Polyethylene Glycol Precipitation Method Research Articles

Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

The cases of tracing the source of patients infected with Omicron variant of SARS-CoV-2 based on wastewater-based epidemiology in Shenzhen

Wastewater-based epidemiology (WBE) is an emerging discipline, which has been applied to drug abuse tracking and infectious disease pathogen surveillance. During the COVID-19 epidemic, WBE has been applied to monitor the epidemic trend and SARS-CoV-2 variants etc. In order to detect hidden COVID-19 cases and prevent transmission in the community, wastewater surveillance system for monitoring SARS-CoV-2 RNA was developed in Shenzhen. The sewage sampling sites were set up in key places such as the port areas, urban villages and residential communities of Futian, Nanshan, Luohu and Yantian districts. From July 26 to November 30, 2022, a total of 369 sewage sampling sites were set up, covering 1.93 million people. Continuous sampling was carried out for 3 hours in the peak period of water use every day. Sewage virus enrichment and SARS-CoV-2 nucleic acid detection were carried out by polyethylene glycol precipitation method and RT-qPCR, and a positive water sample disposal process was molded. This article aims to introduce the case of source tracing of COVID-19 infected patients based on urban sewage in Shenzhen. The sewage monitoring of Honghu water treatment plant in Luohu District played an early warning role, and the source of infection was traced. In the disposal of positive water samples in Futian South Road, Futian District, the important experience of monitoring point layout was obtained. In the sewage monitoring of Nanshan village, Nanshan District, the existence of occult infection was revealed. Sharing the experience of tracing the source of COVID-19 patients to avoid the spread of COVID-19 in the community based on wastewater surveillance of SARS-CoV-2 RNA in Shenzhen, and summarizing the advantages and application prospects of sewage surveillance can provide new ideas for monitoring emerging or re-emerging pathogens that are known to exhibit gastrointestinal excretion in the future.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Prolactin Monomeric Polyethylene Glycol Measurement Method and Study of Reference Value Verification

Purpose: Prolactin in the blood is separated into three types, and over 90% of prolactin presents as a double monomer (23 KDa). Rarely, it can exist in the size of big prolactin (150 KDa), which is called macroprolactin and is known as an autoantibody complex. When macroprolactin accounts for more than 60% of prolactin in the blood, it is called macroprolactinemia. The presence of such macroprolactin was first reported in a patient with hyperprolactinemia but without typical symptoms. Macroprolactinemia is emerging as an important cause of idiopathic hyperprolactinemia. The polyethylene glycol (PEG) precipitation method using the property of precipitating large-molecular-weight proteins is simple and recently has been widely used as a screening test. The results are in good agreement with the results of gel chromatography. The purpose of this study was to confirm the measurement method and reference value verification of monomeric prolactin in blood prolactin using the PEG precipitation method. Materials and Methods: For 40 examinees who visited the Gangnam Center of Seoul National University Hospital in 2021, the prolactin level was verified using radioimmunoassay (RIA). For macroprolactinemia PEG precipitation method, 25% PEG (molecular weight 6000kDa) solution and serum were mixed in equal amounts in a test tube, then left at room temperature for 20 minutes and centrifuged at 4℃ for 30 minutes (1500g). The prolactin level was measured in the supernatant. Results : After confirming that more than 90% of the 40 tested samples within the reference range <25 ng/mL, the same value as the reference value for prolactin was applied. Since the concentration of monomeric prolactin in serum from which macroprolactin has been removed from blood is diluted 1:1 with PEG, our laboratory is currently reporting the result by multiplying the result by a dilution factor of 2. Conclusion: Radioimmunoassay using PEG precipitation method using the property of precipitating large molecular weight proteins is simple and effective for quantitative measurement of monomeric prolactin in blood prolactin.

B-003 Decrease in Serum apoE-rich HDL-cholesterol May Be Associated With Cholesterol Accumulation in the Liver of Rats Fed A High-cholesterol Diet

Abstract Background Free cholesterol accumulation in the liver has been suggested to be one of risk factors for the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis. Therefore, a non-invasive approach to assess hepatic cholesterol accumulation in the early stage without elevation of liver enzymes (AST and ALT) may be useful in clinical setting. In this study, we show that serum apoE-rich HDL-cholesterol level is a possible indicator for predicting hepatic cholesterol accumulation in rats with diet-induced simple liver steatosis. Methods Male Sprague-Dawley rats (8 weeks) were purchased from Charles River Laboratories. Four different diets were obtained from Oriental Yeast; control diet (MF), high-fat diet (HF; 68% MF, 30% palm oil, and 2% cholic acid), high-cholesterol diet (HC; 95.5% MF, 2.5% cholesterol, and 2% cholic acid), and high-fat/high-cholesterol diet (HFC; 81.75% MF, 15% palm oil, 1.25% cholesterol, and 2% cholic acid). The rats were randomly divided into 4 groups and fed as follows. The control and HFC groups received the MF and HFC diets for 8 weeks, respectively. The HF/HC group received the HF diet for the first 4 weeks and the HC diet for the next 4 weeks. The HC/HF group received the HC diet first, then the HF diet. Blood and liver tissue specimens were collected under anesthesia. Total HDL fractions were isolated from whole sera by the polyethylene glycol precipitation method, and applied into a cation-exchange column (HiTrap SPHP, GE healthcare) for determining apoE-rich HDL-cholesterol levels. Correlations between variables were assessed with Spearman’s correlation coefficient (rs). Results Total cholesterol content of the liver was significantly higher in the HFC (11.1-fold, P < 0.001), HF/HC (9.1-fold, P < 0.001), and HC/HF (5.8-fold, P < 0.001) groups than the control group. Hepatic free cholesterol was also higher in the HFC (5.5-fold, P < 0.001), HF/HC (3.9-fold, P < 0.001), and HC/HF (3.3-fold, P < 0.001) groups. Hepatic triglycerides were slightly increased in the HC/HF (1.6-fold, P < 0.01) and HFC (1.5-fold, P < 0.05) groups. Serum apoE-rich HDL-cholesterol was significantly decreased in the HF/HC (0.21-fold, P < 0.001), HC/HF (0.25-fold, P < 0.001) and HFC (0.42-fold, P < 0.01) groups. The HFC group had significantly higher serum ALT (3.0-fold, P < 0.05) and AST (2.3-fold, P < 0.01) levels, compared with the control groups. There was a significant increase in hepatic mRNA expression of TNF-α (24.8-fold, P < 0.01) and TGF-β (3.8-fold, P < 0.001) only in the HFC group, indicating liver inflammation and stellate cell activation. To exclude the effects of hepatocyte injury on apoE-rich HDL metabolism, rats with ALT less than 100 U/mL (n = 16) were included in the following analysis. ApoE-rich HDL-cholesterol levels were significantly and inversely correlated with hepatic total cholesterol (rs = −0.535, P < 0.05) and free cholesterol (rs = −0.532, P < 0.05) levels but not with hepatic triglycerides. Furthermore, the ratio of ALT to apoE-rich HDL-cholesterol was more highly correlated with hepatic total cholesterol (rs = 0.626, P < 0.01) and free cholesterol (rs = 0.632, P < 0.01). There was no such correlation for apoE-poor HDL-cholesterol. Conclusion: The present observations suggest that serum apoE-rich HDL-cholesterol levels may be a useful indicator for cholesterol accumulation in the liver of cholesterol-fed rats.

Human Adenovirus Detection and Genetic Characterization in Irrigation Water from the Riyadh Region, Saudi Arabia

Adenoviruses are considered one of the most persistent enteric pathogens that can penetrate entire aquifer matrices. An ongoing monitoring of irrigation water is essential to mitigate potential public health risks. We investigated the prevalence of human adenoviruses (HAdV) in the groundwater discharge basins of Aluraiji (AW-DB) and Diriyah (DW-DB) and in the irrigation water of Al Harriq (H-IW) and Al Hayer (AH-IW) from January to December 2021. The meteorological impact (temperature, relative humidity, and wind speed) on HAdV prevalence and molecular diversity was investigated by targeting a selected region of the Hexon gene. The samples were concentrated using the polyethylene glycol precipitation (PEG) method. HAdVs were detected using PCR and sequenced by Sanger sequencing, and phylogenetic analysis was performed. The highest HAdV prevalence was recorded in H-IW, AH-IW, and DW-DB (100%). HAdV sequences were found to be closely related to species F (type 41) with a predominance of the 00-2B64 sequence (86.4%). Phylogenetic analysis depicted a close relationship of this study’s isolate 00-2B64 to a Brazilian and Saudi isolate, whereas 08-2B64 was found to be related to a sequence from an AnNazim landfill (LF1; d = 0.00) from Riyadh, Saudi Arabia. A high HAdV prevalence was recorded at a temperature range of 19–28 °C, wind speed was recorded at a range of (16–20 km/h), and relative humidity was recorded at a range of (15–25%). Meteorological variations exhibited no significant effect on the prevalence of HAdVs. The findings provided data on HAdV prevalence and predominant species in the irrigation water of Riyadh, Saudi Arabia and presented information regarding the environmental effects of HAdV persistence. In public health contexts, this will help in the planning of disease control strategies.

Long-term SARS-CoV-2 surveillance in wastewater and estimation of COVID-19 cases: An application of wastewater-based epidemiology

The role of wastewater-based epidemiology (WBE), a powerful tool to complement clinical surveillance, has increased as many grassroots-level facilities, such as municipalities and cities, are actively involved in wastewater monitoring, and the clinical testing of coronavirus disease 2019 (COVID-19) is downscaled widely. This study aimed to conduct long-term wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Yamanashi Prefecture, Japan, using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay and estimate COVID-19 cases using a cubic regression model that is simple to implement. Influent wastewater samples (n = 132) from a wastewater treatment plant were collected normally once weekly between September 2020 and January 2022 and twice weekly between February and August 2022. Viruses in wastewater samples (40 mL) were concentrated by the polyethylene glycol precipitation method, followed by RNA extraction and RT-qPCR. The K-6-fold cross-validation method was used to select the appropriate data type (SARS-CoV-2 RNA concentration and COVID-19 cases) suitable for the final model run. SARS-CoV-2 RNA was successfully detected in 67 % (88 of 132) of the samples tested during the whole surveillance period, 37 % (24 of 65) and 96 % (64 of 67) of the samples collected before and during 2022, respectively, with concentrations ranging from 3.5 to 6.3 log10 copies/L. This study applied a nonnormalized SARS-CoV-2 RNA concentration and nonstandardized data for running the final 14-day (1 to 14 days) offset models to estimate weekly average COVID-19 cases. Comparing the parameters used for a model evaluation, the best model showed that COVID-19 cases lagged 3 days behind the SARS-CoV-2 RNA concentration in wastewater samples during the Omicron variant phase (year 2022). Finally, 3- and 7-day offset models successfully predicted the trend of COVID-19 cases from September 2022 until February 2023, indicating the applicability of WBE as an early warning tool.

Wastewater surveillance of Mpox virus in Baltimore

This study aimed to utilize wastewater surveillance for monitoring Mpox cases at a community level. Untreated wastewater samples were collected once a week from two wastewater treatment plants (A and B) in Baltimore City from July 27, 2022–September 22, 2022. The samples were concentrated via an adsorption–elution (AE) method and Polyethylene Glycol (PEG) precipitation method followed by quantitative polymerase chain reaction (qPCR). Monkeypox virus (MPXV) was detected in 89 % (8/9) samples from WWTP A and 55 % (5/9) samples from WWTP B with at least one concentration method. Higher detection rate in samples concentrated with PEG precipitation compared to AE method was observed, indicating that PEG precipitation is a more effective virus concentration method for MPXV. To our knowledge, this is the first study reporting the detection of MPXV in wastewater in Baltimore. The results highlight that wastewater surveillance could be used as a complementary early warning tool for monitoring future Mpox outbreaks.

Validation of a method to elute viruses from different types of face masks

Due to the SARS-CoV-2 pandemic, it is crucial to study the efficiency of face masks in retaining viruses for the upcoming years. The first objective of this study was to validate a method to elute viruses from polyester and cotton face masks. We observed that deionized water followed by 3% beef glycine (pH 9.5 or pH 7.2) was significantly more efficient (p < 0.05) in eluting the bacteriophage phiX174 virus from polyester (4.73% ± 0.25% to 28.67% ± 1.89%), polyester/cotton (3% ± 0.33%), and cotton (1.7% ± 0.21%) face masks than 3% beef glycine only (pH 9.5 or pH 7.2) as a single eluent (3.4% ± 0.16% to 21.33% ± 0.94% for polyester, 1.91% ± 0.08% for polyester/cotton, and 1.47% ± 0.12% for cotton face masks). Also, deionized water was significantly less efficient as a single eluent for eluting bacteriophage phiX174 from all the studied face mask types. The polyethylene glycol (PEG) precipitation method was substantially more efficient (p < 0.05) as a second step concentration method for the viruses in the eluates than the organic flocculation (OF) method. Higher viral loads were eluted from polyester face masks than cotton ones. We also found varying viral loads in the eluate solutions from different commercial polyester face masks, with the highest percentage seen for the N95 face mask. The second objective was to apply the validated method to study the effect of autoclaving on the different face mask materials. Results of the study did not show any significant differences in the viral loads eluted from the studied face masks before and after one and five autoclaving cycles. Moreover, a scanning electron microscope (SEM) analysis revealed no changes in the yarns, elongation, tensile strength, and contact angle measurements of the polyester or cotton materials after one or five autoclaving cycles.

Very low likelihood that cultivated oysters are a vehicle for SARS-CoV-2: 2021–2022 seasonal survey at supermarkets in Kyoto, Japan

The pandemic caused by novel coronavirus disease of 2019 (COVID-19) is a global threat. Wastewater surveillance in Japan and abroad has led to the detection of SARS-CoV-2, causing concern that SARS-CoV-2 in the feces of infected persons may contaminate the aquatic environment. Bivalves such as oysters cultivated in coastal areas are known to filter and concentrate viruses such as norovirus present in seawater in their bodies; however, whether they do so with SARS-CoV-2 is unknown. Therefore, we examined cultivated oysters sold in Japan for the presence of SARS-CoV-2 between October 2021 and April 2022 to clarify the extent of viral contamination and evaluate the risk of food-borne transmission of SARS-CoV-2. Porcine epidemic diarrhea virus (PEDV), known as pig coronavirus, was used to spike midgut-gland samples as a whole process control. The presence of SARS-CoV-2 and PEDV was investigated using a modified polyethylene glycol precipitation method and RT-qPCR. While all samples spiked with the whole process control were positive, no SARS-CoV-2 was detected in any of the 145 raw oyster samples surveyed, despite a marked increase in infections caused by the Omicron variant from January to April 2022 in Japan. Therefore, our results suggest that with well-developed sewage treatment facilities, consumption of oysters cultivated in coastal areas may not be a risk factor for SARS-CoV-2 outbreaks.

Development of a magnetic nanoparticle-based method for concentrating SARS-CoV-2 in wastewater

Several virus concentration methods have been developed to increase the detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater, as part of applying wastewater-based epidemiology. Polyethylene glycol (PEG) precipitation method, a method widely used for concentrating viruses in wastewater, has some limitations, such as long processing time. In this study, Pegcision, a PEG-based method using magnetic nanoparticles (MNPs), was applied to detect SARS-CoV-2 in wastewater, with several modifications to increase its sensitivity and throughput. An enveloped virus surrogate, Pseudomonas phage φ6, and a non-enveloped virus surrogate, coliphage MS2, were seeded into wastewater samples and quantified using reverse transcription-quantitative polymerase chain reaction to assess the recovery performance of the Pegcision. Neither increasing MNP concentration nor reducing the reaction time to 10 min affected the recovery, while adding polyacrylic acid as a polyanion improved the detection sensitivity. The performance of the Pegcision was further compared to that of the PEG precipitation method based on the detection of SARS-CoV-2 and surrogate viruses, including indigenous pepper mild mottle virus (PMMoV), in wastewater samples (n = 27). The Pegcision showed recovery of 14.1 ± 6.3 % and 1.4 ± 1.0 % for φ6 and MS2, respectively, while the PEG precipitation method showed recovery of 20.4 ± 20.2 % and 18.4 ± 21.9 % (n = 27 each). Additionally, comparable PMMoV concentrations were observed between the Pegcision (7.9 ± 0.3 log copies/L) and PEG precipitation methods (8.0 ± 0.2 log copies/L) (P > 0.05) (n = 27). SARS-CoV-2 RNA was successfully detected in 11 (41 %) each of 27 wastewater samples using the Pegcision and PEG precipitation methods. The Pegcision showed comparable performance with the PEG precipitation method for SARS-CoV-2 RNA concentration, suggesting its applicability as a virus concentration method.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4–52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9–23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

Prevalence of Macroprolactinaemia in Women with Hyperprolactinaemia: A Retrospective Study

Introduction: Macroprolactinaemia causes asymptomatic hyperprolactinaemia in many patients which leads to misdiagnosis, inappropriate investigation and needless treatment in these patients. Though immunoassays for prolactin are sturdy and reliable, they are prone to interference from macroprolactin. Polyethylene Glycol (PEG) precipitation is used as a screening test for macroprolactinaemia. Aim: To find out the prevalence of macroprolactin in women with hyperprolactinaemia this will help in evaluation and treatment of such patients. Materials and Methods: This retrospective study was conducted at a Global Reference Laboratory in Mumbai over a period of three and a half years from January 2018 to May 2021. Total available data of 1,15,149 women with age above 18 years were included in the study. Prolactin concentrations were measured before and after PEG precipitation. Macroprolactinaemia was characterised by percentage recovery and post PEG prolactin concentrations. Continuous variables were expressed as Mean±Standard Deviation (SD), Range and categorical variables as number and percentage. The differences in categorical variables were assessed with Chisquare test or Fisher’s-exact test. Results: A total of 36,247 women (31.48%) women out of the total 1,15,149 were observed to have hyperprolactinaemia Prevalence of macroprolactinaemia using recovery criteria of ≤50% was 7.88%. Amongst the women diagnosed with hyperprolactinaemia highest prevalence of 62.46% (22,639) was found between 18-30 years. Macroprolactin results and age were found to be statistically significant. Infertility, Oligomenorrhoea/amenorrhoea, and thyroid disorders was seen more frequently in hyperprolactinaemia than in macroprolactinaemia. Twelve women with prolactin values above 100 ng/mL were found to have macroprolactinaemia. Conclusion: Macroprolactin determination with the PEG precipitation method might prevent unnecessary tests and treatments during the diagnosis process and follow-up of patients.

Development of SarS-Cov-2 antigen Detection Kit Based on Immunoglobulin Y (Igy) Using Surface Plasmon resonance (SPr)

SARS disease reappeared at the end of 2019 with a new name as Coronavirus Disease 2019 (COVID-19) caused by a new virus called SARS-CoV-2. This virus has spread throughout the world until recently and caused massive deaths and losses. The nucleic acid test in the form of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is very important to diagnose COVID-19 in patients, but this method has several drawbacks such as operators who have to be trained, the diagnosis results appear in a relatively long time, and the examination price relatively expensive. This research was conducted to produce immunoglobulin Y (IgY) extracted from chicken egg yolk targeting the S-protein receptor-binding domain (RBD) on SARS-CoV-2 as a component of the surface plasmon resonance (SPR) SARS-CoV-2 antigen detection kit. This research was started by extracting IgY from hyperimmune chicken egg yolks with the polyethylene glycol (PEG) precipitation method and continued with dialysis. The extracted IgY was further purified using thiophilic adsorption chromatography and concentrated by using Amicon® Ultra-15 ultrafiltration. The IgY activity against SARS-CoV-2 RBD was tested qualitatively using the agar gel precipitation test (AGPT) technique and the total protein content was determined using the Lowry method. IgY was tested for its affinity against SARS-CoV-2 RBD using SPR. The IgY concentration obtained was 11 mg/mL. The AGPT result showed the presence of IgY activity against SARS-CoV-2 RBD isolated from egg yolk and chicken serum after 8 weeks after the first vaccination of chickens. The SDS-PAGE results showed a very clear band of IgY characters. The obtained IgY showed adequate interaction with commercial SARS-CoV-2 RBD on an SPR device. The purified IgY was able to bind with protein-S RBD and showed a fairly good affinity for the SARS-CoV-2 antigen sample. The results of these observations indicate that IgY anti-S-protein SARS-CoV-2 can be produced and purified from chicken egg yolk and can be used as a diagnostic component to detect SARS-CoV-2 antigen, especially on SPR.

Production of IgY polyclonal antibody against diphtheria toxin and evaluation of its neutralization effect by Vero cell assay

BackgroundDiphtheria is a bacterial disease which is caused by Corynebacterium diphtheriae. The symptoms are due to the diphtheria toxin produced by the bacteria. Antibiotic therapy and the use of diphtheria antitoxin is a recommended strategy to control diphtheria. Although mammalian antibodies are used to treat patients, IgY antibody has advantages over mammalian ones, including cost-effectiveness and production through non-invasive means. Moreover, in contrast to mammalian antibodies, IgY does not bind to the rheumatoid factor and does not activate the complement system. The objective of this study was to evaluate the in vitro neutralizing effect of IgY against diphtheria toxin.ResultsAnti-DT IgY was produced by immunization of the laying white leghorn chickens. Indirect enzyme-linked immunosorbent assay revealed successful immunization of the animals, and the IgY was purified with a purity of 93% via polyethylene glycol precipitation method. The neutralizing activity of the purified IgY was evaluated by Vero cell viability assay. This assay confirmed that 1.95 μg (8.6 μg/ml of culture medium) of anti-DT IgY would neutralize 10 fold of cytotoxic dose 99% of DT, which was 0.3 ng (1.33 ng/ml of culture medium).ConclusionThis anti-DT IgY may be applicable for diphtheria treatment and quality controls in vaccine production.

An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater

Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6–56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0–21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation.

Prevalence of Macroprolactinaemia by Polyethylene Glycol Precipitation Method: A Cross-sectional Study

Introduction: Macroprolactin, an immunoreactive molecule resulting from association of monomeric Prolactin (mPRL) and immunoglobulin G is a significant cause of misdiagnosis, unnecessary radiological investigation and treatment for hyperprolactinaemia. Data on its prevalence and clinical manifestation varies regionally. Case presentation can vary with assymptomatic cases to those with galactorrhea and irregular menses. Aim: To find the prevalence and clinical features associated with macroprolactin in cases of hyperprolactinaemia in hospital patients. Materials and Methods: A cross-sectional study was conducted in the Department of Biochemistry, Institute of Postgraduate Medical Education and Research and SSKM hospital, Kolkata, India, from November 2018 to April 2019. Serum samples were assayed for serum PRL levels in 1400 subjects by Chemiluminescence immunoassay (Immulite 1000 siemens) based on presenting symptoms of galactorrhoea, amenorrhoea and infertility. Serum PRL samples (n=240) above the manufacturer’s reference cut-off level (PRL ≥30 ng/mL) were obtained from patients with or without symptoms of hyperprolactinaemia. Retesting for PRL levels were done following precipitation of macroprolactin using Polyethylene Glycol (PEG, MW: 6000). Fourty cases with physiological causes of PRL excess, hypothyroidism, polycystic ovary syndrome, antidopaminergic drug intake, hepatorenal diseases and chest wall disorders were excluded. The results were expressed in terms of Mean±Standard Deviation (SD) and compared using student t-test. Results: Prevalence of macroprolactin was 16 (13.3%) out of 120 among true hyperprolactaemic cases (male=2; female, n=14) based on percentage recovery of PRL in post-PEG cases (Recovery Rate (RR) <40%). The mean pre-PEG and post-PEG values were 52.5 ng/mL and 19.2 ng/mL (RR: 36.5%; p-value <0.05), respectively. The mean pre PEG, PRL values were significantly lower in macroprolactaemic cases than those with true hyperprolactinaemia (52.5 ng/mL versus 74.57 ng/mL; p-value 0.038). Some of the Macro-PRL cases reported with complaints of galactorrhoea, menstrual irregularities and infertility. Conclusion: The results revealed a prevalence rate similar to those reported in other studies worldwide. Clinical features alone are an unreliable tool to distinguish between cases with true high PRL levels and macroprolactinaemia. Macro-PRL cases once diagnosed requires no extended endocrine review and long term management. Hence, in cases with high PRL levels discordant with clinical symptoms/radiological data routine PEG precipitation test is an inexpensive assay for initial screening for presence of macroprolactin and also monitoring of patients already started on dopamine agonists for hyperprolactinaemia of unknown aetiology.

Prolactin Level in Polycystic Ovary Syndrome (PCOS): An approach to the diagnosis and management.

Introduction:In spite of the increasing prevalence of polycystic ovary syndrome, there are restricted studies about different features of PCOS. In this study, we evaluate serum prolactin levels in PCOS patients and compare clinical features and hormone levels between patients with hyperprolactinemia and normal levels of prolactin.Methods:Serum prolactin level in 330 PCOS patients were evaluated. If the prolactin levels were normal (equal to or less than 25 ng/ml) the patient was considered normal. If the prolactin levels were >25 ng/ml, then the level was measured by Polyethylene glycol (PEG) precipitation method to detect macroprolactinemia. Furthermore, brain MRI was done in case of hyperprolactinemia to discover pituitary adenoma.Results:Among 330 patients with PCOS, 208 (63.4%) had normal prolactin levels and 122 (37%) patients had hyperprolactinemia. Among hyperprolactinemic patients, 72 (59%) had normal prolactin levels after PEG precipitation and 33 (27%) patients had pituitary adenoma in their brain MRI and 17 (13%) patients were considered as idiopathic hyperprolactinemia with normal MRI. Further, higher levels of LH and LH/FSH ratio and lower estradiol levels were observed in patients with normal prolactin levels. Also, menstrual disorders were more prevalent among patients with pituitary adenoma.Conclusions:Investigating increased level of prolactin in PCOS patients is recommended to detect the causes of hyperprolactinemia, especially macroprolactinemia. (www.actabiomedica.it)

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters

Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1–2), PEG precipitation (Methods 3–7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2% and 45.0%, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.