Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21–42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). The relative quantity of SARS-CoV-2 RNA fragments in the 24h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and RT-qPCR assay targeted with N2-assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of new and confirmed COVID-19 cases in the sewer network area.Over the 10-month surveillance period, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results), while only 24% of all samples exhibited gene copy (GC) numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7, 18 and 36 cases within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 GC required significantly more COVID-19 cases: the quantification rate was 50%, 75% and 95% when the 14-day incidence was 110, 152 and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 GC quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the GC of SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Validation of the spectrophotometric method for the dosing of some combined capsule

Background: UV-Vis spectrophotometry remains the most accessible spectral method with a high degree of sensitivity and information. The advantage of the method consists in its universality, the ability to combine with other methods, the minimum error, as well as its economic efficiency. The objective of this study was the determination of some validation parameters for the spectrophotometric method of dosing piracetam and nicergoline in combined capsules. Material and methods: Agilent 8453 UV-Vis spectrophotometer, reference standards of piracetam and nicergoline, 0.1 M HCl methanolic solution. Validation of the spectrophotometric method according to the requirements of the ICH guide “Q2R1: For analytical procedures and validation”. Results: Linearity was investigated on concentration ranges 5-40 µg / mL. The regression (R2 ) values were 0.9998 for nicergoline and 0.998 for piracetam, respectively. The limit of detection was 1.737 µg / mL for nicergoline and 0.369 µg / mL for piracetam. Quantification limit values were also calculated as 5.265 and 1.118 µg / mL for nicergoline and piracetam, accordingly. The results obtained showed that the developed spectrophotometric method is accurate, precise and robust, because the value of the relative standard deviation was less than 1.0%. Conclusions: The developed spectrophotometric method showed specificity, linearity, accuracy, precision and robustness, and can be applied on the concentration range between 80-120% of the nominal value of the content of nicergoline and piracetam in the preparation.

Determination of Eleven Veterinary Drugs in Chicken Meat and Liver

Brazil chicken production is around 13 million tons and about a third is exported to over 150 countries, placing Brazil as the world largest chicken meat producer, and therefore it is crucial to follow the legislation of all importer markets. This study conducted a survey by chance in 45 meat industries able to export. Therefore, 2580 chicken meat samples were collected and submitted to 11 analyte extraction and chromatographic verification of compliance in an accredited laboratory. Ten chemical residues (amoxicillin, bacitracin, colistin, dinitolmide + zoalene, spectinomycin, roxarsone, tiamulin, tylosin, trenbolone acetate and virginiamycin) were investigated in chicken meat and one (halofuginone hydrobromide) in chicken liver. The results showed that no compound exceeded the maximum residue limits established by seven legislations. All residue concentrations found were below the method quantification limit, thereby confirming the capability of Brazilian chicken meat industries in complying to foreign markets.

Pesticides Contamination of Cereals and Legumes: Monitoring of Samples Marketed in Italy as a Contribution to Risk Assessment

The evaluation of cereal-based product contamination by pesticide residues is a topic of worldwide importance, and reliable analytical methods for official check analyses and monitoring studies are required for multi-residue analysis at trace levels. In this work, a validated multi-residual analytical method by gas-chromatography and tandem mass spectrometry coupled with a rapid QuEChERS procedure was used for the determination of 37 pesticides (pyrethroids, organophosphorus and organochlorine compounds) in 209 commercially available samples of cereals and 11 legumes, placed on the Italian market in 2018 and 2019, coming from different regions of Italy, eastern Europe, and some non-European countries. No pesticide traces were observed in the analyzed legume samples. A total of 18 cereal samples were found to be contaminated by at least one pesticide, with a concentration level higher than the corresponding quantification limit, but never exceeding the maximum level fixed in the European Regulations. This work is the first part of a surveillance study for pesticide control in food samples.

Development of a Novel Sensor Based on Polypyrrole Doped with Potassium Hexacyanoferrate (II) for Detection of L-Tryptophan in Pharmaceutics

This study describes the development of a new sensor with applicability in the determination and quantification of yjr essential amino acid (AA) L-tryptophan (L-TRP) from pharmaceutical products. The proposed sensor is based on a carbon screen-printed electrode (SPCE) modified with the conductor polymer polypyrrole (PPy) doped with potassium hexacyanoferrate (II) (FeCN). For the modification of the SPCE with the PPy doped with FeCN, the chronoamperometry (CA) method was used. For the study of the electrochemical behavior and the sensitive properties of the sensor when detecting L-TRP, the cyclic voltammetry (CV) method was used. This developed electrode has shown a high sensibility, a low detection limit (LOD) of up to 1.05 × 10−7 M, a quantification limit (LOQ) equal to 3.51 × 10−7 M and a wide linearity range between 3.3 × 10−7 M and 1.06 × 10−5 M. The analytical performances of the device were studied for the detection of AA L-TRP from pharmaceutical products, obtaining excellent results. The validation of the electroanalytical method was performed by using the standard method with good results.

Electrochemical Characterization and Detection of Lead in Water Using SPCE Modified with BiONPs/PANI

The need for constant assessment of river water qualities for both aquatic and other biological survival has emerged a top priority, due to increasing exposure to industrial pollutants. A disposable screen print carbon electrode was modified with a conductive polymer (PANI) and Zn and/or Cu oxides NPs, obtained through bioreduction in citrus peel extracts (lemon and orange), for ultra-sensitive detection of PB2+, in the Crocodile River water sample. The synthesized materials were characterized with Fourier-transform infra-red spectroscopy (FTIR), ultra-violet visible spectroscopy (UV-Vis), and scanning electron microscopy (SEM). The SPC-modified electrodes designated as SPCE/LPE/BiONPs/PANI and SPCE/OPE/BiONPs/PANI were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and eventually deployed in the electrochemical detection of PB2+ in water using square wave voltammetry (SWV) technique. The electrochemical responses of the modified electrodes for both CV and EIS in 0.1 M HCl demonstrated enhanced performance relative to the bare SPCE. A detection and quantification limit of 0.494 ppb and 1.647 were obtained at SPCE/LPE/BiONPs/PANI, respectively, while a detection and quantification limit of 2.79 ppb and 8.91 ppb, respectively, were derived from SPCE/OPE/BiONPs/PANI. The relative standard deviations (RSD) for SPC electrode at a 6.04 µM PB2+ analyte concentration was 4.76% and 0.98% at SPCE/LPE/BiONPs/PANI and SPCE/LPE/BiONPs/PANI, respectively. The effect of copper, zinc, iron, cobalt, nickel, and magnesium on the stripping peaks of PB2+ at SPCE/OPE/BiONPs/PANI, showed no significant change except for cobalt, with about 17.67% peak current drop. The sensors were assessed for possible determination of PB2+ in spiked river water samples. The average percentage recovery and RSD calculated were 94.25% and 3.74% (n = 3) at SPCE/LPE/BiONPs/PANI and, 96.70% and 3.71% (n = 3) at SPCE/OPE/BiONPs/PANI, respectively. Therefore, the fabricated sensor material could be used for environmental assessment of this highly toxic heavy metal in the aquatic system

Enzymatic Electroanalytical Biosensor Based on Maramiellus colocasiae Fungus for Detection of Phytomarkers in Infusions and Green Tea Kombucha

In this work, we developed an enzymatic voltammetric biosensor for the determination of catechin and gallic acid in green tea and kombucha samples. The differential pulse voltammetry (DPV) methodology was optimized regarding the amount of crude enzyme extract, incubation time in the presence of the substrates, optimal pH, reuse of the biosensor, and storage time. Samples of green tea and kombucha were purchased in local markets in the city of Goiânia-GO, Brazil. High performance liquid chromatography (HPLC) and Folin-Ciocalteu spectrophotometric techniques were performed for the comparison of the analytical methods employed. In addition, two calibration curves were made, one for catechin with a linear range from 1 to 60 µM (I = −0.152 * (catechin) − 1.846), with a detection limit of 0.12 µM and a quantification limit of 0.38 µM and one for gallic acid with a linear range from 3 to 60 µM (I = −0.0415 * (gallic acid) − 0.0572), with a detection limit of 0.14 µM and a quantification limit of 0.42 µM. The proposed biosensor was efficient in the determination of phenolic compounds in green tea.

Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

Optimization and Validation of an Extraction Method for Endosulfan Lactone on a Solid Substrate

Endosulfan lactone is a metabolite obtained from the biological oxidation of the insecticide endosulfan by action of the microorganisms present in the soil. This metabolite is more toxic and persistent than the parent compound. Therefore, it is extremely important to be able to determine the presence of this metabolite in the soil. However, accessible methods for extraction of endosulfan lactone in soil were not found in published literature. For this reason, the aim of this study was to evaluate two conventional methods of liquid–solid extraction for the determination of endosulfan lactone in solid substrate using two solvents (ethyl acetate and acetonitrile) and HPLC UV-VIS. The acetonitrile and rotary agitation extraction method was the one with the highest efficiency (97%), optimized using a factorial 32 response surface design, and validated in terms of linearity and precision. The linearity shown was r > 0.999 in a wide spike level (0.15–100 mg kg−1), with the detection limit (DL) of 0.045 mg kg−1 and quantification limit (QL) of 0.15 mg kg−1. The extraction of endosulfan lactone in solid substrate using acetonitrile was more efficient than that used with ethyl acetate, so this method could be used to extract and quantify endosulfan lactone in agricultural soil.

Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: There were no significant differences between the GCN results obtained from uniplex andmultiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.