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Background The present study investigated the formulation, development, and characterization of Fosfomycin pessaries for the treatment of genitourinary tract infections. To achieve higher local concentration and to avoid the systemic exposure of the body towards Fosfomycin, vaginal pessaries have been developed in this research work. Methodology The Fosfomycin pessaries were prepared using the hand rolling method. After the formulation of the Fosfomycin pessaries, pre-formulation studies were conducted to evaluate the drug-excipient compatibility, followed by in vivo studies. Results The pre-formulation studies using FTIR (Fourier Transform Infra-Red) spectrophotometric analysis showed no significant interaction of Fosfomycin with the base and formation of Fosfomycin pessaries. Furthermore, DSC (differential scanning calorimetry) and TGA (thermogravimetric analysis) analysis confirm the formulation stability. The physicochemical analysis of Fosfomycin pessaries, including liquefaction time and breaking force, showed the formulation within the pharmacopeia standard range. Furthermore, the pharmacokinetic and in vitro release studies showed that Fosfomycin pessaries, in contrast to the Fosfomycin suspension, exhibited higher elimination time, higher absorption rate, and area under the curve. The antibacterial results of the Fosfomycin pessaries showed significant activity against E. coli using in vitro and in vivo studies. Conclusion The in vitro and in vivo results revealed that Fosfomycin pessaries have improved pharmacokinetics and increased antibacterial activity.

Antibiotic resistance (AR) is the result of microbes’ natural evolution to withstand the action of antibiotics used against them. AR is rising to a high level across the globe, and novel resistant strains are emerging and spreading very fast. Acinetobacter baumannii is a multidrug resistant Gram-negative bacteria, responsible for causing severe nosocomial infections that are treated with several broad spectrum antibiotics: carbapenems, β-lactam, aminoglycosides, tetracycline, gentamicin, impanel, piperacillin, and amikacin. The A. baumannii genome is superplastic to acquire new resistant mechanisms and, as there is no vaccine in the development process for this pathogen, the situation is more worrisome. This study was conducted to identify protective antigens from the core genome of the pathogen. Genomic data of fully sequenced strains of A. baumannii were retrieved from the national center for biotechnological information (NCBI) database and subjected to various genomics, immunoinformatics, proteomics, and biophysical analyses to identify potential vaccine antigens against A. baumannii. By doing so, four outer membrane proteins were prioritized: TonB-dependent siderphore receptor, OmpA family protein, type IV pilus biogenesis stability protein, and OprD family outer membrane porin. Immuoinformatics predicted B-cell and T-cell epitopes from all four proteins. The antigenic epitopes were linked to design a multi-epitopes vaccine construct using GPGPG linkers and adjuvant cholera toxin B subunit to boost the immune responses. A 3D model of the vaccine construct was built, loop refined, and considered for extensive error examination. Disulfide engineering was performed for the stability of the vaccine construct. Blind docking of the vaccine was conducted with host MHC-I, MHC-II, and toll-like receptors 4 (TLR-4) molecules. Molecular dynamic simulation was carried out to understand the vaccine-receptors dynamics and binding stability, as well as to evaluate the presentation of epitopes to the host immune system. Binding energies estimation was achieved to understand intermolecular interaction energies and validate docking and simulation studies. The results suggested that the designed vaccine construct has high potential to induce protective host immune responses and can be a good vaccine candidate for experimental in vivo and in vitro studies.

We aimed to determine the prevalence of pulmonary tuberculosis (TB) amongst the adult population in 2010-2011 in Pakistan. A nationwide cross-sectional survey with multistage cluster sampling was conducted among adults (≥15 years) in 95 clusters in 2010-2011. All consenting participants were screened for cough and by chest X-ray. Participants with presumptive TB submitted two sputum samples for smear microscopy, culture, and molecular testing if needed. The TB prevalence estimates were adjusted for missing data and the cluster design. Of 131,329 eligible individuals, 105,913 (81%) participated in the survey, of whom 10,471 (9.9%) were eligible for sputum examination. We found 341 bacteriologically positive TB cases of whom 233 had sputum smear-positive TB. The adjusted prevalence estimates for smear and bacteriologically positive TB were 270/100,000 (95% confidence interval (CI) 217-323), and 398/100,000 (95% CI 333-463), respectively. Only 61% of the diagnosed TB cases screened positive on symptoms (cough >2wks), whereas the other TB cases were detected based on X-ray abnormalities. The TB prevalence increased with age and was 1.8 times higher among men than women. The prevalence-to-notification ratio of smear-positive TB was 3.1 (95% CI 2.5-3.7), was higher among men than women, and increased with age. Our data suggest that there is under-detection and/or -notification of TB, especially among men and elderly. TB control should be strengthened specifically in these risk groups. X-ray examination should be combined with symptom screening to enhance case detection.

The ongoing antibiotic-resistance crisis is becoming a global problem affecting public health. Urgent efforts are required to design novel therapeutics against pathogenic bacterial species. Brucella melitensis is an etiological agent of brucellosis, which mostly affects sheep and goats but several cases have also been reported in cattle, water buffalo, yaks and dogs. Infected animals also represent the major source of infection for humans. Development of safer and effective vaccines for brucellosis remains a priority to support disease control and eradication in animals and to prevent infection to humans. In this research study, we designed an in-silico multi-epitopes vaccine for B. melitensis using computational approaches. The pathogen core proteome was screened for good vaccine candidates using subtractive proteomics, reverse vaccinology and immunoinformatic tools. In total, 10 proteins: catalase; siderophore ABC transporter substrate-binding protein; pyridoxamine 5′-phosphate oxidase; superoxide dismutase; peptidylprolyl isomerase; superoxide dismutase family protein; septation protein A; hypothetical protein; binding-protein-dependent transport systems inner membrane component; and 4-hydroxy-2-oxoheptanedioate aldolase were selected for epitopes prediction. To induce cellular and antibody base immune responses, the vaccine must comprise both B and T-cells epitopes. The epitopes were next screened for antigenicity, allergic nature and water solubility and the probable antigenic, non-allergic, water-soluble and non-toxic nine epitopes were shortlisted for multi-epitopes vaccine construction. The designed vaccine construct comprises 274 amino acid long sequences having a molecular weight of 28.14 kDa and instability index of 27.62. The vaccine construct was further assessed for binding efficacy with immune cell receptors. Docking results revealed that the designed vaccine had good binding potency with selected immune cell receptors. Furthermore, vaccine-MHC-I, vaccine-MHC-II and vaccine-TLR-4 complexes were opted based on a least-binding energy score of −5.48 kcal/mol, 0.64 kcal/mol and −2.69 kcal/mol. Those selected were then energy refined and subjected to simulation studies to understand dynamic movements of the docked complexes. The docking results were further validated through MMPBSA and MMGBSA analyses. The MMPBSA calculated −235.18 kcal/mol, −206.79 kcal/mol, and −215.73 kcal/mol net binding free energy, while MMGBSA estimated −259.48 kcal/mol, −206.79 kcal/mol and −215.73 kcal/mol for TLR-4, MHC-I and MHC-II complexes, respectively. These findings were validated by water-swap and entropy calculations. Overall, the designed vaccine construct can evoke proper immune responses and the construct could be helpful for experimental researchers in formulation of a protective vaccine against the targeted pathogen for both animal and human use.

Currently, only 62% of incident tuberculosis (TB) cases are reported to the national programme in Pakistan. Several innovative interventions are being recommended to detect the remaining 'missed' TB cases. One such intervention involved expanding contact investigation to the community using the Xpert MTB/RIF test. This was a before and after intervention study involving retrospective record review. Passive case finding and household contact investigation was routinely done in the pre-intervention period July 2011-June 2013. Four districts with a high concentration of slums were selected as intervention areas; Lahore, Rawalpindi, Faisalabad and Islamabad. Here, in the intervention period, July 2013-June 2015, contact investigation beyond household was conducted: all people staying within a radius of 50 metres (using Geographical Information System) from the household of smear positive TB patients were screened for tuberculosis. Those with presumptive TB were investigated using smear microscopy and the Xpert MTB/RIF test was performed on smear negative patients. All the diagnosed TB patients were linked to TB treatment and care. A total of 783043 contacts were screened for tuberculosis: 23741(3.0%) presumptive TB patients were identified of whom, 4710 (19.8%) all forms and 4084(17.2%) bacteriologically confirmed TB patients were detected. The contribution of Xpert MTB/RIF to bacteriologically confirmed TB patients was 7.6%. The yield among investigated presumptive child TB patients was 5.1%. The overall yield of all forms TB patients among investigated was 22.3% among household and 19.1% in close community. The intervention contributed an increase of case detection of bacteriologically confirmed tuberculosis by 6.8% and all forms TB patients by 7.9%. Community contact investigation beyond household not only detected additional TB patients but also increased TB case detection. However, further long term assessments and cost-effectiveness studies are required before national scale-up.

Efflux pumps are a specialized tool of antibiotic resistance used by Pseudomonas aeruginosa to expel antibiotics. The current study was therefore conducted to examine the expression of MexAB-OprM and MexCD-OprJ efflux pump genes. In this study, 200 samples were collected from Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC) in Peshawar, Pakistan. All the isolates were biochemically identified by an Analytical Profile Index kit and at the molecular level by Polymerase Chain Reaction (PCR) utilizing specific primers for the OprL gene. A total of 26 antibiotics were tested in the current study using the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and high-level resistance was shown to amoxicillin-clavulanic acid (89%) and low-level to chloramphenicol (1%) by the isolates. The antibiotic-resistant efflux pump genes MexA, MexB, OprM, MexR, MexC, MexD, OprJ, and NfxB were detected in 178 amoxicillin-clavulanic acid-resistant isolates. Mutations were detected in MexA, MexB, and OprM genes but no mutation was found in the MexR gene as analyzed by I-Mutant software. Statistical analysis determined the association of antibiotics susceptibility patterns by ANOVA: Single Factor p = 0.05. The in silico mutation impact on the protein structure stability was determined via the Dynamut server, which revealed the mutations might increase the structural stability of the mutants. The docking analysis reported that MexA wild protein showed a binding energy value of −6.1 kcal/mol with meropenem and the mexA mutant (E178K) value is −6.5 kcal/mol. The mexB wild and mutant binding energy value was −5.7 kcal/mol and −8.0 kcal/mol, respectively. Efflux pumps provide resistance against a wide range of antibiotics. Determining the molecular mechanisms of resistance in P. aeruginosa regularly will contribute to the efforts against the spread of antibiotic resistance globally.

Staphylococcus hominis is a Gram-positive bacterium from the staphylococcus genus; it is also a member of coagulase-negative staphylococci because of its opportunistic nature and ability to cause life-threatening bloodstream infections in immunocompromised patients. Gram-positive and opportunistic bacteria have become a major concern for the medical community. It has also drawn the attention of scientists due to the evaluation of immune evasion tactics and the development of multidrug-resistant strains. This prompted the need to explore novel therapeutic approaches as an alternative to antibiotics. The current study aimed to develop a broad-spectrum, multi-epitope vaccine to control bacterial infections and reduce the burden on healthcare systems. A computational framework was designed to filter the immunogenic potent vaccine candidate. This framework consists of pan-genomics, subtractive proteomics, and immunoinformatics approaches to prioritize vaccine candidates. A total of 12,285 core proteins were obtained using a pan-genome analysis of all strains. The screening of the core proteins resulted in the selection of only two proteins for the next epitope prediction phase. Eleven B-cell derived T-cell epitopes were selected that met the criteria of different immunoinformatics approaches such as allergenicity, antigenicity, immunogenicity, and toxicity. A vaccine construct was formulated using EAAAK and GPGPG linkers and a cholera toxin B subunit. This formulated vaccine construct was further used for downward analysis. The vaccine was loop refined and improved for structure stability through disulfide engineering. For an efficient expression, the codons were optimized as per the usage pattern of the E coli (K12) expression system. The top three refined docked complexes of the vaccine that docked with the MHC-I, MHC-II, and TLR-4 receptors were selected, which proved the best binding potential of the vaccine with immune receptors; this was followed by molecular dynamic simulations. The results indicate the best intermolecular bonding between immune receptors and vaccine epitopes and that they are exposed to the host’s immune system. Finally, the binding energies were calculated to confirm the binding stability of the docked complexes. This work aimed to provide a manageable list of immunogenic and antigenic epitopes that could be used as potent vaccine candidates for experimental in vivo and in vitro studies.

Abstract Background Pakistan has the sixth largest population in the world and boasts the fifth greatest burden of tuberculosis. The Government of Pakistan has set the ambitious goal of zero deaths due to tuberculosis and universal access to tuberculosis care by 2020. Successfully reaching these goals is dependent on the country’s capacity to diagnose and successfully treat an estimated 200000 unnotified or missing patients with tuberculosis. Methods A patient-pathway analysis (PPA) was conducted at the national level, as well as for each of the 4 provinces, to assess the alignment between patient care seeking and the availability of tuberculosis diagnostic and treatment services. Results Almost 90% of patients initiated care in the private sector, which accounts for only 15% of facilities with the capacity for tuberculosis diagnosis and treatment. Across the country, nearly 50% of tuberculosis microscopy laboratories were located in public-sector–basic health units and regional health centers. However, very few patients initiated care in these facilities. Overall, tuberculosis case detection was high given the low likelihood of patients reaching facilities with the capacity for tuberculosis service delivery during their first visit. Discussion Improving the engagement of the informal sector and lower-level clinicians will improve the efficiency and timeliness of tuberculosis diagnosis for patients in Pakistan. Concurrently, the apparent strength of the referral networks connecting community-level workers and private clinicians to the public sector for tuberculosis diagnosis and treatment suggests that strengthening the capacity of the public sector could be valuable.

Background: Pakistan is facing challenges regarding the availability of reliable data for malaria surveillance. These include lack of coordination between different reporting systems and fragmented information system. This study aimed to compare the reporting of malaria surveillance systems in Pakistan. Methods: There are two parallel reporting systems for malaria surveillance in Pakistan, the District Health Information System (DHIS) and Malaria Information System (MIS). DHIS reports on all morbidity at health facility level, while MIS is only used for malaria surveillance in the donor supported districts. A cross sectional study was conducted between July-September 2018 by using the retrospective records of DHIS and MIS data reported to the Directorate of Malaria Control (DOMC) Islamabad during 2013-17. Descriptive and inferential analysis was performed to compare the coverage, outcome and impact indicators. Results: During 2013-17, all districts (n=145, 100%) across Pakistan reported on the DHIS. The MIS reporting coverage has gradually increased from 21 (14.5%) to 72 (49.7%) districts. Reported number of suspected screened and confirmed malaria cases were compared. MIS reported twice the number of suspects screened for malaria (100.5%) and confirmed malaria cases (124.4%) as compared to the DHIS. The difference in the reported average annual blood examination rate (ABER) was 3.8, test positivity rate (TPR) was -0.9 and the annual parasite incidence (API) was 4.9/1000 population over five years between two systems. DHIS reported only half the ABER and API as compared to MIS. Conclusion: There is huge under-reporting of suspected and confirmed malaria cases in the DHIS as compared to MIS. Urgent attention is needed to address this, as it is vital to have uniform reporting of true disease burden across the country. An integrated disease surveillance system, improved data validation systems, and use of the online DHIS-2 are potential options for better integrity and coherence of reported data.

Efflux pumps are a specialized tool of antibiotic resistance used by Pseudomonas aeruginosa to expel antibiotics. The current study was therefore conducted to examine the expression of MexAB-OprM and MexCD-OprJ efflux pump genes. In this study, 200 samples were collected from Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC) in Peshawar, Pakistan. All the isolates were biochemically identified by an Analytical Profile Index kit and at the molecular level by Polymerase Chain Reaction (PCR) utilizing specific primers for the OprL gene. A total of 26 antibiotics were tested in the current study using the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and high-level resistance was shown to amoxicillin-clavulanic acid (89%) and low-level to chloramphenicol (1%) by the isolates. The antibiotic-resistant efflux pump genes MexA, MexB, OprM, MexR, MexC, MexD, OprJ, and NfxB were detected in 178 amoxicillin-clavulanic acid-resistant isolates. Mutations were detected in MexA, MexB, and OprM genes but no mutation was found in the MexR gene as analyzed by I-Mutant software. Statistical analysis determined the association of antibiotics susceptibility patterns by ANOVA: Single Factor p = 0.05. The in silico mutation impact on the protein structure stability was determined via the Dynamut server, which revealed the mutations might increase the structural stability of the mutants. The docking analysis reported that MexA wild protein showed a binding energy value of −6.1 kcal/mol with meropenem and the mexA mutant (E178K) value is −6.5 kcal/mol. The mexB wild and mutant binding energy value was −5.7 kcal/mol and −8.0 kcal/mol, respectively. Efflux pumps provide resistance against a wide range of antibiotics. Determining the molecular mechanisms of resistance in P. aeruginosa regularly will contribute to the efforts against the spread of antibiotic resistance globally.