Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.

The Prevalence and Clinical Implications of Rectal SARS-CoV-2 Shedding in Danish COVID-19 Patients and the General Population

Background: SARS-CoV-2 has resulted in a global pandemic since its outbreak in Wuhan, 2019. Virus transmission primarily occurs through close contact, respiratory droplets, and aerosol particles. However, since SARS-CoV-2 has been detected in fecal and rectal samples from infected individuals, the fecal-oral route has been suggested as another potential route of transmission. This study aimed to investigate the prevalence and clinical implications of rectal SARS-CoV-2 shedding in Danish COVID-19 patients.Methods: Hospitalized and non-hospitalized adults and children who were recently tested with a pharyngeal COVID-19 test, were included in the study. A rectal swab was collected from all participants. Hospitalized adults and COVID-19 positive children were followed with both pharyngeal and rectal swabs until two consecutive negative results were obtained. RT-qPCR targeting the envelope gene was used to detect SARS-CoV-2 in the samples. Demographic, medical, and biochemical information was obtained through questionnaires and medical records.Results: Twenty-eight of 52 (53.8%) COVID-19 positive adults and children were positive for SARS-CoV-2 in rectal swabs. Seven of the rectal positive participants were followed for more than 6 days. Two of these (28.6%) continued to test positive in their rectal swabs for up to 29 days after the pharyngeal swabs had turned negative. Hospitalized rectal positive and rectal negative adults were comparable regarding demographic, medical, and biochemical information. Furthermore, no difference was observed in the severity of the disease among the two groups.Conclusions: We provided evidence of rectal SARS-CoV-2 shedding in Danish COVID-19 patients. The clinical importance of rectal SARS-CoV-2 shedding appears to be minimal.

Multiplex Lateral Flow Immunoassay for the Detection of Expanded-Spectrum Hydrolysis and CTX-M Enzymes

Background: Early detection of expanded-spectrum cephalosporinase (ESC) hydrolyzing ß-lactamases is essential for antibiotic stewardship. Here we have developed a multiplex lateral flow immunoassay (LFIA) that detects cefotaxime-hydrolyzing activity as well as the most prevalent ESC-hydrolyzing ß-lactamases: the CTX-M-like. Methods: The Rapid LFIA ESC test was evaluated retrospectively on 188 (139 Enterobacterales, 30 Pseudomonas spp. and 14 Acinetobacter spp.) agar-grown bacterial isolates with well-characterized ß-lactamase content. One single colony was resuspended in 150 µL extraction buffer containing cefotaxime, incubated at room temperature for 30 min prior to loading on the LFIA for reading within 10 min. Results: Out of the 188 isolates, all 17 that did not express a β-lactamase hydrolyzing cefotaxime gave negative results, and all 171 isolates expressing a β-lactamase known to hydrolyze cefotaxime, gave a positive test result. In addition, all 86 isolates expressing a CTX-M-variant belonging to one of the five CTX-M-subgroups were correctly identified. The sensitivity and specificity was 100% for both tests. Conclusions: The results showed that the multiplex LFIA was efficient, fast, low cost and easy to implement in routine laboratory work for the confirmation of ESC hydrolyzing activity and the presence of CTX-M enzymes.

Exploring Higher-Order Conceptual Learning in an Arthropod with a Large Multisensory Processing Center

Comparative cognition aims to understand the evolutionary history and current function of cognitive abilities in a variety of species with diverse natural histories. One characteristic often attributed to higher cognitive abilities is higher-order conceptual learning, such as the ability to learn concepts independent of stimuli—e.g., ‘same’ or ‘different’. Conceptual learning has been documented in honeybees and a number of vertebrates. Amblypygids, nocturnal enigmatic arachnids, are good candidates for higher-order learning because they are excellent associational learners, exceptional navigators, and they have large, highly folded mushroom bodies, which are brain regions known to be involved in learning and memory in insects. In Experiment 1, we investigate if the amblypygid Phrynus marginimaculatus can learn the concept of same with a delayed odor matching task. In Experiment 2, we test if Paraphrynus laevifrons can learn same/different with delayed tactile matching and nonmatching tasks before testing if they can transfer this learning to a novel cross-modal odor stimulus. Our data provide no evidence of conceptual learning in amblypygids, but more solid conclusions will require the use of alternative experimental designs to ensure our negative results are not simply a consequence of the designs we employed.

Effectiveness of Public Spending before and during COVID-19 Pandemic

The pandemic of COVID-19 influences all sectors of the economy. It caused decreasing in produced Gross domestic product (GDP) and higher unemployment. As it is known, to overcome this negative tendency, it is possible to put in practice monetary and fiscal instruments. During the pandemic, the government tried to slow down negative economic results through public spending. With them, the government looks to be increased aggregate demand in the economy and as a result-GDP raises and unemployment reduces. The research is based on created original model for testing the impact of total public spending, capital, salary, social insurance and care, for maintenance by a consolidated fiscal program on the value of GDP. The changes of GDP measure the effectiveness of public spending. The period of research is before and during the COVID-19 crisis (2019-2020) in the case of Bulgaria. Before the pandemic the analysis shows coefficient of determination for capital spending is more significant compare with all other types of public expenditure and these cost predetermine economic growth. During the pandemic of COVID-19 public spending has used as the main instrument to overcome the negative results for the economy. For this period it found an extremely strong impact of labor costs and social care expenditure on aggregate demand. They bring more positive results to be solved health issues, but not for faster recovery of the economy.

Clinical Improvement in Patients with Morbus Hansen Borderline Tuberculoid Lesions with 3-Month Multibacillary Regimen Therapy

Background/Aim: Morbus Hansen (MH) is a chronic infectious disease caused by Mycobacterium leprae. The disease has an average incubation period of 3-5 years. Morbus Hansen (MH) has various clinical, microbiological and histopathological features. In the following, a new case of MH is reported with a clinical manifestation of Borderline Tuberculoid (BT) type MH. The patient was treated with the MDT MB regimen for 3 months and had clinical improvement. Case Report: A 23-year-old boy with complaints of sudden feeling of heat, tingling and cramping from the tip of his right foot to the sole of his right foot. He also complaining about black lesions on both hands, right elbow, face, chest and back. The diagnosis of gout, fungus and dermatitis can be ruled out after microscopic examination of AFB on the right, left ear lobe, and foot lesions showed negative results and histopathological examination results showed Morbus Hansen type BT. The risk factor for contracting Morbus Hansen is the patient has lived in a dormitory for 2.5 years and it is known that there are people who suffer from a similar disease in that environment. The patient was treated using a multibacillary regimen for 3 months and the lesions improved and the numbness decreased. Treatment plan during the case for 12-18 months. This case is unique due to the clinical manifestation has not significant characteristic for MH, AFB is not found from ear lobe smear. MH established after skin biopsy done. Conclusion: It was reported that one MH case with TB for 3 months had clinical improvement, with plans to continue treatment for 12-18 months. Keywords: Borderline Tuberculoid; Morbus Hansen, Multibacillary.

Investigating Reversibility of Steps in Petri Nets

In reversible computations one is interested in the development of mechanisms allowing to undo the effects of executed actions. The past research has been concerned mainly with reversing single actions. In this paper, we consider the problem of reversing the effect of the execution of groups of actions (steps). Using Petri nets as a system model, we introduce concepts related to this new scenario, generalising notions used in the single action case. We then present properties arising when reverse actions are allowed in place/transition nets (PT-nets). We obtain both positive and negative results, showing that allowing steps makes reversibility more problematic than in the interleaving/sequential case. In particular, we demonstrate that there is a crucial difference between reversing steps which are sets and those which are true multisets. Moreover, in contrast to sequential semantics, splitting reverses does not lead to a general method for reversing bounded PT-nets. We then show that a suitable solution can be obtained by combining split reverses with weighted read arcs.

Epidemiology of SARS-CoV-2 infection in Italy using real-world data: methodology and cohort description of the second phase of web-based EPICOVID19 study.

Digital technologies have been extensively employed in response to the SARS-CoV-2 pandemic worldwide. This study describes the methodology of the two-phase internet-based EPICOVID19 survey, and the characteristics of the adult volunteers respondents who lived in Italy during the first (April - May 2020) and the second wave (January - February 2021) of the epidemic. Validated scales and ad-hoc questionnaires were used to collect socio-demographic, medical and behavioural characteristics, as well as information on COVID-19. Among those who provided email addresses during phase I (105,355), 41,473 participated in phase II (mean age 50.7 years +/- 13.5 SD, 60.6% females). After a median follow-up of ten months, 52.8% had undergone naso-pharyngeal swab (NPS) testing and 13.2% had positive result. More than 40% had undergone serological test (ST) and 11.9% were positive. Out of the 2,073 participants with at least one positive ST, 72.8% had only negative results from NPS or never performed it. These results indicate that a large fraction of individuals remained undiagnosed, possibly contributing to the spread of the virus in the community. Participatory online surveys offer a unique opportunity to collect relevant data at individual level from large samples during confinement.

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae

Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss.