Genomic Insights into and In Vitro Evaluation of Antimicrobial Combination Therapies for Carbapenem-Resistant Acinetobacter baumannii.

Background and Objectives: Acinetobacter baumannii (A. baumannii), particularly carbapenem-resistant A. baumannii (CRAB), represents a grave concern in healthcare settings and is associated with high mortality. This study aimed to conduct molecular, mutational, and phylogenetic analyses of specific genes in CRAB and evaluate the synergistic effects of selected antimicrobial combinations. Materials and Methods: Phenotypic characterization was performed on six CRAB strains by using the Modified Hodge Test (MHT) and IMP-EDTA Double-Disc Synergy Test (IMP-EDTA DDST). Carbapenemase- and metallo-beta-lactamase-encoding genes were amplified by using Polymerase Chain Reaction. Phylogenetic analysis using the MEGA 11 tool was used to determine the evolutionary relatedness of these genes. Mutational analysis was performed by using I-Mutant, MUPro, and PHD-SNP bioinformatics tools to predict mutations in the carbapenemase-encoding genes. Microdilution checkerboard titration assessed the synergistic effects of antimicrobial combinations (azithromycin-meropenem, rifampicin-meropenem, meropenem-colistin, and azithromycin-colistin) on these CRAB isolates. Results: The phenotypic characterization of six CRAB isolates revealed positive results for MHT and IMP-EDTA DDST. The molecular characterization revealed that carbapenemase- and MBL-encoding genes were present in all isolates with varying frequencies, including blaOXA-51 (100%) and blaIMP (0%). The sequence analysis revealed high evolutionary relatedness to sequences in the NCBI database. The mutational analysis identified 16 mutations, of which 1 mutation (P116L) in the blaOXA-58 gene predicted a change in the protein product, potentially contributing to carbapenem resistance. The checkerboard titration method did not reveal any synergism among the tested antimicrobial combinations against CRAB. Conclusion: This study's findings underscore the significant challenges posed by CRAB isolates harboring multiple resistant genes in treatment. This highlights the urgent need for novel antimicrobial agents, a crucial step towards reducing mortality rates not only in Pakistan but also globally.

Molecular characterization and risk factors of oral high-risk human papillomavirus among females in Punjab, Pakistan.

BACKGROUND: The development of oral and oropharyngeal cancers is associated with human papillomavirus (HPV) infection. However, debate exists about the prevalence of high-risk oral HPV (HR-HPV) and its associated risk factors. This study evaluated the distribution of genotypes and associated risk factors of oral HR-HPV infection in the normal oral mucosa of the Pakistani female population with general gynecological problems. METHODS: A total of 200 females from a regional gynecological hospital in Pakistan were included in this cross-sectional study. A self-administered questionnaire was carried out, followed by the study participants' oral and dental examinations. Whole saliva was collected by a drooling method. The viral DNA extraction and oral HR-HPV genotyping were detected via real-time polymerase chain reaction. RESULTS: Among the 200 females (mean age, 37.76 years), 22 females (11%) were positive for oral HR-HPV DNA. Single oral HR-HPV infection was seen in 8%, whereas co-infection was observed in 3% with HPV16 and HPV18 most common genotypes. Comparison of the risk factors like oral sex history odds ratio (OR 8.21, 95% confidence interval [CI] 1.85-28.16; p < 0.005), and open-mouth kissing (OR, 5.17; 95% CI 1.48-27.41; p = 0.005) with oral HR-HPV infection was found to be significantly associated. CONCLUSION: In Pakistan, oral HR-HPV-positive infection is not uncommon; HPV16 and HPV18 are the most frequent genotypes. Furthermore, this study also provides data that oral HR-HPV positive infection was significantly associated with sexual behaviors. Therefore, preventive health efforts, like health education and vaccination, should be practiced.

Detection of antibacterial activities of Miswak, Kalonji and Aloe vera against oral pathogens & anti-proliferative activity against cancer cell line.

BACKGROUND: Emerging drug resistance and hindrance of treatment is provoking scientists to search new, less expensive medicinally active compounds. Dental diseases caused by oral pathogens are very frequent chronic infections around the world. The medical potentials of a lot of Pakistani local herbs and herbal combinations is relatively unknown, hence attempted to explore. A study was designed to investigate potential role of local medicinal herbs for example Miswak, Kalonji & Aloe vera as antimicrobial, antioxidant and anti-proliferative agents against oral pathogens and cancer cell line. METHODS: Medicinal extracts were prepared in solvents of different polarities. Their antimicrobial activity was determined alone and in combination against oral pathogens. Antioxidant activity was evaluated through Catalase and Superoxide dismutase assay and anti-proliferative activity was evaluated through 3-(4, 5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) assay. RESULTS: Plant extracts alone and in combinations were found significantly effective as antimicrobial agent against standard ATCC strains of C. albicans and S. aureus (P ˂0.001). Especially Miwak extract was found highly significant against fungus. Extracts of Kalonji were found significant in inhibiting growth of HeLa cell lines. Miswak and Kalonji showed significant levels of antioxidant activity. CONCLUSION: Medicinal herbs Miswak and Kalonji have potential to be used for therapeutic purposes. Results suggested that herbal medicinal composition can be prepared using these extracts after applying scientific standardization methods.

Exploring the Concordance Between High-Risk Human Papillomavirus Infections in Cervical and Oral Sites Among Females: A Cross-Sectional Study in Punjab, Pakistan.

PURPOSE: Human papillomavirus (HPV) is widely recognized as a key contributing factor in cervical and oropharyngeal cancers. However, there has been limited research on the prevalence of concurrent HPV infections in various anatomic regions. This study aimed to investigate the prevalence and specific types of high-risk HPV (HR-HPV) infections in the cervical and oral regions of females in Punjab, Pakistan. METHODS: We conducted a cross-sectional study involving women seeking care for general gynecologic issues at the gynecologic Outpatient Department of Lady Wallington Hospital in Lahore. After interviews and clinical examinations, we collected whole-saliva samples and high vaginal swabs from each participant. HR-HPV detection and genotyping were performed using real-time polymerase chain reaction at both the anatomic sites. RESULTS: In this study, 170 females, averaging 35.36 ± 8.305 years, participated. HR-HPV infection was more prevalent in the cervix (83/170 [48.8%]) than in the oral cavity (19/170 [11.2%]). Concordant HPV infections occurred in 10/170 participants (5.9%). HPV 16 was the most common genotype in cervical and oral locations, at rates of 21.8% and 5.3%, respectively, among concordant HR-HPV types. Socioeconomic status (P = .013), age at first sexual intercourse (P = .015), and history of oral sex (P = .01) were significantly associated with concurrent HR-HPV infection in both regions. CONCLUSION: This study suggests that HR-HPV cervical infections may increase the risk of oral transmission, especially during orogenital sexual practices. Thus, it is important to recognize that HPV infections may be linked in both areas. We emphasize the importance of comprehensive cervical and oral examinations and HPV vaccination in young women irrespective of their sexual practices.

Coagulase-Negative Staphylococci in Neonatal Blood: How Concerning?

Objective Coagulase-negative staphylococci (CoNS) are being implicated as one of the leading causes of bloodstream infection (BSI). To study the spectrum, prevalence, and antimicrobial susceptibility of CoNS causing BSI in neonates. Materials and Methods A cross-sectional study was done in level III neonatal intensive care unit (NICU). Blood samples in automated culture bottles were processed as per the standard technique. Previously validated methods were followed for the characterization of CoNS and for AST of standard antibiotics by Kirby Bauer disk diffusion and vancomycin by agar dilution. The prevalence of causative organisms and susceptibility of CoNS were statistically analyzed. Categorical variables were compared by chi-square or Fisher's exact probability tests. Result In total, 1,365 blood samples (1,365 neonates) were studied, of which 383 (28.05%) were positive and 982 (71.94%) were negative. Gram-positive organisms (GPC) predominated ( n = 238; 62.14%) ( p < 0.001) with 41.77% (160/383) S. aureus and 13.83% (53/383) CoNS. CoNS included S. epidermidis (19, 38%), S . haemolyticus (7, 14%), S. hominis (6, 12%), S. simulans (6,12%), S. capitis (5,10%), S. cohnii (4, 8%), S. warneri (1, 2%), and S. xylosus (1, 2%). The susceptibility to netilmicin, linezolid, and vancomycin was 100% ( p ≤ 0.001), and 54% ( n = 27) had vancomycin MIC of 0.125 µg/mL but methicillin-resistant CoNS (MRCoNS) was 70%. Methicillin-susceptible (MS) CoNS had lower MIC of vancomycin ( p < 0.05) than MRCoNS. Conclusion The spectrum of pathogens causing BSI in neonates is changing with predominance of GPC and among CoNS, S. epidermidis . Considerable proportion of MRCoNS with the emergence of MIC creep for vancomycin requires immediate attention.

Whole-genome analysis and evolutionary characterization of cervical and oral human papillomavirus 16.

High-throughput genome-wide sequencing has revealed high genomic variability of HPV16 in different geographic regions which is the most predominant genotype in human papillomavirus (HPV)-associated malignancies. Analysis of the HPV16 by whole-genome sequence (WGS) is an advanced method for the identification of mutations in the genome. There is limited information about HPV16 diversity in Pakistan, especially at the genomic level. Till now, WGS for HPV16 has not been previously reported in Pakistan. The current study has sequenced three HPV16 viral genomes, from two cervical and one oral cavity positive sample of women presented with general gynecological problems without any evidence of precancerous or cancerous lesions using an ion ampliseq customized panel. Sequencing analysis detected 38 variations, including single-nucleotide polymorphisms (SNPs) and two Indels, across three samples with the highest number of SNPs present in E1, E2, and L2, respectively. A total of 20 non-synonymous and 11 synonymous mutations with amino acid substitutions (T1421C, G1515A, T2223C, T1389C, G1483A, and T2191C) were identified. The phylogenetic analysis revealed the genomes of HPV16 are closely associated with those reported from Thailand and the United States. These are the first HPV16 WGS from Pakistan. However, more research is needed with a large sample size from diversified areas to assess the carcinogenic consequences and impact of HPV vaccinations.

Distribution of High-risk Human Papillomavirus Genotypes in Cervical Secretions in Punjab.

OBJECTIVE: To determine the high risk-human papillomavirus (HR-HPV) prevalence, genotype distribution, and associated risk factors in women with general gynaecological problems. STUDY DESIGN: Descriptive, cross-sectional study. PLACE AND DURATION OF STUDY: Lady Wallingdon Hospital, Lahore from August 2018 to May 2019. METHODOLOGY: One hundred females with general gynaecological problems constituted the sample. Real-time polymerase chain reaction and liquid base cytology were performed for HR-HPV detection, genotyping, and cytological changes in cervical samples. RESULTS: The overall prevalence of HR-HPV was found to be 57%, with the most frequent HR-HPV genotypes HPV 16 (18%), HPV 18 (6%), and HPV 45 (1%). The total prevalence of single HR-HPV infection was 25%; whereas, multiple co-infection of HR-HPV was seen in 32% of cases. In total, 81% of these women had normal cytology and 19% had cytologic abnormalities. A significant association was observed between HR-HPV infection and general gynaecological complaints and between HR-HPV infection and cytological abnormalities (p < 0.001). CONCLUSION: The HR-HPV prevalence was 57% with the most prevalent genotypes were HPV 16 and HPV 18. The results will also aid the identification of HR-HPV 51 and 31 genotypes, which should be added in the multi-type HPV vaccines. Key Words: Cancer of cervix, Human papillomavirus, Papanicolaou test, Pakistan.

Staphylococcus aureus carrying lukS/F Panton-Valentine Leukocidin (PVL) toxin genes in hospitals of Lahore city.

INTRODUCTION: Panton Valentine-Leukocidin (PVL) toxin is secreted by Staphylococcus aureus and is mostly associated with skin and soft tissue infections (SSTI). This study aims to find out the prevalence of lukS/F-PV gene, which encode PVL toxin from strains of SSTI, burn wounds and nasal colonizers of out-patients and to measure the antimicrobial susceptibility of S. aureus isolates. METHODOLOGY: This is an analytical observational cross-section study and was conducted from July 2014 to June 2015 at four tertiary care hospitals and PCSIR Laboratories Complex, Lahore, Pakistan. A total of 376 random clinical swabs were collected from SSTI (n = 179), nasal nares (n = 134) and burn wounds (n = 63) from out-patients' departments (OPD). The specimens were cultured on nutrient and mannitol salt agar (MSA) and the organism was identified by catalase, coagulase, and DNase tests. Antimicrobial susceptibility, methicillin, inducible clindamycin, and high-level mupirocin (HLMR) resistance were determined as per CLSI guidelines. Molecular identification of mecA and lukS/F-PV genes was performed by PCR. RESULTS: We isolated 127 S. aureus, where 41 (32.3%) were MRSA and 86 (67.7%) were MSSA. All MRSA carried mecA gene whereas lukS/F-PV gene was found in 21 MRSA and 31 MSSA strains. Overall, a high antimicrobial resistance was found against MRSA and lukS/F-PV positive MSSA. Inducible clindamycin and high-level mupirocin resistance (HLMR) was 23.6% and 19.5% respectively. CONCLUSIONS: A high rate of PVL toxin gene was detected among S. aureus strains and a high prevalence of antimicrobial resistant strains was observed.

Effect of cream containing Melia azedarach flowers on skin diseases in children.

A herbal cream containing a methanolic HPLC-standardized extract of Melia azedarach flowers has been prepared and found potent against bacterial skin diseases like cellulitis, pustules, pyogenic infections, etc. in children. The results obtained are comparable to those with neomycin.