Survival of Salmonella spp. and Pathogenic Escherichia coli in Food Matrixes and Its Relevance in the Development of Proficiency Testing Samples.

Monitoring of food products by government agencies for their compliance to regulatory limits is an essential step in controlling foodborne outbreaks. For monitoring purposes, an extensive setup of the surveillance system is used, which involves ISO 17025:2017 accredited laboratories for food testing. Participation in proficiency testing (PT) programs is a requirement of ISO 17025:2017, which ensures data accuracy and analyst competency. Participation in PT schemes is costly for laboratories in developing countries as most of the commercial suppliers are situated in the United States and Europe. The literature or data available on creation of microbiological proficiency testing is scanty as much of the data available with commercial suppliers are trade secrets, and there is only 0.06% of research articles available in the Scopus database on the topic. In this review article, an attempt is made to understand the factors impacting the survival of two important foodborne pathogens, i.e., Escherichia coli and Salmonella spp., by extracting information available from growth studies and root-cause analysis of various food safety incidents and recalls. Utilization of this information in the development of PT samples is discussed in this review article along with a focus on the availability of PT samples and associated ISO standards to formulate homogeneous and stable PT samples. This review article elaborates on the focus areas that can be considered by PT providers (PTP)-for example, initial inoculum level and preparation, strain type, microbial growth phase, the impact of different types of food matrixes including low-moisture food, antimicrobial components, pH, presence of competitor microbes, and environmental conditions involving storage temperature, time, and relative humidity. These focus areas can be used to successfully create PT samples by PTP in developing countries.

Motion-compensated online object tracking for activity detection and crowd behavior analysis.

It is a nontrivial task to manage crowds in public places and recognize unacceptable behavior (such as violating social distancing norms during the COVID-19 pandemic). In such situations, people should avoid loitering (unnecessary moving out in public places without apparent purpose) and maintain a sufficient physical distance. In this study, a multi-object tracking algorithm has been introduced to improve short-term object occlusion, detection errors, and identity switches. The objects are tracked through bounding box detection and with linear velocity estimation of the object using the Kalman filter frame by frame. The predicted tracks are kept alive for some time, handling the missing detections and short-term object occlusion. ID switches (mainly due to crossing trajectories) are managed by explicitly considering the motion direction of the objects in real time. Furthermore, a novel approach to detect unusual behavior of loitering with a severity level is proposed based on the tracking information. An adaptive algorithm is also proposed to detect physical distance violation based on the object dimensions for the entire length of the track. At last, a mathematical approach to calculate actual physical distance is proposed by using the height of a human as a reference object which adheres more specific distancing norms. The proposed approach is evaluated in traffic and pedestrian movement scenarios. The experimental results demonstrate a significant improvement in the results.

Case Reports on Black Fungus of the Gastrointestinal Tract: A New Complication in COVID-19 Patients / 대한소화기학회지

Gastrointestinal mucormycosis is a rare disease with a significant mortality rate, even when promptly diagnosed and treated. An unusual complication was observed in India during the second wave of coronavirus disease 2019 (COVID-19). Two incidences of gastric mucormycosis were found. A 53-year-old male patient with a history of COVID-19 one month earlier came into the intensive care unit. After admission, the patient developed hematemesis, which was initially treated with blood transfusions and digital subtraction angiography embolization. Esophagogastroduodenoscopy (EGD) revealed a large ulcer with a clot in the stomach. During an exploratory laparotomy, the proximal stomach was necrotic. Histopathological examination confirmed mucormycosis. The patient was started on antifungals, but despite rigorous therapy, the patient died on the tenth postoperative day. Another patient, an 82-year-old male with a history of COVID-19, arrived with hematemesis two weeks earlier and was treated conservatively. EGD revealed a large white-based ulcer with abundant slough along the larger curvature of the body. Mucormycosis was verified by biopsy.He was treated with amphotericin B and isavuconazole. He was discharged after two weeks in a stable condition. Despite quick detection and aggressive treatment, the prognosis is poor. In the second case, prompt diagnosis and treatment saved the patient’s life.

Primary peritoneal carcinoma and ovarian carcinoma - a ten-year comparative analysis.

BACKGROUND: Primary peritoneal carcinoma (PPC) at presentation often masquerades as epithelial ovarian carcinoma (OC) but behaves different with respect to treatment response, recurrence patterns and has inferior outcomes. The objective of this study is to compare the clinicopathological features and survival outcomes of PPC and OC. METHODS: Prospectively maintained database of patients presenting to the gynecologic oncology department at a tertiary hospital was reviewed between 1st January 2010 and 31st December 2020. A comparative analysis of high-grade serous stage III/IV PPC and OC was done. Demographics, treatment details, complications and survival outcomes were collected from electronic medical records. RESULTS: 151 OC and 69 PPC patients were included. A higher proportion of women with PPC had reduced performance status prior to hysterectomy with salpingo-oophorectomy, a shorter symptom to treatment interval, and large volume ascites. A significantly lower number of women with PPC (4.3 vs. 46.1%; P < 0.001) underwent primary cytoreduction, had a lower median surgical complexity score (3 vs. 4; P < 0.001) but higher recurrence rates (66.7 vs. 47.0%; P = 0.041) as compared to the patients with OC. The median progression-free survival (PFS) was 18 (15-20) months in PPC and 23 (17-28) months in OC patients (log-rank P = 0.034), while the median overall survival (OS) was similar (44 vs. 48 months; log-rank P = 0.696). The presence of extraperitoneal disease and interval cytoreduction was associated with shorter PFS. Suboptimal cytoreduction and delay in adjuvant chemotherapy beyond 6 weeks post-surgery was associated with reduced OS. CONCLUSION: PPC is an aggressive disease with lower PFS compared to OC. Commonly presenting with large volume carcinomatosis, it is not amenable for primary cytoreduction, making the usage of neoadjuvant chemotherapy a common practice and pragmatic approach.

The association of ABO blood group with the asymptomatic COVID-19 cases in India

The COVID-19 pandemic has resulted several waves of infection in many countries worldwide. The large variations in case fatality ratio among different geographical regions suggests that the human susceptibility against this virus varies substantially. Several studies from different parts of the world showed a significant association of ABO blood group and COVID-19 susceptibility. It was shown that individuals with blood group O are at the lower risk of coronavirus infection. To establish the association of ABO blood group in SARS-CoV-2 susceptibility, we for the first time analysed SARS-CoV-2 neutralising antibodies as well as blood groups among 509 random individuals from three major districts of Eastern Uttar Pradesh region of India.. Interestingly, we found neutralising antibodies in significantly higher percentage of people with blood group AB (0.36) followed by B (0.31), A (0.22) and lowest in people with blood group O (0.11). This indicates that people with blood group AB are at comparatively higher risk of infection than other blood groups. Further, in line to previous reports we too observed that people with blood group O have significantly decreased risk of SARS-CoV-2 infection. Thus, among the asymptomatic SARS-CoV-2 infected individuals with blood group AB has highest, whilst blood group O has lowest risk of infection.

Estimation of real-infection and immunity against SARS-CoV-2 in Indian populations

Infection born by Coronavirus SARS-CoV-2 has swept the world within a time of a few months. It has created a devastating effect on humanity with social and economic depression. Europe and America were the hardest hit continents. India has also lost lives, making the country fourth most deadly worldwide. However, the infection and death rate per million and the case fatality ratio in India were substantially lower than in many developed nations. Several factors have been proposed including genetics. One of the important facts is that a large chunk of Indian population is asymptomatic to the SARS-CoV-2 infection. Thus, the real infection in India is much higher than the reported number of cases. Therefore, the majority of people are already immune in the country. To understand the dynamics of real infection as well as the level of immunity against SARS-CoV-2, we have performed antibody testing (serosurveillance) in the urban region of fourteen Indian districts encompassing six states. In our survey, the seroprevalence frequency varied between 0.01-0.48, suggesting high variability of viral transmission between states. We also found out that the cases reported by the government were several fold lower than the real incidence of infection. This discrepancy is mainly driven by the higher number of asymptomatic cases. Overall, we suggest that with the high level of immunity developed against SARS-CoV-2 in the majority of the districts, the case fatality rate of second wave in India will be minor than first wave.

Sonographic Optic Nerve Sheath Diameter Following Seizure Prophylaxis in Pre-Eclamptic Parturients With Severe Features: A Prospective, Observational Study.

OBJECTIVES: Neurological manifestations, including raised intracranial pressures, are a hallmark of worsening pre-eclampsia. Invasive methods for measuring intracranial pressure, though a gold standard, are not always a viable option. Maternal ocular sonography is a promising bedside tool, which serves as a noninvasive, cost-effective means for measuring optic nerve sheath diameter (ONSD), a surrogate marker of raised intracranial pressures. We studied the ultrasonographically measured ONSD in severely pre-eclamptic women, and the effect of magnesium sulfate therapy on its values. METHODS: Thirty severely pre-eclamptic women at ≥28 weeks gestation were included. We recorded baseline ONSD values, serum magnesium levels, neurological symptoms, vitals, and repeated them at 4 and 12 hours following magnesium sulfate therapy, and then at 24 hours postpartum. An ONSD value >5.8 mm was suggestive of raised intracranial pressure. Primary outcome measure was to evaluate changes in ultrasonographically measured ONSD following seizure prophylaxis with magnesium sulfate. RESULTS: Women, 73.3%, had baseline ONSD >5.8 mm, with mean diameter being significantly high (6.02 ± 0.77 mm). There was a statistically nonsignificant decline in mean ONSD values at 4 and 12 hours, as well as at 24 hours postpartum. Patients with neurological symptoms declined significantly (from 70 to 10%; p value <.001) following magnesium sulfate therapy. CONCLUSIONS: Majority of severely pre-eclamptic parturients had high ONSD value suggestive of raised intracranial pressures, which persisted in the postpartum period and was unaffected by magnesium sulfate therapy. Ultrasound can thus serve as a point-of-care, cost-effective, easily available bedside tool for indirectly measuring intracranial pressures in this high-risk population.

Phosphate starvation controls lactose metabolism to produce recombinant protein in Escherichia coli.

Phosphate is one of the major constituents in growth media. It closely regulates central carbon and energy metabolism. Biochemical reactions in central carbon metabolism are known to be regulated by phosphorylation and dephosphorylation of enzymes. Phosphate scarcity can limit microbial productivity. However, microorganisms are evolved to grow in phosphate starvation environments. This study investigates the effect of phosphate-starved response (PSR) stimuli in wild-type and recombinant Escherichia coli cells cultivated in two different substrates, lactose, and glycerol. Phosphate-starved E. coli culture sustained bacterial growth despite the metabolic burden that emanated from recombinant protein expression albeit with altered dynamics of substrate utilisation. Induction of lactose in phosphate-starved culture led to a 2-fold improvement in product titre of rSymlin and a 2.3-fold improvement in product titre of rLTNF as compared with phosphate-unlimited culture. The results obtained in the study are in agreement with the literature to infer that phosphate starvation or limitation can slow down the microbial growth rate in order to produce recombinant proteins. Further, under PSR conditions, gene expression analysis demonstrated that while selected genes (gapdh, pykF, ppsA, icdA) in glycolysis and pentose phosphate pathway (zwf, gnd, talB, tktA) were up-regulated, other genes in lactose (lacY, lacA) and acetate (ackA, pta) pathway were down-regulated. We have demonstrated that cra, crp, phoB, and phoR are involved in the regulation of central carbon metabolism. We propose a novel cross-regulation between lactose metabolism and phosphate starvation. UDP-galactose, a toxic metabolite that is known to cause cell lysis, has been shown to be significantly reduced due to slow uptake of lactose under PSR conditions. Therefore, E. coli employs a decoupling strategy by limiting growth and redirecting metabolic resources to survive and produce recombinant protein under phosphate starvation conditions. KEY POINTS: • Phosphate starvation controls lactose metabolism, which results in less galactose accumulation. • Phosphate starvation modulates metabolic flow of central carbon metabolism. • Product titre improves by 2-fold due to phosphate starvation. • The approach has been successfully applied to production of two different proteins.

Microaerobic fermentation alters lactose metabolism in Escherichia coli.

Microaerobic fermentation has been shown to improve lactose transport and recombinant protein production in Escherichia coli. Mechanistic correlation between lactose and dissolved oxygen has been studied and it has been demonstrated that E. coli can switch its genetic machinery upon fluctuations in dissolved oxygen levels and thereby impact lactose transport, resulting in product formation. Continuous induction of lactose in microaerobic fermentation led to a 3.3-fold improvement in product titre of rLTNF oligomer and a 1.8-fold improvement in product titre of rSymlin oligomer as compared with traditional aerobic fermentation. Transcriptome profiling indicated that ribosome synthesis, lactose transport and amino acid synthesis genes were upregulated during microaerobic fermentation. Besides, novel lactose transporter setB was examined and it was observed that lactose uptake rate was 1.4-fold higher in microaerobic fermentation. The results indicate that microaerobic fermentation can offer a superior alternative for industrial production of recombinant therapeutics, industrial enzymes and metabolites in E. coli. KEY POINTS: • Microaerobic fermentation results in significantly improved protein production • Lactose transport, ribosome synthesis and amino acid synthesis are enhanced • Product titre improves by 1.8-3.3-fold.

Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production.

Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression.

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster®/B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

Multicystic peritoneal mesothelioma: not always a benign disease

Mesothelioma is a slow-growing insidious lesion of neoplastic aetiology arising from the pleural, peritoneal or pericardial mesothelium. It shows a predilection for the surfaces of the pelvic viscera and has a high rate of recurrence after excision. Cystic mesotheliomas are not associated with asbestos exposure. We report a case of cystic mesothelioma of the peritoneum encasing the ovary, which presented as a cystic adnexal mass. As highlighted in this case and other recent reports, a cystic mesothelioma should not be referred to as a benign cystic mesothelioma, as it has potential for locoregional invasion, as well as distant nodal and serosal metastases. This tumour should be treated with aggressive cytoreductive surgery and appropriate chemotherapy. We review the differential diagnosis of this rare entity and suggest guidelines for its differentiation.