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.

Identifying and elucidating the resistance of Staphylococcus aureus isolated from hospital environment to conventional disinfectants.

BACKGROUND: Staphylococcus aureus is a nosocomial pathogen, detection and elucidation of its resistance mechanisms to conventional disinfectants may aid in limiting its spread on environmental surfaces in health care settings. In the current study, disinfectant susceptibility of S. aureus strains isolated from the hospital environment as well as possible associations between the presence of disinfectant-resistance genes and reduced susceptibility to disinfectants was investigated. METHODS: A total of 245 samples were collected from the hospital environmental surfaces. The minimum inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants against S. aureus isolates were determined using the micro-broth dilution method. The qac genes (qacA, qacE, and qacΔE1) were detected by PCR and confirmed by sanger sequencing. RESULTS: A total of 47 S. aureus strains were isolated, with more than 85% of them showing methicillin resistance. The qacA, qacE, and qac∆E1 genes were found in 23.4%, 29.7%, and 4.2% isolates respectively. All the isolates with qac genes had higher MIC and MBC values to selected disinfectants. CONCLUSIONS: Significant methicillin resistant S. aureus (MRSA) contamination in the hospital environment was detected. Furthermore, higher qac gene frequencies were found in MRSA isolates that also correlated with higher MIC/MBC values to different disinfectants. The study proposes that hospitals should develop policies to determine disinfectant MICs against the common environmental isolates to contain the spread of resistant strains.

A modified hand washing method for resource limited settings.

The Good Microbiological Practices & Procedures (GMPP) is the most significant risk control measure as per the fourth edition of the WHO laboratory biosafety manual. Among GMPP, one of the best practices is hand washing. WHO and other public health agencies have published several guidance documents on hand washing, that describe closing the tap using a disposable paper towel/tissue paper at the end of hand washing as one of the critical steps. In resource-limited settings, where disposable paper towels cannot be provided at all times, the staff is left with ambiguous instructions on how to close the tap. In this paper, a modified hand washing method is documented that doesn't necessitate the use of disposable paper towels. In this method, both hands and faucets remain in contact with soap for at least 40-60 s. The method was validated by the use of Glo Germ. A survey questionnaire was also designed and conducted for the lab staff (n = 12) of the two laboratories, where this method was implemented, to assess whether this hand washing method brought any improvement in their hand washing practices and implementation. All (100%) of the survey respondents reported that this method of hand washing is more applicable and implementable than the WHO-recommended hand washing technique. Eighty three percentage reported that this modified method of hand washing raised their hand washing compliance. The authors suggest that this hand washing method can be used in resource-limited laboratory settings as an effective GMPP to ensure infection control.

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.

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.

Assessment of biosafety implementation in clinical diagnostic laboratories in Pakistan during the COVID-19 pandemic.

Laboratory diagnostic capacity is crucial for an optimal national response to a public health emergency such as the COVID-19 pandemic. Preventing laboratory-acquired infections and the loss of critical human resources, especially during a public health emergency, requires laboratories to have a good biorisk management system in place. In this study, we aimed to evaluate laboratory biosafety and biosecurity in Pakistan during the COVID-19 pandemic. In this cross-sectional study, a self-rated anonymous questionnaire was distributed to laboratory professionals (LPs) working in clinical diagnostic laboratories, including laboratories performing polymerase chain reaction (PCR)-based COVID-19 diagnostic testing in Punjab, Sindh, Khyber Pakhtunkhwa, and Gilgit-Baltistan provinces as well as Islamabad during March 2020 to April 2020. The questionnaire assessed knowledge and perceptions of LPs, resource availability, and commitment by top management in these laboratories. In total, 58.6% of LPs performing COVID-19 testing reported that their laboratory did not conduct a biorisk assessment before starting COVID-19 testing in their facility. Only 31% of LPs were aware that COVID-19 testing could be performed at a biosafety level 2 laboratory, as per the World Health Organization interim biosafety guidelines. A sufficiently high percentage of LPs did not feel confident in their ability to handle COVID-19 samples (32.8%), spills (43.1%), or other accidents (32.8%). These findings demonstrate the need for effective biosafety program implementation, proper training, and establishing competency assessment methods. These findings also suggested that identifying and addressing gaps in existing biorisk management systems through sustainable interventions and preparing LPs for surge capacity is crucial to better address public health emergencies.

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.

Immune System Activation in Rheumatic Heart Disease.

OBJECTIVE: To assess activation of immune system in rheumatic heart disease (RHD) patients in the form of AECA, ACL and anti GBM antibodies. STUDY DESIGN: Descriptive, observational study. PLACE AND DURATION OF STUDY: Department of Immunology, University of Health Sciences (UHS), Lahore, and Outpatient Department, Punjab Institute of Cardiology, from February 2015 to January 2016. METHODOLOGY: Clinically suspected patients of RHD and confirmed by echocardiography were included. AECA, ACL and anti GBM antibodies were investigated in the sera of RHD patients. RESULTS: Eighty-six RHD patients were included in the study; the mean age of the patients was 30 ±9.3 years. Among these patients, 59 (68.6%) were females and 27 (31.4%) were males. AECA was most commonly detected autoantibody i.e. in 17 (19.8%) patients; whereas, ACL was detected in only 2 (2.3%) subjects. Another 2 (2.3%) patients had both AECA and ACL antibodies. AGBM was not detected in any of the patients. ACL was seen in females with isolated MR. AECA were seen in mixed valvular heart disease patients. CONCLUSION: Immune system gets activated in RHD patients leading to formation of different antibodies, and they are also related to the type of lesion. ACL antibodies are present in females with isolated mitral regurgitation, while AECA are present in both the genders with mixed valvular heart disease. Anti GBM antibodies are not seen in RHD patients.

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.

Relationship between percentage of regulatory t-cells and dental amalgam fillings / Relación entre el porcentaje de células t-reguladoras y obturaciones de amalgama

Abstract: introduction: regulatory T-cells are the main component of peripheral tolerance and their level is decreased in autoimmunity. In dental amalgam, a mixture of metals is used as a restorative material. During daily a ctivities, these metals are ingested and affect renal, neurosensory and immune systems. Studies have demonstrated an increased risk of autoimmune diseases in patients with dental amalgam fillings. It was hypothesized that the percentage of regulatory T-cells decreases in individuals with amalgam fillings. Therefore this study was designed to determine and compare the percentage of regulatory T-cells in individuals with and without amalgam fillings. Material and Methods: This was a cross-sectional study. Subjects were divided into two groups with each group consisting of 40 individuals. Group I (study group) comprised individuals with amalgam fillings, and Group II (control group), individuals without amalgam fillings in their teeth. Blood samples of all the participants were collected and tagged with CD4 FITC, CD25 PE and CD127 PerCP-Cy monoclonal antibodies for the detection of regulatory T-cells, FACSCalibur was used for this purpose. Results: The percentage of regulatory T-cells in the control group was high (77.77 +/- 5.54 percent) compared to the study group (76.09 +/- 7.68 percent), however, on comparison, the difference was not statistically significant (p=0.25). Conclusion: Dental amalgam fillings did not show a declining effect on the percentage of regulatory T-cells.

Resumen: introducción: las células T reguladoras son el principal componente de la tolerancia periférica y su nivel se reduce en la autoinmunidad. En las obturaciones de amalgama, una mezcla de metales se utiliza como un material de restauración. Durante las actividades diarias, estos metales se ingieren y afectan el sistema renal, neurosensorial e inmunológico. Los estudios han demostrado un aumento del riesgo de enfermedades autoinmunes en pacientes con amalgamas dentales. Se planteó la hipótesis que el porcentaje de células T reguladoras disminuye en individuos con obturaciones de amalgama. Por tanto, este estudio fue diseñado para determinar y comparar el porcentaje de células T reguladoras en individuos con y sin obturaciones de amalgama. Material y Métodos: Se realizó un estudio de corte transversal. Los sujetos fueron divididos en dos grupos, cada uno con 40 individuos. El grupo I (de estudio) estuvo conformado por individuos con obturaciones de amalgama y el grupo II (de control) por individuos sin obturaciones de amalgma. Se colectaron muestras de sangre, las que fueron marcadas con anticuerpos monoclonales CD4 FITC, CD25 PE y CD127 PerCP-C para detectar las células T reguladoras, se utilizó FACSCalibur para este propósito. Resultados: El porcentaje de células T reguladoras en el grupo control fue alta (77,77 +/- 5,54 por ciento) en comparación con el grupo de estudio (76,09 +/- 7,68 por ciento), pero esta diferencia no fue estadísticamente significativa (p=0,25). Conclusión: Las obturaciones de amalgama no se asociaron con una disminución en el porcentaje de células T reguladoras.

Immune mechanisms in type-2 diabetic retinopathy.

OBJECTIVE: To enumerate CDR+CD25+ Treg cells and determine serum IL-6 and IL-17 in type 2 diabetes mellitus patients with retinopathy. METHODS: The case-control study was conducted at the Department of Immunology, University of Health Sciences, Lahore, from November 2009 to January 2012 and comprised diabetic patients and healthy controls who were divided into three groups. Group 1 had controls, while Group 2 had diabetic patients without retinopathy and Group 3 had diabetic patients with retinopathy. Flowcytometre and enzyme-linked immunosorbent assay were used for CD4+CD25+ Tregs and serum IL-6 and IL-17 respectively. SPSS 20 was used for statistical analysis. RESULTS: Of the 212 subjects in the study, 30(14%) were Group 1, 30(14%) in Group 2 and 152(72%) in Group 3.There were 25 (83%) women in Group 2 and 101 (66%) in Group 3 compared to 9 (30%) in Group 1. Higher mean age was in Group 3 (50.88 ± 8.9 years) and Group 2 (49.46 ± 9.94 years) compared to Group 1 (34.66 ± 8.78 years) while longer mean disease duration was in Group 3 (10.51 ± 5.24 years) than Group 2 (7.76 ± 4.14 years). Highest median ratio of IL- 6 was in Group 1 (1468.62) (Q1-Q3: 1229.9-1543.35), followed by Group 2 (1455.32) (Q1-Q3:1214.22-158.9) and Group 3 (469.84) (Q1-Q3: 206.53-1231.33) whereas IL-17 was the highest in Group 1 (339.38) (QT-Q3: 159.89- 1174.93), followed by Group 3 (216.60) (Q1-Q3:141.87-410.25) and Group 2 (174.17) (Q1-Q3: 138.77-458.17). Higher percentage of Tregs was in Group 2 (3.07 ± 0.43) followed by Group 1 (2.91 ± 0.04) and Group 3 (2.88 ± 0.38). Significant difference was observed in gender, age, disease duration, level of IL-6 and IL-17 (p < 0.05 each), while no difference was found in glycated haemoglobin, CD4+CD25+ and Tregs (p > 0.05 each). CONCLUSION: Age, gender and duration of diabetes contributed to diabetic retinopathy, while CD4+CD25+ T cells and Treg cells did not. Serum IL-6 and IL-17 were inversely associated with diabetic retinopathy.

Negative association of serum IL-6 and IL-17 with type-II diabetes retinopathy.

BACKGROUND: Diabetes mellitus (DM) is a health concern which leads to complications such as retinopathy. Pakistan has 6.9 million people living with DM and this toll will be doubled by 2025. OBJECTIVE: To determine serum IL-6 and IL-17 of type 2 diabetes mellitus (T2DM) patients with retinopathy. METHODS: In this cross-sectional case-control study, 212 subjects enrolled which were categorized into 3 groups. Group-I included 30 subjects without diabetes, group-II consisted of 30 subjects with T2DM without retinopathy and group-III consisted of 152 subjects with T2DM and retinopathy. Serum IL-6 and IL-17 levels were determined by ELISA. Data was analysed using SPSS 17.0 and one way ANOVA to observe group mean differences. RESULTS: Longer mean duration of disease was detected in group-III than group-II (p=0.007). Highest IL-6 level was detected in group-II and highest IL-17 level was detected in group-I. For IL-6, significant differences were detected among groups in total, between Group-I and Group-III and between Group-II and Group-III (p<0.0001 each). Regarding IL-17, significant differences were found among groups in total (p=0.002) and between Group-I and Group-III (p=0.001). No significant difference in the percentages of HbA1c observed between groups. CONCLUSIONS: Age, gender and duration of diabetes contribute to T2DM retinopathy. Serum IL-6 and IL-17 were inversely associated with T2DM retinopathy.