The possible protective role of HLA B27 and relevant immune markers in Juvenile Idiopathic Arthritis patients.

Background & Objectives: JIA is a disease with different immunological characteristics and a complicated genetic foundation. HLA B27 is a risk factor for the development of JIA, and its impact on immunopathogenesis of the disease is also an area of interest. To determine whether HLA B27 and immune markers varied between JIA patients and healthy population. Methods: This comparative cross-sectional study was conducted at Immunology Department of University of Health sciences (UHS), Lahore from February 2018 till August 2021. A total of (71) JIA patients and (34) healthy controls were enrolled. B cells were enumerated by flowcytometry, ELISA was used for serum cytokines estimation and HLA B27 allele was detected by SPSS polymerase chain reaction. Results: The HLA B27 allele was significantly more in the control group than in the patient group, suggesting it is a protective allele to prevent JIA. Peripheral blood B cell counts and percentages were significantly lower in the HLA B27 positive group than in the HLA B27 negative group of control population. Serum cytokine levels were not significantly different between the HLA B27 positive and HLA B27 negative allele of the two study populations. Conclusion: In this study B cells are different between the two groups of control population however; serum cytokines are comparable between the study groups. Though, it was indicated that HLA B27 may be a preventive allele in the onset of JIA.

Cervical cancer screening practices in HIV positive females - a missing link in health care delivery in Pakistan.

Objectives: To probe cervical cancer screening practices in local women positive for human immunodeficiency virus, and to determine the cervical cytological changes in them. METHODS: The serial cross-sectional study was conducted at the Jinnah Hospital and Services Hospital, Lahore, Pakistan, from April 2019 to October 2020, and comprised female patients aged 18-45 years who were positive for human immunodeficiency virus or acquired immunodeficiency syndrome and were registered with the relevant programme being run by the provincial government in Punjab. Blood samples of all the patients were collected for the determination of human immunodeficiency virus viral load and cluster of differentiation 4+ count. Cervical smears were taken for cytopathological analysis, while the swabs were analysed for culture sensitivity. The same individuals were subjected to the same testing one year later, and the status of the disease and clinical stability or disease progression was explored. Data was analysed using SPSS 25. RESULTS: There were 150 women with mean age 32.08±7.13 years (range: 21-45 years). Age at marriage/sexual activity was 17.33±4.73 years in 15(10%) subjects. Cytological examination showed atypical squamous cells of undetermined significance in 6(4%) of the cases whereas 3(2%) cases showed atypical squamous cells, which cannot rule out high grade squamous intraepithelial lesion on cytology, while the rest were classified as negative for intraepithelial lesion or malignancy. Cervical microbial changes revealed methicillin-resistant staphylococcus aureus infection in 9(6%) cases, extended-spectrum beta-lactamase in 15(10%) cases, whereas fungal infection and trichomonas vaginalis infection were found in 30(20%) smears. There was a significant association between cluster of differentiation 4+ cell count and stability of high-risk patients (p<0.001). After one year, 84(56%) patients remained clinically stable, while 51(34%) developed some chronic illness. There was a significant association between cluster of differentiation 4+ cell count <200/mm3 and the risk of developing a chronic illness (p<0.001). CONCLUSIONS: There was a dire need to educate healthcare workers to offer regular cervical screening to patients with high-risk sexually-transmitted infections to prevent them from the morbidity and mortality related to cervical cancer.

Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon.

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon. Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software. Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index. Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Epidemiology, Clinico-Pathological Characteristics, and Comorbidities of SARS-CoV-2-Infected Pakistani Patients.

SARS-CoV-2 is a causative agent for COVID-19 disease, initially reported from Wuhan, China. The infected patients experienced mild to severe symptoms, resulting in several fatalities due to a weak understanding of its pathogenesis, which is the same even to date. This cross-sectional study has been designed on 452 symptomatic mild-to-moderate and severe/critical patients to understand the epidemiology and clinical characteristics of COVID-19 patients with their comorbidities and response to treatment. The mean age of the studied patients was 58 ± 14.42 years, and the overall male to female ratio was 61.7 to 38.2%, respectively. In total, 27.3% of the patients had a history of exposure, and 11.9% had a travel history, while for 60% of patients, the source of infection was unknown. The most prevalent signs and symptoms in ICU patients were dry cough, myalgia, shortness of breath, gastrointestinal discomfort, and abnormal chest X-ray (p < 0.001), along with a high percentage of hypertension (p = 0.007) and chronic obstructive pulmonary disease (p = 0.029) as leading comorbidities. The complete blood count indicators were significantly disturbed in severe patients, while the coagulation profile and D-dimer values were significantly higher in mild-to-moderate (non-ICU) patients (p < 0.001). The serum creatinine (1.22 µmol L-1; p = 0.016) and lactate dehydrogenase (619 µmol L-1; p < 0.001) indicators were significantly high in non-ICU patients, while raised values of total bilirubin (0.91 µmol L-1; p = 0.054), C-reactive protein (84.68 mg L-1; p = 0.001), and ferritin (996.81 mg L-1; p < 0.001) were found in ICU patients. The drug dexamethasone was the leading prescribed and administrated medicine to COVID-19 patients, followed by remdesivir, meropenem, heparin, and tocilizumab, respectively. A characteristic pattern of ground glass opacities, consolidation, and interlobular septal thickening was prominent in severely infected patients. These findings could be used for future research, control, and prevention of SARS-CoV-2-infected patients.

Novel acetamide derivatives of 2-aminobenzimidazole prevent inflammatory arthritis in rats via suppression of pro-inflammatory mediators.

Benzimidazole ring system is an important pharmacophore with diverse pharmacological activities. In this study, we explored the anti-arthritic effects of newly synthesized acetamide derivatives of 2-aminobenzimidazole (N1 and N2) in rats. FTIR and NMR spectroscopies were used to characterize these compounds. Carrageenan (CRG) induced paw edema model was used to test the acute anti-inflammatory activity of various doses (10, 20 and 30 mg/kg) of N1 and N2 compounds. Based on acute anti-inflammatory effects, the most potent dose of each compound was selected and investigated in complete freund's adjuvant (CFA) induced inflammatory arthritis (RA) model (n = 4 in each group). Histopathological, hematological, radiographic, and RT-qPCR analyses were performed to assess the progression or resolution of inflammatory arthritis. The tested compounds produced a dose-dependent anti-inflammatory activity against CRG induced paw inflammation and similarly reduced edema in CFA induced inflammatory arthritis model. Histopathological and X-ray analyses of ankle joints revealed minimal inflammation and normal joint structures in N1 and N2 treated groups. The tested compounds also reduced the levels of autoantibodies and restored hematological parameters. Interestingly, the tested compounds did not elevate aspartate aminotransferase and alanine transaminase levels and displayed a better safety profile than methotrexate. N1 and N2 compounds also attenuated the transcript levels of IRAK1, NF-kB1, TNF-α, IL-1ß, IL17 and MMP1. In addition, N1 displayed a greater inhibition of mRNA levels of COX1, COX2, mPGES1 and PTGDS as compared to N2. Our findings demonstrate that N1 and N2 compounds possess strong anti-arthritic activity which can be attributed to the suppression of pro-inflammatory mediators.

Four year trend of antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus in a tertiary care hospital, Lahore.

OBJECTIVE: To determine the susceptibility pattern of methicillin-resistant staphylococcus aureus to different antibiotics. METHODS: The descriptive study was conducted at the Microbiology Department of the University of Health Sciences, Lahore, Pakistan, from August 2016 to July 2019, and comprised staphylococcus aureus samples that were processed and identified using colony morphology on blood agar, gram stain, catalase, coagulase and deoxyribonuclease testing. Screening for methicillin-resistant staphylococcus aureus was done using cefoxitin disc 30µg and oxacillin disc 1mg. Antimicrobial susceptibility was tested using the modified Kirby-Bauer disc diffusion method in line with the Clinical and Laboratory Standards Institute guidelines 2019. Data was analysed using SPSS 24. RESULTS: Of the 2704 strains processed, 402(14.86%) were found to be methicillin-resistant staphylococcus aureus. Of them, 204(50.74%) were recovered from pus, while 10(2.48%) were recovered from urine. All 402(100%) isolates were sensitive to vancomycin and linezolid, and resistant to penicillin, followed by erythromycin 306(76.11%) and sulfamethoxazole/ trimethoprim 295(73.38%). Overall, lower resistance was seen with doxycycline 145(36.06%) and clindamycin 160(39.80%). Inducible clindamycin resistance was seen in 142(35.23%) isolates. CONCLUSIONS: An efficacious susceptibility pattern of methicillin-resistant staphylococcus aureus was seen with vancomycin and linezolid, moderate susceptibility with doxycycline and clindamycin, while high resistance was observed for penicillin, erythromycin and trimethoprim/sulfamethoxazole.

SLC11A1 genetic variation and low expression may cause immune response impairment in TB patients.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis. Host genetic factors are important for the detection of TB susceptibility. SLC11A1 is located in monocyte phagolysosomes that help to limit M. tuberculosis growth by transferring divalent cations across the membrane. Genetic variation in SLC11A1 may alter its expression and increase the susceptibility of individuals to TB. The current study aimed to provide insight into host genetic variations and gene expression in TB patients. A total of 164 TB patients and 85 healthy controls were enrolled in this study. SLC11A1 polymorphisms were detected by PCR-RFLP. Real-time qPCR was used for SLC11A1 gene expression, and ELISA was used for protein estimation. GTEx Portal was used for quantitative trait loci analysis, while the STRING (v.11) web platform was used for gene interactive network construction. Data were analyzed using SPSS, GraphPad Prism, Haploview, and SNPstats. SLC11A1 polymorphisms and combinatorial genotypes were strongly associated with TB susceptibility, which may explain the greater prevalence of tuberculosis in the local population. Polymorphisms in SLC11A1 have also been linked to gene expression variation. Furthermore, the expression of SLC11A1 was downregulated in TB patients, which may influence the function of other associated genes and may impair the immunological response to tuberculosis.

Decreased classical monocytes and CD163 expression in TB patients: an indicator of drug resistance.

Tuberculosis (TB) is a disease instigated by Mycobacterium tuberculosis. Peripheral blood monocytes represent highly efficient effector cells of innate immunity against TB. Little is known about monocyte subsets and their potential involvement in the development of M. tuberculosis drug resistance in patients with TB. This study was conducted to investigate alterations in monocyte subsets, CD163 expression on monocytes, and its serum level in patients without and with rifampicin resistance TB (RR-TB) and healthy controls. A total of 164 patients with TB (84 without RR-TB and 80 patients with RR-TB) and 85 healthy controls were enrolled in this study. The percentages of various monocyte subsets and surface expression of CD163 on monocytes were quantitatively determined using flow cytometry. The serum level of CD163 was determined by commercially available ELISA kits. Decreased frequency of classical monocytes was detected in patients with RR-TB. Non-classical monocytes were decreased in patients without RR-TB; however, intermediate monocytes were raised in patients with RR-TB. The serum level of CD163 was decreased in patients of RR-TB that showsed a positive correlation with the frequency of CD14++CD16-CD163+ and CD14++CD16+CD163+ monocytes. It is concluded that decreased classical monocytes and sCD163 in patients with RR-TB could be an indicator of drug resistance.

Assessment of Vancomycin MIC Creep Phenomenon in Methicillin-Resistant Staphylococcus aureus isolates in a Tertiary Care Hospital of Lahore.

OBJECTIVE: To assess vancomycin MIC creep phenomenon in methicillin-resistant Staphylococcus aureus isolated from clinical specimens. METHODS: This descriptive study was conducted in Microbiology department of University of Health Sciences, Lahore from January 2016- December 2019. In this study, vancomycin MICs were revealed by E test method for clinical MRSA strains. For the final evaluation, a single isolate from each patient was taken. The reported vancomycin MICs results were used and the values were not rounded up to the next upward value. For every study year, MIC50, MIC90, median and geometrical mean MIC, percentages of susceptible and resistant strains were calculated. RESULTS: A total of 352 MRSA strains were isolated out of 2704 staphylococcal isolates. Our study showed elevated vancomycin MIC among MRSA isolates. The majority of isolates showed MIC values ≥1.5µg/ml. MIC50, MIC 90 was constant throughout four years period. However, geometric mean MIC increased gradually during the study period. The MIC greater than base year median was overall 17.3%. A complete shift can be observed between MIC "1.0" and "2.0" the percent of cases with MIC "1.0" decreased and with MIC "2.0" increased over time crossing each other in 2017. CONCLUSION: Vancomycin MIC creep was identified in clinical isolates of MRSA, during four years of study period. Even though there is an absence of VISA and VRSA strains; this significant increase in vancomycin MIC trend is indeed worrying for the clinicians about the threat of potential failure of treatment in MRSA infections.

Salivary Levels of IL-6 and IL-17 Could Be an Indicator of Disease Severity in Patients with Calculus Associated Chronic Periodontitis.

Background/Purpose. Chronic periodontitis is an inflammatory disease of gums that causes loss of supporting structures of teeth, that is, gingiva, periodontal ligament, cementum, and alveolar bone. Levels of various cytokines in the serum, gingival tissues, and gingival crevicular fluid in patients with chronic periodontitis have been studied, but limited data are available on the level of cytokines in saliva. Therefore, a study was designed to determine levels of salivary IL-6 and IL-17 in patients with calculus associated chronic periodontitis. Materials and Methods. It was a comparative, cross-sectional study that is comprised of 41 healthy controls and 41 calculus associated chronic periodontitis patients (CP patients). According to the degree of attachment loss, CP patients were subcategorized as mild (CAL 1-2 mm), moderate (CAL 3-4 mm), and severe (CAL > 5 mm) forms of periodontitis. Salivary levels of IL-6 and IL-17 were determined using enzyme-linked immunosorbent assay (ELISA) technique. Data was analyzed using SPSS 20.0. Results. Between healthy controls and CP patients (moderate and severe disease), a statistically significant difference was observed in the concentrations of IL-6 and IL-17. In CP patients, the highest mean ± SD of salivary IL-6 and IL-17 was observed in severe CP, followed by moderate and mild CP. Regarding level of IL-6, a statistically significant difference was observed between mild and severe disease and between moderate and severe subcategories of CP patients. Similarly, statistically significant difference was observed in the level of IL-17 between mild and moderate, mild and severe disease, and moderate and severe disease. Conclusion. The levels of salivary IL-6 and IL-17 were increased significantly in calculus associated CP patients as compared to healthy controls and these levels increased with the progression of CP. Clinical Significance. Salivary levels of IL-6 and IL-17 may help in the subcategorization of CP.

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus.

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known. To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against. We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.

Percentage of CD4+ and CD8+ T-lymphocytes in blood of tuberculosis patients.

BACKGROUND: Tuberculosis is a fatal infectious disease, mainly caused by Mycobacterium tuberculosis. Spread of TB is controlled by cell-mediated immunity. Purpose of this study was to determine CD4+ and CD8+ T cell percentages in TB patients. METHODS: 77 subjects consisted of 39 patients of active tuberculosis and 37 normal healthy individuals were recruited for the study. Among patients, 27 were at different stages of anti-tuberculous therapy while rests of the patients were not taking treatment. Sixteen patients were sputum positive for AFB while other patients were sputum negative for AFB. T cells percentages were determined by flow cytometer. RESULTS: In TB patients CD4+ and CD8+ T cells percentages were 34.4 +/- 9.8 and 32.0 +/- 9.8 while in controls these were 37.1 +/- 6.9 and 30.2 +/- 7.2 respectively but the difference was statistically insignificant. CD4+ T cell percentage in newly diagnosed TB patients was 28.8 +/- 8.7 while it was 37.9 +/- 8.9 in TB patients who were on therapy and difference was statistically significant whereas difference in CD8+ T-cell percentages was statistically insignificant. A negative correlation between CD8+ T-cells percentage and the duration of ATT was found. CONCLUSION: CD4+ and CD8+ T-cells percentages may help to find out the immune status of TB patients before and after the completion of ATT.

In vitro phagocytosis of methicillin resistant and methicillin sensitive Staphylococcus aureus by human polymorphonuclear leucocytes.

BACKGROUND: Staphylococcus aureus is a gram positive bacterium that causes a number of diseases such as abscesses, infective endocarditis, septic arthritis, etc. It is acquiring resistance against many antibiotics like methicillin; therefore its control is becoming increasingly difficult. Peripheral blood phagocytes particularly polymorphonuclear leucocytes play an important role in the protective mechanisms against these organisms. Phagocytes interact with bacteria and phagocytose these microorganisms to kill them. METHODS: Phenotypically different isolates of Staphylococcus aureus including methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA) were collected from various hospitals of Lahore, Pakistan. Fresh polymorphonuclaer leucocytes were obtained from healthy individuals by centrifugation using Ficol-Hypaque gradient combined with dextran sedimentation. Microbiological method was used for the determination of phagocytic index of phenotypic variants of Staphylococcus aureus. RESULTS: A significant difference was observed between the phagocytic index of both bacterial groups. MSSA group showed the Mean +/- SD of 79.46% +/- 3.9 while MRSA group showed 72.350% +/- 2.5. CONCLUSION: Significant difference in phagocytic index indicates that it can be one of the mechanisms of MRSA to evade host immune system as compare to MSSA.