A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Knowledge Gaps and Determination of Attitude and Practice among Medical Students toward Hepatitis B Infection: A Nationwide Cross-Sectional Study.

Introduction: As reported by the World Health Organization (WHO), annually, there are 1.5 million new infections, with more than 290 million people living with chronic hepatitis B diseases in 2019. Medical students (MSs), a subgroup of health care workers (HCWs), are at high occupational risk for HBV infection since HCWs have a 2-10 times greater risk of getting the virus than the general population. Therefore, they must have a broad understanding of hepatitis B infection. In this study, we aimed to assess the level of knowledge, attitude, and practices among MSs, and to explore its predictors. Methodology. A descriptive cross-sectional research was done among MSs, those who finished their infectious disease course. MSs were asked to participate in a self-administered online-structured questionnaire. Results: 612 MSs were enrolled in our study. 67.5% were females, and 36.9% were in their 6th year. Around half of the participants held a high level of knowledge, attitude, and practices toward HBV infection. MSs in the last year of the study who encountered HBV patients showed to have better knowledge. Male MSs who encountered HBV patients and took extra HBV courses showed better attitudes. High practice level among MSs was associated with being in the 6th year and taking extra HBV courses. Conclusions: This study demonstrated a satisfactory level of knowledge, attitude, and practices toward HBV infection among MSs. However, awareness must be enhanced in some areas especially transmission routes of HBV. Thus, it will strengthen the level of attitude and practices to omit the effect of the stigma while delivering care to HBV patients.

Sociodemographic Determinants of Knowledge, Attitudes, and Practices Toward Hepatitis B Infection Among Pregnant Women: A Cross-National Study in Jordan.

Background Mother-to-child transmission (MTCT) of the hepatitis B virus (HBV) is significant, as most infants infected at birth go on to develop chronic hepatitis B. Vaccination and antiviral treatment during pregnancy could primarily prevent vertical transmission. Therefore, the purpose of this study is to assess pregnant Jordanian females' knowledge, attitude, and practices (KAP) toward HBV. In addition, to explore the relationship between the level of KAP toward HBV infection and its predictors. Methods Our cross-sectional study was conducted among pregnant women in Jordan. We enrolled 621 participants between January and April 2023. Our survey was derived from a previously validated tool that was used to investigate a similar aim as our study. The survey was done via Google Forms (Google LLC, Mountain View, California, United States) and it contained questions divided into four main sections: participants' demographics, knowledge section, attitudes section, and practices section. Results The majority of participants have neither a personal nor family history of HBV, and only 91 (14.7%) of the participants had a medical degree. The overall knowledge, attitude, and practice (KAP) scores were low, as only 176 (28.3%), 315 (50.7%), and 244 (39.3%) of participants achieved high levels of knowledge, attitude, and practice scores, respectively. A significant association was found between knowledge level, practice assessment, and the following variables: age, educational level, job, study field, history of HBV in the family, and source of knowledge. Regarding attitude, a significant association was found with the job, study field, and source of knowledge. Conclusion This study found that pregnant women in Jordan had a low level of awareness of HBV infection. Thus, more efforts should be made to raise awareness about HBV among high-risk groups, especially pregnant women.

Attitudes and Practices Regarding Helicobacter Pylori Infection Among the Public in Jordan: A Cross-Sectional Survey.

BACKGROUND: Helicobacter pylori is a major infection that can cause a variety of complications, including stomach cancer and peptic ulcers. There is a scarcity of research on the awareness of H. pylori in the general population in Jordan. Because public awareness and behavioral changes are powerful tools in curbing transmission rates, this study evaluated Jordanians' beliefs and behaviors about H. pylori infection. METHODS: The study was carried out in 2021 between May and July. Those who met the requirements for inclusion were asked to fill out a questionnaire through interviews. The questionnaire had three sections: sociodemographic data, participants' attitudes regarding H. pylori infection, and daily practices that could affect H. pylori transmission. RESULTS: Responses were collected from 767 participants, 50.7% were females, 65.8% were married, and 65.1% had a high educational level. Only 31.6% of the participants held a positive attitude. The female gender was significantly associated with better attitudes regarding H. pylori infection. One-third of the interviewed participants showed good practices. The female gender and being 50 years old and above were significantly associated with better practices. CONCLUSION: This study demonstrated that attitudes and practices regarding H. pylori infection in Jordan were unsatisfactory. Subsequently, public health efforts should be aimed at modifying those behaviors to decrease the disease burden.

Enhanced recovery concepts in paediatric patients who underwent appendectomy: a retrospective cohort study at a tertiary university hospital.

OBJECTIVE: To investigate if the implementation of enhanced recovery after surgery (ERAS) pathways could be effective in paediatric patients undergoing appendectomy. METHODS: This retrospective study analysed the data from patients aged ≤18 years that underwent appendectomies. The data collected included age, sex, application of enhanced recovery concepts (ERCs) and clinical outcomes. RESULTS: A total of 93 paediatric patients were retrospectively categorized into two groups: patients in group 1 did not experience the use of ERCs during their appendectomies; and patients in group 2 had ERCs applied during their appendectomies. Patients in both groups were exposed to some elements of ERAS, including preoperative patient/parent counselling, limited fasting durations, antibiotic prophylaxis and no bowel preparations. Duration of hospital stay was significantly longer in group 1 compared with group 2 (3.47 ± 1.81 days versus 2.24 ± 1.52 days, respectively. There were no significant differences between the two groups in term of postoperative pain control, hospital cost, readmissions, reoperations and emergency room visits. CONCLUSION: Implementing ERCs in paediatric patients undergoing appendectomies provided a significant improvement in patient recovery with a shorter hospital stay, with no increase in postoperative pain, hospital cost, rate of readmission and reoperation.

Knowledge, Attitude, and Practices toward Hepatitis B Infection among Healthcare Students-A Nationwide Cross-Sectional Study in Jordan.

The World Health Organization has estimated that around 66 thousand HBV infection cases are caused by needlestick injuries annually. Healthcare students should be aware of HBV transmission routes and preventive measures. This study assessed the knowledge, attitudes, and practices toward HBV among Jordanian healthcare students and its associated factors. A cross-national study was conducted from March to August 2022. The questionnaire was composed of four sections: participants' sociodemographics, knowledge, attitudes, and practices about HBV, and 2322 participants were enrolled. The collected responses were analyzed with SPSS software (version 25 (IBM Corp., Armonk, NY, USA)) using descriptive statistics, unpaired t-tests, chi-square tests, and multivariate regression analyses. A p-value ≤ 0.05 was considered statistically significant. The results showed that 67.9% were females, 26.4% were medical students, and 35.9% were in their 3rd year. Overall, 40% of the participants held high levels of knowledge and attitudes. Further, 63.9% of participants had good practices toward HBV. Gender, year of study, encountering HBV patients, college, and having extra HBV courses were associated with high levels of KAP. This study demonstrated insufficient knowledge and attitudes toward HBV; however, the practice level toward HBV among healthcare students was promising. Therefore, public health efforts should modify the knowledge and attitude gaps to reinforce awareness and minimize the risk of infection.

Knowledge and information sources towards Helicobacter pylori in Jordan.

In 2017, the Jordanian Ministry of Health reported that gastric cancer was one of Jordan's most diagnosed cancers. Gastric cancer is often linked to Helicobacter pylori, one of the foremost risk factors. Despite the high prevalence of H. pylori in Jordan, no information is available regarding the general population's awareness of the harmful effects of this pathogen. The study aims to assess the knowledge and the impact of the source of knowledge on H. pylori among the general population in Jordan. A cross-sectional study involving 933 participants was conducted between May and July of 2021. After meeting the inclusion criteria and consenting to participate in this study, participants completed the questionnaire. An interview-based questionnaire covered the following sections: sociodemographic data and knowledge related to H. pylori infection. 63% of the participants had a high level of education, 70.5% got their information about H. pylori infection from non-medical sources, and 68.7% had a low level of knowledge. Working in the medical field, attaining information from medical resources, and having a history of self-reported or family member of H. pylori infection showed a significant association with a high level of knowledge. The Mann-Whitney U test showed that the mean ranks of all knowledge items in the medical source group significantly exceed those of the non-medical source group (p-values < 0.05). In Jordan, the awareness of H. pylori was unsatisfying, like in other countries. Nevertheless, misconceptions in knowledge about H. pylori were identified and further awareness must be spread and advocated. Close observation of the non-medical sources of information is essential for delivering sufficient amount of knowledge to the general population.

Anti-SARS-Cov-2 S-RBD IgG Formed after BNT162b2 Vaccination Can Bind C1q and Activate Complement.

Background: The ability of vaccine-induced antibodies to bind C1q could affect pathogen neutralization. In this study, we investigated C1q binding and subsequent complement activation by anti-spike (S) protein receptor-binding domain (RBD) specific antibodies produced following vaccination with either the mRNA vaccine BNT162b2 or the inactivated vaccine BBIBP-CorV. Methods: Serum samples were collected in the period of July 2021-March 2022. Participants' demographic data, type of vaccine, date of vaccination, as well as adverse effects of the vaccine were recorded. The serum samples were incubated with S protein RBD-coated plates. Levels of human IgG, IgA, IgM, C1q, and mannose-binding lectin (MBL) that were bound to the plate, as well as formed C3d, and C5b-9 were compared between different groups of participants. Results: A total of 151 samples were collected from vaccinated (n = 116) and nonvaccinated (n = 35) participants. Participants who received either one or two doses of BNT162b2 formed higher levels of anti-RBD IgG and IgA than participants who received BBIBP-CorV. The anti-RBD IgG formed following either vaccine bound C1q, but significantly more C1q binding was observed in participants who received BNT162b2. Subsequently, C5b-9 formation was significantly higher in participants who received BNT162b2, while no significant difference in C5b-9 formation was found between the nonvaccinated and BBIBP-CorV groups. The formation of C5b-9 was strongly correlated to C1q binding and not to MBL binding, additionally, the ratio of formed C5b-9/bound C1q was significantly higher in the BNT162b2 group. Conclusion: Anti-RBD IgG formed following vaccination can bind C1q with subsequent complement activation, and the degree of terminal complement pathway activation differed between vaccines, which could play a role in the protection offered by COVID-19 vaccines. Further investigation into the correlation between vaccine protection and vaccine-induced antibodies' ability to activate complement is required.

Knowledge, attitudes, and practices toward tuberculosis among Jordanian university students.

Background: Tuberculosis (TB) is one of the leading causes of death from infectious diseases worldwide with numerous undiagnosed and untreated cases, emphasizing the need for TB awareness to minimize transmission and initiate early treatment. Data regarding the knowledge, attitudes, and practices (KAP) toward TB among Jordanians is lacking but requires attention given the massive migration spells to Jordan from neighboring countries in the past decade. Methods: A descriptive cross-sectional study was conducted from May to June 2022. An online questionnaire was developed following World Health Organization (WHO) recommendations for TB KAP surveys and was distributed to Jordanian university students. The questionnaire documented sociodemographic data and measured participants' KAP toward TB. Descriptive and analytic statistics were used to report KAP levels and highlight relevant sociodemographic factors associated with better KAP. Results: 602 participants completed the survey; most were females (60.8%), in their first 3 years of school (84.4%), and from a healthcare field of study (57.0%). The knowledge section median score was 27 out of 51. Knowledge gaps in TB treatment, and to a lesser extent, TB transmission routes were identified. The attitudes section median score was 6 out of 9, attitudes were generally positive toward TB patients with no indication of a social stigma. The practice section median score was 6 out of 8, most participants would take the correct measures if they suspected being infected, yet around 41.0% were not confident that masks are important in preventing airborne diseases. Students in healthcare specialties had significantly better KAP scores and identifying as a smoker was associated with a lower practice score. Conclusion: Although university students displayed satisfactory KAP scores, the focus should be aimed at informing students from non-healthcare fields on TB transmission routes, treatment options, and the role of masks in preventing disease transmission.

Medical students and risk of COVID-19 infection: A descriptive cross-sectional study from the University of Jordan.

BACKGROUNDS: The coronavirus disease-2019 (COVID-19) pandemic with increasing morbidity and mortality has impacted the lives of the global population, including medical education. With the return of on-site medical education in Jordan, it is important to know whether this would pose any risk of COVID-19 infection in medical students. OBJECTIVE: To investigate COVID-19 infection infection rates among medical students and whether there is difference between preclinical and clinical students' infection rate. METHODS: The study is a cross-sectional study, designed to (1) determine the incidence of COVID-19 in clinical and preclinical medical students (2) determine if there is a difference in infection rates between clinical and preclinical students. The study was situated at the University of Jordan and its affiliated hospitals. Data were collected from the 4th of December 2020 till the 17th of February 2021through a structured web-based questionnaire. RESULTS: A total of 1,830 responses were retrieved. Thirty-nine percent were males, and (61.5%) were females. Overall, 237(13%) of students reported testing positive for COVID-19 infection by PCR, of which 123 were clinical students (15.2%) and 114 were pre-clinical students (11.2%), representing a relative risk of 1.36 of COVID-19 infection among clinical students compared to pre-clinical students. This difference is statistically significant (P = 0.010). Rates of COVID-19 in females 13%; CI 7.5,18.4), were very close to those of males (12.9%; 95% CI 6.0,19.8). Positive COVID-19 cases peaked in November and October forming 36.3% and 32.9% of the cases, respectively. CONCLUSION: The incidence of positive COVID-19 tests was found to be higher among clinical students as compared to pre-clinical students. Commitment to general health safety precautions did not appear to be protective enough for clinical students. It is fundamental that additional strategies, including access to vaccines, are set, and deficiencies in current protections are identified to maintain students' safety and well-being.

Transmission dynamics study of tuberculosis isolates with whole genome sequencing in southern Sweden.

Epidemiological contact tracing complemented with genotyping of clinical Mycobacterium tuberculosis isolates is important for understanding disease transmission. In Sweden, tuberculosis (TB) is mostly reported in migrant and homeless where epidemiologic contact tracing could pose a problem. This study compared epidemiologic linking with genotyping in a low burden country. Mycobacterium tuberculosis isolates (n = 93) collected at Scania University Hospital in Southern Sweden were analysed with the standard genotyping method mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and the results were compared with whole genome sequencing (WGS). Using a maximum of twelve single nucleotide polymorphisms (SNPs) as the upper threshold of genomic relatedness noted among hosts, we identified 18 clusters with WGS comprising 52 patients with overall pairwise genetic maximum distances ranging from zero to nine SNPs. MIRU-VNTR and WGS clustered the same isolates, although the distribution differed depending on MIRU-VNTR limitations. Both genotyping techniques identified clusters where epidemiologic linking was insufficient, although WGS had higher correlation with epidemiologic data. To summarize, WGS provided better resolution of transmission than MIRU-VNTR in a setting with low TB incidence. WGS predicted epidemiologic links better which could consolidate and correct the epidemiologically linked cases, avoiding thus false clustering.

A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis.

Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.

Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.

Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.

Impaired CXCR1-dependent oxidative defence in active tuberculosis patients.

Much of the pronounced host inflammatory response that occurs in tuberculosis (TB) is related to failed immunity against the invading pathogen. The G-protein coupled receptors CXCR1 and CXCR2 are implicated in important signal transduction pathways in lung inflammatory responses. We investigated the expression and function of these receptors in a simple whole blood model from 24 patients with pulmonary TB and in subjects with latent TB infection (LTBI). Healthy controls were recruited from close contacts to the pulmonary index patients. We found that pulmonary TB patients had significantly increased CXCR1 expression on blood cells compared to LTBI subjects and controls (p < 0.001). In contrast, LTBI subjects had a significant increase in CXCR2 expression compared to pulmonary TB patients (p < 0.001) and controls (p < 0.01). Leukocyte function, measured as oxidative capacity, was decreased in pulmonary TB patients compared to LTBI and controls (p < 0.001) and correlated with the increased CXCR1 expression. Leukocyte recruitment, measured as the expression of microRNA-223 was increased in pulmonary TB patients compared to LTBI (p < 0.05). We found that variations in receptor expression are linked to disease progression and affect the immune response against Mycobacterium tuberculosis (Mtb).

Mycobacteria bypass mucosal NF-kB signalling to induce an epithelial anti-inflammatory IL-22 and IL-10 response.

The mechanisms by which mycobacteria subvert the inflammatory defence to establish chronic infection remain an unresolved question in the pathogenesis of tuberculosis. Using primary epithelial cells, we have analysed mycobacteria induced epithelial signalling pathways from activation of TLRs to cytokine secretion. Mycobacterium bovis bacilli Calmette-Guerin induced phosphorylation of glycogen synthase kinase (GSK)3 by PI3K-Akt in the signalling pathway downstream of TLR2 and TLR4. Mycobacteria did not suppress NF-κB by activating the peroxisome proliferator-activated receptor γ. Instead the pro-inflammatory NF-κB was bypassed by mycobacteria induced GSK3 inhibition that promoted the anti-inflammatory transcription factor CREB. Mycobacterial infection did not thus induce mucosal pro-inflammatory response as measured by TNFα and IFNγ secretion, but led to an anti-inflammatory IL-10 and IL-22 production. Apart from CREB, MAP3Ks p38 and ERK1/2 activated the transcription factor AP-1 leading to IL-6 production. Interestingly, blocking of TLR4 before infection decreased epithelial IL-6 secretion, but increased the CREB-activated IL-10 production. Our data indicate that mycobacteria suppress epithelial pro-inflammatory production by suppressing NF-κB activation thereby shifting the infection towards an anti-inflammatory state. This balance between the host immune response and the pathogen could determine the outcome of infection.