aspergillus flavus and A. parasiticus fungal species. Furthermore, the aflatoxins (AFs) production was also significantly inhibited when compared with the Fe- and Cu-NPs. In contrast, the adsorption results of NPs with aflatoxin B1 (AFB1) were observed as following order Fe->Cu->Ag-NPs. The Langmuir isotherm model well described the equilibrium data by the sorption capacity of Fe-NPs (105.3 ng mg-1), Cu-NPs (88.5 ng mg-1) and Ag-NPs (81.7 ng mg-1). The adsorption was found feasible, endothermic and follow the pseudo-second order kinetic model as revealed by the thermodynamic and kinetic studies. The present findings suggests that the green synthesis of metal NPs is a simple, sustainable, non-toxic, economical and energy-effective as compared to the others conventional approaches. In addition, synthesized metal NPs might be a promising AFs adsorbent for the detoxification of AFB1 in human and animal food/feed.

Antibacterial, anticoagulant and cytotoxic evaluation of biocompatible nanocomposite of chitosan loaded green synthesized bioinspired silver nanoparticles.

Present work reports the green synthesis of chitosan functionalized silver nanoparticles (CS-AgNPs) using ethanolic buds extract of Sygyzium aromaticum. CS-AgNPs were characterized physically, evaluated for antibacterial, anticoagulant and antiplatelet activities, and toxicity profile. The physical characterization of CS-AgNPs was done by UV/vis, SEM, TEM, FTIR and EDX. The sphericity was found uniform. FTIR and EXD showed noninterfering few impurities. The antibacterial activity against VRSA (ZI, 23.2 ± 0.51 mm) and MRSA (ZI, 25.8 ± 0.32 mm) were determined. The rise in bleeding and thromboplastin was observed highly significant while increased in prothrombin and activated partial prothrombin time in significant manner at both the doses of CS-AgNPs (0.025 mg/kg and 0.05 mg/kg). Reduction in the levels of fibrinogen was also highly significant. Platelet aggregation decreased at high dose of CS-AgNPs i.e. 55.14 ± 8.25% (arachidonic acid) and 13.06 ± 2.17% (collagen). Thrombin antithrombin (TAT) complex activity was found highest for CS-AgNPs. Cytotoxicity was assessed using HeLa cell lines (LC50; 125 µg/ml) and brine shrimp lethality tests (LC50; 518 µg/ml). The work suggests that green synthesized chitosan functionalized silver nanoparticles may be utilized as an effective antibacterial agent and anticoagulant with low toxicity. The current findings will open a new window for nanomedicine development and future clinical application.

Synergistic Nanocomposites of Different Antibiotics Coupled with Green Synthesized Chitosan-Based Silver Nanoparticles: Characterization, Antibacterial, in vivo Toxicological and Biodistribution Studies.

PURPOSE: The present study reports chitosan functionalized green synthesized CS-AgNPs, conjugated with amoxicillin (AMX), cefixime (CEF), and levofloxacin (LVX) for safe and enhanced antibacterial activity. METHODS: The CS-AgNPs and conjugates CS-AgNPs+AMX CS-AgNPs+CEF, and CS-AgNPs+LVX were characterized by UV-Vis, FTIR, SEM, TEM, EDX spectroscopy. The size distribution and zeta potential were measured using the dynamic light scattering (DLS) technique. The interaction between CS-AgNPs and antibiotic molecules was also investigated using UV-Vis spectroscopy at the concentrations of 5, 50, 500, and 5000 µM for each antibiotic. Antibacterial activity and synergism were assessed by the Fractional Inhibitory Concentration (FIC) index. The mechanism for synergistic activity was investigated by the detection of hydroxyl species based on the chemiluminescence of luminol. The biocompatibility index (BI) was calculated from IC50 using the HeLa cell line. In vivo toxicity and tissue distribution of silver ions were evaluated on Sprague Dawley rats. Physical interactions of antibiotics and significant (P<0.05) antibacterial activity were observed after loading on CS-AgNPs surfaces. RESULTS: The spherical shape nanocomposites of CS-AgNPs with different antibiotics were prepared with mean size ranges of 80-120 nm. IC50 of antibiotics-conjugated CS-AgNPs decreased compared to CS-AgNPs. The biocompatibility (BI) index showed that antibiotics-conjugated CS-AgNPs have high antibacterial potential and low toxicity. Highly significant (P<0.005) increase in the generation of hydroxyl species indicated the radical scavenging mechanism for synergistic activity of CS-AgNPs after combined with different antibiotics. Biochemical analysis and histopathological examinations confirmed low toxicity with minor hepatotoxicity at higher doses. After oral administration, extensive distribution of Ag ion was observed in spleen and liver. CONCLUSION: The study demonstrates positive attributes of antibiotics-conjugated CS-AgNPs, as a promising antibacterial agent with low toxicity.

Aflatoxin M1 in fresh milk collected from local markets of Karachi, Pakistan.

During 2016-2017, 156 samples of fresh milk samples were collected from local markets of Karachi, Pakistan and analysed for aflatoxin M1 (AFM1) contamination using ELISA technique. AFM1 was detected in 143 (91.7%) samples, ranged from 20 to 3090 ng L-1 with a mean level of 346.2 ng L-1. In 125 (80.1%) samples, the AFM1 contamination was greater than the maximum limit (ML = 50 ng L-1) set by EU. However, in 51 (32.7%) samples, the AFM1 level was higher than the ML of 500 ng L-1 as assigned by the USA. Statistical analysis showed that the AFM1 level in milk samples from summer was significantly (p < 0.05) higher than that obtained in winter. It was concluded that the AFM1 levels in the tested samples appear to be a serious public health problem. Therefore, immediate measures should be taken and re-evaluation done for the procedures for farming, transportation, refrigeration, and storage for the control of AFM1 level in milk samples.

Aflatoxins and ochratoxin A in export quality raisins collected from different areas of Pakistan.

During 2012-2014, 170 samples of export quality raisins were collected from different vendors in Pakistan. The collected samples were analysed for the presence of aflatoxins (AFs) and Ochratoxin A (OTA) contamination using high-performance liquid chromatography technique. The limit of detection and limit of quantification of AFs/OTA were 0.12/0.10 and 0.36/0.30 µg kg(-1), respectively. Only 5% of the samples were contaminated with AFs, ranging 0.15-2.58 µg kg(-1) with a mean of 0.05 ± 0.26 µg kg(-1). None of the raisin samples exhibited AFs contamination above the maximum limit (ML = 4 µg kg(-1)) as set by the European Union (EU). About 72% of the samples were contaminated with OTA, ranging 0.14-12.75 µg kg(-1) with a mean of 2.10 ± 1.9 µg kg(-1). However, in 95.3% of the tested samples, OTA level was lower than the ML of 10 µg kg(-1) as regulated by the EU. Apparently, a strict and continuous monitoring plan, including regulatory limits, improves food safety and quality for all types of commodities.

Aflatoxins in composite spices collected from local markets of Karachi, Pakistan.

This survey was carried out to evaluate the occurrence of total aflatoxins (AFs; B1+B2+G1+G2) in unpacked composite spices. A total of 75 samples of composite spices such as biryani, karhai, tikka, nihari and korma masalas were collected from local markets of Karachi, Pakistan, and analysed using HPLC technique. The results indicated that AFs were detected in 77% (n = 58) samples ranging from 0.68 to 25.74 µg kg(-1) with a mean of 4.63 ± 0.95 µg kg(-1). In 88% (n = 66) samples, AFs level was below the maximum limits (ML = 10 µg kg(-1)) as imposed by EU. Furthermore, 61% (n = 46) tested samples contained AFs level between 1 and 10 µg kg(-1), 9% (n = 7) exhibited AFs contamination ranged 10-20 µg kg(-1) and only 3% (n = 2) of the investigated samples contained AFs levels higher than the ML of 20 µg kg(-1) for total aflatoxins as set by the USA. It was concluded that there is need to establish a strict and continuous national monitoring plan to improve safety and quality of spices in Pakistan.

Fungal flora and aflatoxin contamination in Pakistani wheat kernels (Triticum aestivum L.) and their attribution in seed germination.

This study aimed to isolate fungal pathogens and to subsequently quantify aflatoxin (AF; B1 + B2 + G1 + G2) contamination in wheat crops grown in Pakistan. Accordingly, a total of 185 wheat samples were collected from different areas of Pakistan and numerous potent fungal pathogens were isolated. AF contamination attributed to the presence of intoxicating fungal pathogens and resulting metabolic activities were quantified using a high performance liquid chromatography-fluorescence detector coupled with postcolumn derivatization. Additionally, the effect of fungal pathogens on seed germination was also examined. The results obtained showed that 50% of tested wheat samples were found to be contaminated with a diverse range of fungal species. The rate of recurrence of fungal pathogens were Aspergillus 31%, Penicillium 9%, Fusarium 8%, Rhizopus 3%, and Alternaria 2%. The presence of Tilletia indica and Claviceps purpurea species was found to be inevident in all tested wheat samples. AFB1 contamination was detected in 48 (26.0%) samples and AFB2 in 13 (7.0%) samples. AFG1 and AFG2 were not found in any of the tested samples. The contamination range of AFB1 and AFB2 was 0.05-4.78 µg/kg and 0.02-0.48 µg/kg, respectively. The total amount of AFs (B1 + B2) found in 48 (26.0%) samples had a mean level of 0.53 ± 0.40 µg/kg and a contamination range of 0.02-5.26 µg/kg. The overall results showed that in 137 (74.0%) samples, AFs were not found within detectable limits. Furthermore, in 180 (97.2%) samples, AF levels were found to be below the maximum tolerated levels (MTL) recommended by the European Union (4 µg/kg). In five (2.7%) samples, AF contamination was higher than the MTL of the European Union. However, these samples were fit for human consumption with reference to the MTL (20 µg/kg) assigned by the USA (Food and Drug Administration and Food and Agriculture Organization) and Pakistan (Pakistan Standards and Quality Control Authority). Germination rates in healthy and contaminated wheat kernels were 84.6% and 45.2%, respectively. Based on the obtained results, it was concluded that the levels of fungal pathogen and AF contamination in Pakistani-grown wheat are not a potential threat to consumer health. However, control procedures along with a strict monitoring policy are mandatory to further minimize the prevalence of fungal carriers and the potency of AFs in crops cultivated in Pakistan.

Occurrence of aflatoxins contamination in brown rice from pakistan.

BACKGROUND: The objective of this study was to determine the distribution of an economically-important class of mycotoxins, the aflatoxins (AFs) in Pakistani Brown Rice. METHODS: A total of 262 of brown rice samples were collected from different vendors during July 2006 to June 2011. Samples were analyzed for the occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) by thin layer chromatography (TLC) technique. RESULTS: AFB1 was detected in 250 (95.4%) samples, whereas AFB2 was detected in 20 (7.6%) samples. Furthermore, AFG1 and AFG2 were not found in any sample. The contamination range of AFB1 and AFB2 was found 1.07-24.65 µg/kg and 0.52-2.62 µg/kg, respectively. Total AFs were quantified in 250 (95.4%) samples with an average of 3.89 µg/kg and contamination range was noted to be between 1.07-27.27 µg/kg. The overall results indicated that in 12 (4.6%) samples, AFs were not found within detectable limits. Furthermore, in 188 (71.7%) samples, AFs level was found below than maximum tolerated levels (MTL) as recommended by the European Union (4 µg/kg). Moreover, in 61 (23.3%) samples, AFs range was found between 4-20 µg/kg, which were fit for human consumption as per MTL (20 µg/kg) assigned by USA (FDA and FAO) and Pakistan (PSQCA). While only one sample (27.27 µg/kg) exceeded the above mention regulation limits. CONCLUSION: Low level of AFs occurs frequently in brown rice, and can be improved using proper harvesting practices, storage and transportation conditions. The small quantities of AFs warrant performing further investigation, monitoring and routine analysis on regular basis.