Seroprevalence and molecular detection of brucellosis among Pakistani women with spontaneous abortion.

Background: Human brucellosis is a neglected disease transmitted to humans from animals such as cattle, goats, dogs, and swine. The causative agents are bacteria of the genus Brucella, intracellular pathogens usually confined to the reproductive organs of their animal hosts causing sterility and abortions. The objective of the study was to determine the seroprevalence of brucellosis among women with spontaneous abortions (SAW) and compare this seroprevalence with that of healthy pregnant women (HPW). Methods: The case-control study was designed to determine the seroprevalence and molecular detection of brucellosis in women who suffered from spontaneous abortion and healthy pregnant women of the Haripur District of Pakistan. A total of 770 blood samples (n = 385 for each group) were collected from 9 public and 11 private hospitals in Haripur District from December 2021-March 2023. Data on demographic features, epidemiological variables, and risk factors were collected from each participant by structured questionnaires. Initial screening for brucellosis was performed by Rose Bengal Plate Test followed by qRT-PCR for molecular detection of the genus-specific BCSP-31 gene of Brucella. Results: The study showed that anti-Brucella antibodies were more found in SAW 23.63% (91/385) than in HPW 1.29% (5/385). Brucella specific DNA was amplified in 89.01% (81/91) seropositive samples of SAW. Demographic features and risk factors such as age, urbanicity, socioeconomic status, education, occupation, and animal contact were found significantly associated with brucellosis (p ≤ 0.05). Consumption of unpasteurized raw milk (OR = 18.28, 95%CI: 8.16-40.94) was found highly concomitant with seroprevalence. Conclusion: This study reports the first evidence of involvement of brucellosis in spontaneous abortions in women of Pakistan. The study can be used to develop strategies for risk management during pregnancy, to raise awareness for brucellosis, and develop control programs.

A hidden dilemma; post COVID-19 first detection of Varicella zoster M4 genotype from Pakistan.

OBJECTIVES: During the COVID-19 pandemic, the risk of childhood infectious diseases was increased. Post-COVID-19 escalation of chickenpox cases, becoming an emerging public health concern. Thus, the study was designed to compare chickenpox prevalence and Varicella zoster virus (VZV) genotypes circulating before, during, and post-COVID-19 in Pakistan. METHODS: A total of 267 lesion specimens collected from tertiary care hospitals, and chickenpox outbreaks from Pakistan were analysed by a two-amplicon approach with phylogenetic analysis. RESULTS: Among suspected cases, overall 178/267 were VZV positive. Majority (84.2 %; 150/178) cases were of post-COVID-19 pandemic time. Small outbreaks occurred soon after COVID-19 in Rawalpindi and Islamabad (Pakistan), 40 positive cases out of 178 cases were outbreak cases. There was first time detection of the M4 genotype, which was significantly associated with disease severity (p = 0.0006) and post-COVID-19 chickenpox outbreaks in 2021 (77.9 %; 46/59; p < 0.00001). However, in pre-COVID-19 only M2 genotype was detected. The M2 prevalence varied from 2019 (100 %; 19/19) to 2022 (3.2 %; 3/91). However, the most prevalent strain of 2022 belonged to the M1 genotype (64.8 %; 59/91). CONCLUSION: A significant rise in chickenpox cases detected soon after COVID-19 in Pakistan, and oscillation of different VZV genotypes with first time detection of M4 genotype is an alarming situation. This demands further detailed genotypic studies on transmission dynamics of a rare M4 with other genotypes to protect the local population and restrict spread in other regions.

Human monkeypox: An updated appraisal on epidemiology, evolution, pathogenesis, clinical manifestations, and treatment strategies.

Monkeypox (Mpox) is a zoonotic viral disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus. The recent occurrence of Mpox infections has become a significant global issue in recent months. Despite being an old disease with a low mortality rate, the ongoing multicountry outbreak is atypical due to its occurrence in nonendemic countries. The current review encompasses a comprehensive analysis of the literature pertaining to MPXV, with the aim of consolidating the existing data on the virus's epidemiological, biological, and clinical characteristics, as well as vaccination and treatment regimens against the virus.

Phenotype versus genotype discordant rifampicin susceptibility testing in tuberculosis: implications for a diagnostic accuracy.

IMPORTANCE: An accurate diagnosis of drug resistance in clinical isolates is an important step for better treatment outcomes. The current study observed a higher discordance rate of rifampicin resistance on Mycobacteria Growth Indicator Tube (MGIT) drug susceptibility testing (DST) than Lowenstein-Jenson (LJ) DST when compared with the rpoB sequencing. We detected a few novel mutations and their combination in rifampicin resistance isolates that were missed by MGIT DST and may be useful for the better management of tuberculosis (TB) treatment outcomes. Few novel deletions in clinical isolates necessitate the importance of rpoB sequencing in large data sets in geographic-specific locations, especially high-burden countries. We explored the discordance rate on MGIT and LJ, which is important for the clinical management of rifampicin resistance to avoid the mistreatment of drug-resistant TB. Furthermore, MGIT-sensitive isolates may be subjected to molecular methods of diagnosis for further confirmation and treatment options.

Presence of T3SS (exoS, exoT, exoU and exoY), susceptibility pattern and MIC of MDR-Pseudomonas aeruginosa from burn wounds.

INTRODUCTION: The emergence of resistance is a major public health and clinical issue, particularly in pathogens causing nosocomial infections. Recently, there is the emergence of Pseudomonas aeruginosa resistance to different broad-spectrum antibiotics. METHODOLOGY: The current study was designed to find out the prevalence of multi-drug resistant (MDR) P. aeruginosa in burn patients, the antibiotic susceptibility pattern of MDR Pseudomonas, and to determine the Minimum Inhibitory Concentration (MIC) of the effective antimicrobials. The assessment of virulence genes (exoT, exoS, exoY and exoU) was also achieved through PCR. In the current study wound swabs were collected from 160 burn patients from two burn units (MTI-Govt. Lady Reading Hospital and MTI-Khyber Teaching Hospital). RESULTS: Out of these 160 samples, 26 samples (16.25%) were positive for P. aeruginosa. Per patients, one isolate was included in the current study. Antibiotic susceptibility pattern showed all P. aeruginosa isolates were 100% resistant to amoxicillin-clavulanic acid, 84.62% resistance to Cefepime, and Ceftazidime, and 76.92% resistance to Amikacin, Aztreonam, and Ciprofloxacin. Whereas the lowest resistance was observed to Imipenem and Piperacillin-Tazobactam (53.85%), Colistin Sulfate (23.08%), and Polymyxin-B (15.38%). Regarding the prevalence of MDR, 22 (84.61%) isolates out of 26 were found to be MDR-P. aeruginosa. For MDR-P. aeruginosa, the MIC range was 1-2 µg/mL against Polymyxin-B, 2-8 µg/mL against Colistin sulfate, 16-1024 µg/mL against Imipenem and 128-1024 µg/mL against Piperacillin-Tazobactam. 100% of the isolates carried exoT, 88.46% carried exoY, and 57.69% and 38.46% carried exoU and exoS, respectively. CONCLUSIONS: These findings further emphasize the need for antibiotic discipline and to follow the recommended hospital antibiotic policy to prevent the proliferation of MDR strains of P. aeruginosa in the community.

Epidemiology of Staphylococcus aureus Non-Susceptible to Vancomycin in South Asia.

Staphylococcus aureus is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens among which multidrug resistance has emerged. Resistance to methicillin has resulted in clinicians using the antibiotic of last resort, vancomycin, to treat infections caused by methicillin-resistant S. aureus (MRSA). However, excessive use and misuse of vancomycin are major causes of resistance among S. aureus strains. South Asia encompasses ~25% of the world's population, and countries in South Asia are often characterized as low- and middle-income with poor healthcare infrastructure that may contribute to the emergence of antibiotic resistance. Here, we briefly highlight the mechanism of vancomycin resistance, its emergence in S. aureus, and the molecular epidemiology of non-susceptible S. aureus to vancomycin in the South Asian region.

Profiling of potential pathogenic candida species in obesity.

PURPOSE OF RESEARCH: The aim of the current study was gut profiling of culturable Candida species and their possible pathogenic potential to asses role in obesity. METHODS: This case control study includes stool samples from 75 obese individuals and 50 controls. Isolation and identification of various Candida species was carried out by standard microbiological techniques. For pathogenic profiling, extracellular enzymatic assays, biofilm forming ability and resistance to azole were analyzed. RESULTS: Culturable gut profiling identified comparative higher abundance and diversity of Candida species among obese compared to controls. The most abundant specie among both groups was C.kefyr. A comparatively higher pathogenic potential as more hydrolases expression was detected in C.kefyr, C.albicans and Teunomyces krusei from obese group. Majority isolates from obese group were strong biofilm formers (47.1%) compared to control group (35.4%) suggesting it as strong risk factor for obesity. Fluconazole resistance was highest among C.kefyr (51%) followed by Teunomyces krusei and C.albicans. All the isolates from different species were voriconazole sensitive except C.kefyr displaying a 4.2% resistance in obese group only. A significant association of dominant colonizing species with meat, fruit/vegetable consumption and residence area was present (p < 0.05). CONCLUSION: The presence of hydrolytic enzymes in gut Candida species showed strong association with protein's degradation and enhanced pathogenicity. C.kefyr and Teunomyces krusei has emerged as potential pathogen showing increased colonization as result of protein rich and low carb diet. Thus presenting it as a bad choice for weight loss in obese individuals.

Emergence of hypermucoviscous colistin-resistant high-risk convergent Klebsiella pneumoniae ST-2096 clone from Pakistan.

Klebsiella pneumoniae convergent clones are considered a threat to healthcare settings. Here we report a comprehensive genomic profiling of an emerging colistin-resistant K. pneumoniae ST-2096 convergent clone from Pakistan. Methods: Whole-genome sequencing was performed and raw reads were assembled antimicrobial resistance and virulence genes were predicted using various online tools. Results & conclusion: The phenotypically multidrug-resistant (MDR) and hypermucoviscous (hv) colistin-resistant K. pneumoniae (hvCRKP-10718), which, intriguingly, possessed a wide range of antimicrobial resistance (blaTEM-1A, blaOXA-1, blaOXA-232, blaCTX-M-15, blaSHV-106, oqxA, oqxB, aac(6')-Ib-cr, aadA2, aac(6')-Ib-cr, armA, tetD, mphE, msrE, fosA, dfrA1, dfrA12, dfrA14, catB3, sul1) and virulence determinants (RmpA/RmpA2, yersiniabactin [ybt], aerobactin [iuc/iut], enterobactin). Furthermore, the acquisition of various mobile genetic elements (MDR/virulent plasmids, type II integron gene cassette, insertional sequences, transposases) and associated hv capsular type made this MDR/hv isolate a convergent clone belonging to a high-risk lineage (ST-2096). Based on core-genome multilocus sequence typing and single-nucleotide polymorphism analysis, this isolate showed ≥99% nucleotide identity with MDR K. pneumoniae isolates from India, depicting its evolutionary background. This study provides a comprehensive genomic profiling of this high-risk convergent K. pneumoniae ST-2096 clone from Pakistan. Comparative genomics of MDR/hv colistin-resistant K. pneumoniae isolates with other MDR convergent strains from the Indian subcontinent indicated the emergence of this evolving superbug.

TLR-2 Germ Line Variants as a Risk for Obesity in Local Pakistani Population.

BACKGROUND: Obesity is increasing rapidly affecting half billion adult's population. Pathophysiology of obesity involves low grade inflammation sustained by Toll like receptor 2 (TLR-2) the innate immune adapters. This study was conducted for detection and association of TLR-2 gene mutations with obesity. METHODS: In this case-control study 228 individuals with obesity and 228 controls were enrolled based on Body Mass Index (BMI) ≥25 and 18-24 kg/m2 respectively. The variations in TLR-2 gene were detected by Sanger sequencing. These identified TLR-2 variants were further analyzed in silico for change in miRNA binding and mRNA strucutre. RESULTS: Four novel single base substitutions (153688371 T >C, 153702295 T >C, 153703504 T >C and 153705074 C >A) were identified in exon 3 and 4 of TLR-2 gene affecting splice site and poly-A tail. The genotypic and allelic frequencies of the variants were strongly associated with increasing obesity susceptibility. Only variant 153703504 T >C was significantly associated with preobesity. Despite variations in gene sequence, no change in miRNA binding except for variant 153688371 T >C of Exon 3 where a novel binding site for hsa-miR-4523 was created. Furthermore, mRNA stability and secondary structure were also compromised in identified variants. CONCLUSION: All detected variants of TLR-2 gene were significantly associated with and posed risk for development of obesity. Furthermore, in silico analysis revealed generation of new miRNA (hsa-miR-4523) binding site and change in mRNA structure/stability which needs to be further investigated for possible role in altering TLR-2 gene regulation/expression in obesity.

Elucidation of molecular mechanism for colistin resistance among Gram-negative isolates from tertiary care hospitals.

Antimicrobial resistance is a growing concern of global public health. The emergence of colistin-resistance among carbapenem-resistant (CPR) Gram-negative bacteria causing fear of pan-resistance, treatment failure, and high mortality across the globe. AIM: To determine the genotypic colistin-resistance mechanisms among colistin-resistant (CR)Gram-negative clinical isolates along with genomic insight into hypermucoviscous(hv)-CR-Klebsiella pneumoniae. METHODS: Phenotypic colistin-resistance via broth-microdilution method. PCR-based detection of plasmid-mediated colistin resistance genes(mcr-1,2,3). Characterization of selected hvCR-K. pneumoniae via Whole-genome sequencing. RESULTS: Phenotypic colistin-resistance was 28% among CPR-Gram-negative isolates of which 90% of CR-isolates displayed MDR profile with overall low plasmid-mediated colistin resistance (mcr-2 = 9.4%;mcr-3 = 6%). Although K. pneumoniae isolates showed the highest phenotypic colistin-resistance (51%) however, relatively low plasmid-mediated gene-carriage (mcr-2 = 11.5%;mcr-3 = 3.4%) pointed toward other mechanisms of colistin-resistance. mcr-negative CR-K. pneumoniae displaying hv-phenotype were subjected to WGS. In-silico analysis detected 7-novel mutations in lipid-A modification genes includes eptA(I38V; V50L; A135P), opgE(M53L; T486A; G236S), and arnD(S164P) in addition to several non-synonymous mutations in lipid-A modification genes conferring resistance to colistin. Insertion of 6.6-kb region harboring putative-PEA-encoding gene(yjgX) was detected for the first time in K. pneumoniae (hvCRKP4771). In-silico analysis further confirmed the acquisition of not only MDR determinants but several hypervirulent-determinants displaying a convergent phenotype. CONCLUSION: overall high prevalence of phenotypic colistin resistance but low mcr-gene carriage suggested complex chromosomal mediated resistance mechanism especially in K. pneumoniae isolates. The presence of novel mutations in lipid-A modification genes and the acquisition of putative-PEA-encoding gene by hvCR-K. pneumoniae points toward the role of chromosomal determinants conferring resistance to colistin in the absence of mcr-genes.

Dysbiosis in salivary bacterial diversity of postpartum females and its association with oral health problems and APOs.

The present study investigates the dysbiosis in salivary bacterial diversity by culture-dependent and independent methods. Culturable aerobic and facultative anaerobic bacterial diversity was studied in saliva collected from 267 postpartum and 54 nonpregnant females by using standard microbiological methods. For unculturable bacterial diversity, DNA from saliva samples of four selected females was sequenced by targeting V4 region of 16S rRNA. In postpartum females, S. mutans was significantly more prevalent. Its colonization was also seen significant among females having gingivitis (P < 0.01), dental caries (P < 0.01), and in those giving birth to low weight baby. In postpartum group, 65.16% females were culture positive for Staphylococcus, 12.73% Gram positive rods, 10.48% N. meningitides, 6.36% K. pneumoniae, 5.61% Enterobacter species and 2.62% E. coli. Isolates showed high biofilm forming ability and antibiotic resistance. Upon analysis of unculturable bacterial diversity, a total of 16 phyla and 156 genera were observed. Alpha diversity was decrease in postpartum female having oral health issues with pre-term low weight birth, compared to females with full term birth. Bray-Curtis dissimilarity was highest between female with dental issues and different pregnancy outcomes. Bacterial diversity and abundance altered among females with different oral health conditions and pregnancy outcomes, and also have pathogenic potential.

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan.

INTRODUCTION: Rifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. METHOD: A total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. RESULT: Among the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. CONCLUSION: Mutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

Association of endothelial nitric oxide synthase gene variants with preeclampsia.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology. The endothelial nitric oxide synthase (eNOS) gene and nitric oxide (NO) levels has been reported to be associated with PE predisposition in various populations. Therefore, present study was designed to investigate the role of NO levels and eNOS gene variants in preeclamptic women in Pakistan. METHODS: A total of 600 women were evaluated, 188 of PE with mild features, 112 of PE with severe features and 300 normotensive pregnant women. NO levels were detected by Greiss reaction method and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. RESULTS: Reduced concentrations of NO were reported in all PE groups (p < 0.05) as compared to controls. The frequency of c.894 T (p.298Asp) and g.-786C alleles were significantly associated with PE. In addition, novel homozygous variant g.2051G > A was also significantly associated with PE when compared to normotensive women. Dynamic simulation studies revealed that Glu298Asp mutation destabilize the protein molecule and decrease the overall stability of eNOS protein. Molecular docking analysis of mutant promoter with transcription factors STAT3 and STAT6 proposed changes in protein regulation upon these reported mutations in upstream region of the gene. CONCLUSION: Considering the results of current study, the functional alterations induced by these variants may influence the bioavailability of NO and represents a genetic risk factor for increased susceptibility to PE. However, large studies or meta-analysis are necessary to validate these findings.

Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology characterized by increased hypertension and proteinuria after 20 weeks of gestation. The present study was directed to determine the role of eNOS in susceptibility to PE and the association of c.894G > T (p.(Glu298Asp), intron 4b/4a, g.-786 T > C and other possible variants of eNOS gene with preeclampsia in Pakistani population. Computational analysis of identified variants in the coding and non-coding region of the eNOS gene was also conducted to determine the change in gene regulation and further protein stability. A total of 600 women were evaluated, 188 with mild and 112 with PE with severe features PE with 300 normotensive pregnant women. NO levels and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. Data from the current study suggest that there might be other risk variants of the eNOS gene (g.2051G > A and g.1861G > A) and lower levels of serum NO that confers in an increased risk of PE. The detailed computational investigation further confirmed the deformities and changes in protein flexibility upon Glu298Asp. These structural alterations might be associated with preeclampsia. Variants in the promoter region of the eNOS gene further validate the change in gene regulation for the onset of disease. Identification of key structural and functional features in eNOS protein and gene regulatory region might be used for designing specific drugs for therapeutic purpose.

Keystone salivary mycobiome in postpartum period in health and disease conditions.

Despite of known pathogenic potential of human mycobiome in initiation and progression of oral disorders, it is poorly characterized and understudied due to its small number in oral cavity. In the present study, salivary mycobiome of three postpartum females along with one healthy non-pregnant female was investigated by targeting ITS region. A total of 55 genera and 92 species were detected with predominant genera: Candida (12.2%) followed by Saccharomyces (9.27%), Phialosimplex (9.19%), Termitomyces (6.96%), Penicillium (6.85%), Aspergillus (6.56%), Olpidium (5.15%), Cochliobolus (4.78%), Malassezia (4.61%), Neurospora (4.3%), and Cristinia (3.04%) in all samples. Diversity increase was observed in postpartum group as compared to non-pregnant female. Stachybotrys, Geotrichum, Talaromyces, Leucosporidium, Acremonium, Wallemia, Eupenicillium, Septoria, Zymoseptoria, Coniosporium, Phialophora, and Mycosphaerella were genera detected only in postpartum group. Postpartum female with gingivitis and dental caries showed greater abundance of genus Saccharomyces, Phialosimplex, Candida, Olpidium, Cochliobolus, Malaseezia, Hyphodontia, Debaryomyces, Mrakia, and Nakaseomyces as compared to those postpartum females with good oral health. Among postpartum group female with oral health issues as well as who had preterm low weight birth (PLWB), showed reduced richness, evenness with elevated levels of Saccharomyces, Candida, Hyphodontia and Malassezia compared to the female having full term birth (FTB). These findings showed that, pregnancy with or without oral health issues is associated with oral microbial diversity change and there might be an association of changing fungal diversity with adverse pregnancy outcomes (APOs) like pre-term birth (PTB) and low weight birth (LWB).

Utilization of an Industry Byproduct, Corymbia maculata Leaves, by Aspergillus terreus to Produce Lovastatin.

Due to its ability to lower cholesterol levels, simvastatin is a leading drug for the prevention of strokes and heart disease: it also lowers the incidence of neurodegenerative diseases. Simvastatin is made from lovastatin, a precursor produced by the industrial fungus, Aspergillus terreus. In this study, Corymbia maculata leaves were tested as a novel substrate for the growth of a new isolate of A. terreus and a lovastatin-resistant strain of A. terreus to produce lovastatin. Corymbia maculata (spotted gum) is well utilized by forest industries as a source of timber because of its high strength, durability and smooth texture. However, the leaves are a major waste product. Growth of A. terreus on Corymbia maculata leaves, in solid-state fermentation resulted in the production of lovastatin. Fermentation of media using fresh leaves of Corymbia maculata produced more lovastatin (4.9 mg g-1), than the sun-dried leaves (3.2 mg g-1). Levels of lovastatin were further increased by the lovastatin-resistant strain of A. terreus (Lvs-r), which produced twice the amount of the parental strain. The production of lovastatin was confirmed by HPLC and LC-MS/MS studies. The study suggests that the utilization of a cheap substrate for the production of lovastatin can have a potential economic benefit.