RelQ Mediates the Expression of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

An induced stringent response, which is established by an increased level of (p)ppGpp, is required for the expression of β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). However, it is not clear whether RSH (enzyme mediating stringent response to amino acid starvation) or small alarmone synthetases (SASs) are involved in the maintenance of (p)ppGpp level in response to β-lactams. Since the S. aureus genome encodes two active SASs (RelP and RelQ), their contribution to the expression of β-lactam resistance in MRSA was investigated. It was determined that relQ deletion renders community-associated MRSA (CA-MRSA) sensitive to β-lactams by negatively affecting the expression of mecA, and induction of (p)ppGpp synthesis by mupirocin bypasses the requirement of relQ for the expression of high-level β-lactam resistance. Surprisingly, relP deletion increased the level of β-lactam resistance. Such contradictory observations could be attributed to the fact that relQ promoter is ~5-fold stronger than the relP and is induced by oxacillin as well as deletion of either of the SASs, while relP promoter responds only to oxacillin. The stronger promoter activity of relQ, coupled with the inducibility of the relQ promoter in response to the lack of relP, results in efficient expression of relQ in the relP-deleted background. This positively affects mecA expression and renders the ΔrelP strain highly resistant. These findings indicate an important role for RelQ in the expression of high-level β-lactam resistance in MRSA

Protocol for the Addressing the Social Determinants and Consequences of Tuberculosis in Nepal (ASCOT) pilot trial.

The World Health Organization's End TB (tuberculosis) Strategy advocates social and economic support for TB-affected households but evidence from low-income settings is scarce. We will evaluate the feasibility and acceptability of a locally-appropriate socioeconomic support intervention for TB-affected households in Nepal. We will conduct a pilot randomised-controlled trial with mixed-methods process evaluation in four TB-endemic, impoverished districts of Nepal: Pyuthan, Chitwan, Mahottari, and Morang. We will recruit 128 people with TB notified to the Nepal National TB Program (NTP) and 40 multisectoral stakeholders including NTP staff, civil-society members, policy-makers, and ASCOT (Addressing the Social Determinants and Consequences of Tuberculosis) team members. People with TB will be randomised 1:1:1:1 to four study arms (n=32 each): control; social support; economic support; and combined social and economic (socioeconomic) support. Social support will be TB education and peer-led mutual-support TB Clubs providing TB education and stigma-reduction counselling. Economic support will be monthly unconditional cash transfers during TB treatment with expectations (not conditions) of meeting NTP goals. At 0, 2, and 6 months following TB treatment initiation, participants will be asked to complete a survey detailing the social determinants and consequences of TB and their feedback on ASCOT. Complementary process evaluation will use focus group discussions (FGD), key informant interviews (KII), and a workshop with multi-sectoral stakeholders to consider the challenges to ASCOT's implementation and scale-up. A sample of ~100 people with TB is recommended to estimate TB-related costs. Information power is estimated to be reached with approximately 25 FGD and 15 KII participants.  The ASCOT pilot trial will both generate robust evidence on a locally-appropriate, socioeconomic support intervention for TB-affected households in Nepal and inform a large-scale future ASCOT trial, which will evaluate the intervention's impact on catastrophic costs mitigation and TB outcomes. The trial is registered with the ISRCTN ( ISRCTN17025974). [Abstract copyright: Copyright: © 2022 Rai B et al.

Comparing cross-sectional and longitudinal approaches to Tuberculosis Patient Cost Surveys using Nepalese data.

The World Health Organization has supported the development of national tuberculosis (TB) patient cost surveys to quantify the socio-economic impact of TB in high-burden countries. However, methodological differences in study design (e.g. cross-sectional vs longitudinal) can generate different estimates making the design and impact evaluation of socioeconomic protection strategies difficult. The objective of the study was to compare the socio-economic impacts of TB estimated by applying cross-sectional or longitudinal data collections in Nepal. We analysed data from a longitudinal costing survey (patients interviewed at three-time points) conducted between April 2018 and October 2019. We calculated both mean and median costs from patients interviewed during the intensive (cross-sectional 1) and continuation phases of treatment (cross-sectional 2). We then compared costs, the prevalence of catastrophic costs and the socio-economic impact of TB generated by each approach. There were significant differences in the costs and social impacts calculated by each approach. The median total cost (intensive plus continuation phases) was significantly higher for the longitudinal compared to cross-sectional 2 (US$119.42 vs 91.63, P < 0.001). The prevalence of food insecurity, social exclusion and patients feeling poorer or much poorer were all significantly higher applying a longitudinal approach. In conclusion, the longitudinal design captured important aspects of costs and socioeconomic impacts which were missed by applying a cross-sectional approach. If a cross-sectional approach is applied due to resource constraints, our data suggest the start of the continuation phase is the optimal timing for a single interview. Further research to optimize methodologies to report patient incurred expenditure during TB diagnosis and treatment is needed

Comparing cross-sectional and longitudinal approaches to Tuberculosis Patient Cost Surveys using Nepalese data : Tuberculosis cost survey approaches

The World Health Organization has supported the development of national tuberculosis (TB) patient cost surveys to quantify the socio-economic impact of TB in high-burden countries. However, methodological differences in study design (e.g. cross-sectional vs longitudinal) can generate different estimates making the design and impact evaluation of socioeconomic protection strategies difficult. The objective of the study was to compare the socioeconomic impacts of TB estimated by applying cross-sectional or longitudinal data collections in Nepal. We analysed data from a longitudinal costing survey (patients interviewed at three-time points) conducted between April 2018 and October 2019. We calculated both mean and median costs from patients interviewed during the intensive (cross-sectional 1) and continuation phases of treatment (cross-sectional 2). We then compared costs, the prevalence of catastrophic costs and the socio-economic impact of TB generated by each approach. There were significant differences in the costs and social impacts calculated by each approach. The median total cost (intensive plus continuation phases) was significantly higher for the longitudinal compared to cross-sectional 2 (US$119.42 vs 91.63,

固有法與繼受法：「公同共有」的社會學考察（上）

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions

PCR and genotyping for HPV in cervical cancer patients

Aims: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). Settings and Design: Cross-sectional observational study. Materials and Methods: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. Statistical Analysis Used: Calculation of percentage from the Microsoft Excel Software. Results: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. Conclusions: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

BACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis. AIM AND OBJECTIVE: The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species. MATERIALS AND METHODS: Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test. RESULTS: The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells. CONCLUSION: Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor

Protocol for the Addressing the Social Determinants and Consequences of Tuberculosis in Nepal (ASCOT) pilot trial

BACKGROUND:  The World Health Organization’s End TB (tuberculosis) Strategy advocates social and economic support for TB-affected households but evidence from low-income settings is scarce. We will evaluate the feasibility and acceptability of a locally-appropriate socioeconomic support intervention for TB-affected households in Nepal. METHODS:  We will conduct a pilot randomised-controlled trial with mixed-methods process evaluation in four TB-endemic, impoverished districts of Nepal: Pyuthan, Chitwan, Mahottari, and Morang. We will recruit 128 people with TB notified to the Nepal National TB Program (NTP) and 40 multisectoral stakeholders including NTP staff, civil-society members, policy-makers, and ASCOT (Addressing the Social Determinants and Consequences of Tuberculosis) team members. People with TB will be randomised 1:1:1:1 to four study arms (n=32 each): control; social support; economic support; and combined social and economic (socioeconomic) support. Social support will be TB education and peer-led mutual-support TB Clubs providing TB education and stigma-reduction counselling. Economic support will be monthly unconditional cash transfers during TB treatment with expectations (not conditions) of meeting NTP goals. At 0, 2, and 6 months following TB treatment initiation, participants will be asked to complete a survey detailing the social determinants and consequences of TB and their feedback on ASCOT. Complementary process evaluation will use focus group discussions (FGD), key informant interviews (KII), and a workshop with multi-sectoral stakeholders to consider the challenges to ASCOT’s implementation and scale-up. A sample of ~100 people with TB is recommended to estimate TB-related costs. Information power is estimated to be reached with approximately 25 FGD and 15 KII participants. CONCLUSIONS:  The ASCOT pilot trial will both generate robust evidence on a locally-appropriate, socioeconomic support intervention for TB-affected households in Nepal and inform a large-scale future ASCOT trial, which will evaluate the intervention’s impact on catastrophic costs mitigation and TB outcomes. The trial is registered with the ISRCTN ( ISRCTN17025974 )

Protocol for the Addressing the Social Determinants and Consequences of Tuberculosis in Nepal (ASCOT) pilot trial

BACKGROUND: The World Health Organization’s End TB (tuberculosis) Strategy advocates social and economic support for TB-affected households but evidence from low-income settings is scarce. We will evaluate the feasibility and acceptability of a locally-appropriate socioeconomic support intervention for TB-affected households in Nepal. METHODS: We will conduct a pilot randomised-controlled trial with mixed-methods process evaluation in four TB-endemic, impoverished districts of Nepal: Pyuthan, Chitwan, Mahottari, and Morang. We will recruit 128 people with TB notified to the Nepal National TB Program (NTP) and 40 multisectoral stakeholders including NTP staff, civil-society members, policy-makers, and ASCOT (Addressing the Social Determinants and Consequences of Tuberculosis) team members. People with TB will be randomised 1:1:1:1 to four study arms (n=32 each): control; social support; economic support; and combined social and economic (socioeconomic) support. Social support will be TB education and peer-led mutual-support TB Clubs providing TB education and stigma-reduction counselling. Economic support will be monthly unconditional cash transfers during TB treatment with expectations (not conditions) of meeting NTP goals. At 0, 2, and 6 months following TB treatment initiation, participants will be asked to complete a survey detailing the social determinants and consequences of TB and their feedback on ASCOT. Complementary process evaluation will use focus group discussions (FGD), key informant interviews (KII), and a workshop with multi-sectoral stakeholders to consider the challenges to ASCOT’s implementation and scale-up. A sample of ~100 people with TB is recommended to estimate TB-related costs. Information power is estimated to be reached with approximately 25 FGD and 15 KII participants. CONCLUSIONS: The ASCOT pilot trial will both generate robust evidence on a locally-appropriate, socioeconomic support intervention for TB-affected households in Nepal and inform a large-scale future ASCOT trial, which will evaluate the intervention’s impact on catastrophic costs mitigation and TB outcomes. The trial is registered with the ISRCTN (ISRCTN17025974).</ns3:p