Identification of immune biomarkers in recent active pulmonary tuberculosis.

Tuberculosis (TB) has remained an unsolved problem and a major public health issue, particularly in developing countries. Pakistan is one of the countries with the highest tuberculosis infection rates globally. However, methods or biomarkers to detect early signs of TB infection are limited. Here, we characterized the mRNA profiles of immune responses in unstimulated Peripheral blood mononuclear cells obtained from treatment naïve patients with early signs of active pulmonary tuberculosis without previous history of clinical TB. We identified a unique mRNA profile in active TB compared to uninfected controls, including cytokines such as IL-27, IL-15, IL-2RA, IL-24, and TGFß, transcription factors such as STAT1 and NFATC1 and immune markers/receptors such as TLR4, IRF1, CD80, CD28, and PTGDR2 from an overall 84 different transcripts analyzed. Among 12 significant differentially expressed transcripts, we identified five gene signatures which included three upregulated IL-27, STAT1, TLR4 and two downregulated IL-24 and CD80 that best discriminate between active pulmonary TB and uninfected controls with AUC ranging from 0.9 to 1. Our data identified a molecular immune signature associated with the early stages of active pulmonary tuberculosis and it could be further investigated as a potential biomarker of pulmonary TB.

Expression profile of KIR3DS1/KIR3DL1 receptors in association with immunological responses in TB, HIV and HIV/TB infected patients.

Several studies investigated KIR3DS1 and KIR3DL1 in the context of various infections. However, none of the studies were performed on KIR3DS1/L1 in association with IFN-É£/IL-10 in TB, HIV-1, and their confections. We aimed to evaluate KIR3DS1/KIR3DL1 expression in association with IFNÉ£/IL-10 in HIV-1 and TB mono-infections and HIV-1/TB confection and compared with uninfected controls using RTq PCR. We also performed correlation analysis between KIR3DS1, KIR3DL1, IFN-É£ and IL-10 in the respective cohorts. The overall expression of KIR3DS1 was found to be downregulated in all groups, whereas in HIV-1 and HIV-1/TB, the frequency of KIR3DS1(+) expression was significantly (p < 0.05) associated with undetected HIV-1 viral load. However, expression of KIR3DL1 was found to be significantly (p < 0.05) upregulated in HIV-1 only. In addition, IFNÉ£ expression was significantly (p < 0.05) decreased in TB, whereas in HIV-1/TB, IFNÉ£ expression was significantly (p < 0.05) increased. In contrast, IL-10 expression was significantly (p < 0.05) increased in HIV-1 and HIV-1/TB but not in TB. Also, we found significant positive correlation (p < 0.05, r = 0.61) between KIR3DL1 and IFNÉ£ expression in TB and negative correlation (p < 0.05, r = - 0.62) between KIR3DS1 and IL-10 in HIV-1/TB. In conclusion, we suggest that expression of KIR3DS1/L1 is associated with IFNÉ£/IL-10 responses and it is involved in modulating disease severity in HIV-1 and TB infections.

Thin Layer Immunoassay: An Economical Approach to Diagnose Helicobacter pylori Infection in Gastroduodenal Ulcer Disease Patients of Pakistan, a Comparative Analysis.

Helicobacter pylori is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. The majority of H. pylori-associated patients live in underdeveloped areas, facing the problem of lack of proper diagnostic facility. Hence, a simple and economical assay is required to handle the majority of gastric patients. Serum samples from gastroduodenal ulcers and gastritis patients were screened for H. pylori infection by thin layer immunoassay. A polystyrene plate coated with H. pylori sonicate whole cell antigen (10 µg/mL). Two-fold diluted patient's serum was allowed to react at 37 °C, incubated at 60 °C for 1 min over a water bath and the water condensation pattern for the H. pylori antibody was recorded. ELISAs were used as reference assays to evaluate the efficacy of the developed thin layer immunoassay (TLI). Gastric patients' blood samples (62% male and 6% female) tested positive for H. pylori, while age-wise, 15-25-year-old males (36%) and 65-75-year-old females (50%) showed the highest number of H. pylori infections. TLI showed sensitivity (72-67%), specificity (100%), accuracy (94-69%) and κ value (0.493-0.357) in comparison with wELISA (Surface whole cell ELISA), sELISA (sonicate whole cell ELISA) and kELISA (commercial KIT ELISA). We conclude that thin layer immunoassay is a low cost, fast, simple and clinically reliable method for H. pylori diagnosis at initial stages in patients in under-developed countries.

Diversity of Rotavirus Strains among Children with Acute Diarrhea in Karachi, Pakistan.

One of the common viral pathogens in infectious diarrhea is Rotavirus; in developing countries, it is a primary cause of deaths in children less than five years of age. This study was planned to find out the etiologic agents of acute watery diarrhea. In this study, 1465 stool samples were analyzed with the symptoms of acute diarrhea. Demographic data analysis showed no. of episodes of diarrhea, vomiting, and fever. All samples were checked by ELISA technique for the presence of Rotavirus circulating strains. More than 6% patients were found to be positive with Rotavirus. Common Rotavirus genotypes, including G2P4, G2P6, G3P4, G8P4, G8P6, G9P4, and G10P4, were detected in patients through RT-PCR. This study concluded that detection of rotavirus strain diversity and management of diarrheal patients may identify assortment of emerging strains and reduce emergence of antimicrobial resistance and repeated episodes of diarrhea, which may also help to avoid and manage the essential nutrients lost leading to malnutrition and stunted growth, as well as to reduce high mortality rate in young children less than five years.

Association of trinucleotide repeat polymorphisms CAG and GGC in exon 1 of the androgen receptor gene with male infertility: a cross-sectional study.

BACKGROUND: : Infertility is a global problem that brings about serious sexual and social consequences that strain the health sector and society. The expansion of CAG and GGC repeats in androgen receptor (AR) gene (Ensembl number ENSG00000169083) may lead to reduced fertility. Our objective was to determine the association of CAG and GGC repeats with altered sperm parameters in male infertile subjects. METHODS: This was a cross-sectional study conducted at Aga Khan University, Karachi, Pakistan. A total of 376 males were recruited, out of which group A (N = 208) and group B (N = 168) were comprised of subjects with normal and altered sperm parameters, respectively, from 18 to 60 years. The numbers of CAG and GGC repeats were determined by using PCR amplification and sequence analysis using the Molecular Evolutionary Genetic Analysis (MEGA) software version 6.0. Statistical analysis was performed using the SPSS version 20 and the P-value of <0.05 was considered significant. RESULTS: The mean androgen receptor gene CAG repeats were significantly longer in males with altered sperm parameters as compared to male subjects with normal sperm parameters (P < 0.001). There was no significant difference found for GGC repeats for subjects with altered sperm parameters. DISCUSSION: Longer CAG length corresponded to greater severity of spermatogenic defect and may lead to subfertility recommendations.

A comparative study on production of extracellular hydrolytic enzymes of Candida species isolated from patients with surgical site infection and from healthy individuals and their co-relation with antifungal drug resistance.

BACKGROUND: Surgical site infection (SSI) is a crucial dilemma of surgery. Patients with SSIs not only face difficulty in treatment but also bear extra cost with high mortality rate. Resistant strains of Candida have emerged as an important nosocomial pathogen. Proteinase and phospholipase are exo- enzymes of Candida species, have importance with respect to their contribution in diseases. This study focused on prevalence of Candida species in surgical wound, their resistance to antifungal drugs, co-relation of these resistance with virulence potential of Candida species and comparison of production level of exo-enzymes of Candida species isolated from patients with SSIs and healthy individuals to highlights their role in SSIs. RESULTS: A total of (n = 555) swab samples were investigated. (n = 450) samples were collected from patients with SSIs and (n = 105) were collected from healthy individuals. Samples were subjected for the identification of Candida species which were subsequently investigated for antifungal susceptibility, MICs and enzymatic activity of Candida species. Out of 128 strains of Candida spp. isolated from SSIs, 54(42.18%) were identified as C. albicans followed by C. glabrata 32(25%), C. parapsilosis 17(13.28%), C. krusei 13(10.16%) and C. tropicalis 12(9.38%). C. albicans isolates showed 100% susceptibility to voriconazole and amphotericin B followed by itraconazole 98% and fluconazole 89%. Out of 6 fluconazole resistant C. albicans 5(83.33%) were able to produce phospholipase while out of 48 fluconazole-susceptible strains 17(35.42%) were found to be phospholipase producer. Out of 54 C. albicans isolated from surgical wound 46(85.18%) and 49(90.74%) were found to be phospholipase and proteinase producer respectively, whereas out of 20 C. albicans isolates from healthy subjects 14(70%) produce proteinase and 12(60%) produce phospholipase. There were significant statistical differences found between the level of enzyme production by C. albicans, in relation to both sites (P = 0.014). CONCLUSION: Study revealed that prevalence of Candida species is high in SSIs. Phospholipase and proteinase activity were more pronounced in Candida Species from surgical wound in contrast to species from healthy individuals suggests these enzymes may have been responsible for the severity of infection in surgical wound patients.

Helicobacter pylori severely reduces expression of DNA repair proteins PMS2 and ERCC1 in gastritis and gastric cancer.

Gastric cancers are the third leading cause of cancer mortality in the world. Helicobacter pylori causes over 60 % of all stomach cancers. Colonization of the gastric mucosa by H. pylori results in increased DNA damage. Repair of DNA damage may also be reduced by H. pylori infection. Reduced DNA repair in combination with increased DNA damage can cause carcinogenic mutations. During progression to gastric cancer, gastric epithelium goes through stages of increasing pathology. Determining the levels of DNA repair enzymes during progression to gastric cancer could illuminate treatment approaches. Our aim is to determine the level of gastric expression of DNA repair proteins ERCC1 (a nucleotide excision repair enzyme) and PMS2 (a mismatch repair enzyme) in the presence of H. pylori infection at successive stages of gastric pathology and in gastric cancers. We analyzed gastric tissues of 300 individuals, including 30 without dyspepsia, 200 with dyspepsia and 70 with gastric cancers. The presence of H. pylori, gastric pathology and expression of DNA repair proteins ERCC1 and PMS2 were evaluated. Infection by H. pylori carrying the common cagA gene reduced median nuclear expression of ERCC1 and PMS2 to less than 20 % and 15 % of normal, respectively, in all pathologic stages preceding cancer. ERCC1 and PMS2 nuclear expression was 0-5 % of normal in gastric cancers. H. pylori can cause deficiency of ERCC1 and PMS2 protein expression. These deficiencies are associated with gastric pathology and cancer. This reduction in DNA repair likely causes carcinogenic mutations. Substantially reduced ERCC1 and PMS2 expression appears to be an early step in progression to H. pylori-induced gastric cancer.

The antibiofilm activity of Acetylsalicylic acid, Mefenamic acid, Acetaminophen against biofilms formed by P. aeruginosa and S. epidermidis.

OBJECTIVE: To evaluate the antibacterial activity of Aspirin, Mefenamic acid and Acetaminophen against Pseudomonas aeruginosa and Staphylococcus epidermidis biofilms. METHODS: The study was conducted AKU Karachi in collaboration with DIHE Karachi from March 2018 to December 2018.Quantitative spectrophotometric method was used to study the reduction and removal of the Pseudomonas aeruginosa and Staphylococcus epidermidis formed biofilms. Statistical tests were performed using Graph Pad Prism software. . RESULTS: Acetaminophen showed maximum biofilm reduction activity against the biofilms formed by Pseudomonas aeruginosa, and Staphylococcus epidermidis. Mefanamic acid showed maximum biofilm removal potential against Pseudomonas aeruginosa, while Aspirin and Mefanamic acid were equally effective in removing biofilms formed by Staphylococcus epidermidis as well. CONCLUSIONS: There is a continuous need to look for non-antibiotic agents for their potential antimicrobial and antibiofilm potential.

Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection.

OBJECTIVES: Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study. MATERIALS AND METHODS: 285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy. RESULTS: Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. CONCLUSION: P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.

Study of class 1 integrons in multidrug-resistant uropathogenic Escherichia coli isolated from different hospitals in Karachi.

OBJECTIVES: Escherichia coli is the key pathogen in the family producing ESBL (extended spectrum ß-lactamase) and associated with community-acquired infections. Therefore, this study was planned to determine the antibiotic susceptibility pattern of uropathogenic E. coli, prevalence of the ESBL gene group and class 1 integrons. MATERIALS AND METHODS: Clinical isolates of uropathogenic E. coli were isolated from different hospitals of Karachi. Antibiotic susceptibility test was performed by Kirby-Bauer Methods. Presence of ß- lactamases genes (CTX, TEM, and SHV) and integron 1 were identified by polymerase chain reaction (PCR). RESULTS: Out of 500, 105 isolates were identified as multi-drug resistant (MDR) uropathogenic E. coli. The subject MDR isolates showed the highest resistance to aztreonam, amoxil/ clavulanic acid, ampicillin, cotrimoxazole, ceftriaxone, cefipime, and cefuroxime. Genetic analysis showed that the majority of the MDR E. coli carry CTX M1 (57.1%) followed by TEM (33.3%) and SHV (9.5%). Moreover, 79% of MDR E. coli harbored class 1 integrons, whereas all three conserved genes for class 1 integrons were present in 58% of MDR E. coli. CONCLUSION: This study is helpful to provide information regarding the antibiotic susceptibility pattern, distribution ESBLs and class 1 integrons among uropathogenic E. coli.

Combination of Interleukin 1 Polymorphism and Helicobacter pylori Infection: an Increased Risk of Gastric Cancer in Pakistani Population.

Helicobacter pylori is one of the major risk factors involved in the development ofgastritis and gastric cancer (GC). H. pylori infection leads to increased production of pro-inflammatory cytokines by the host. Carriage of specific polymorphisms in cytokine genes may be associated with host susceptibility to the development of GC. We investigated the role of host genetic factors including polymorphisms of IL-1B and IL-1RN in correlation with gastritis and GC in H. pylori infected Pakistani population. A total of 230 gastritis cases and 100 GC cases were genotyped for IL 1B-511 and IL-1RN penta-allelic variable number of tandem repeats (VNTRs). A combination of IL-1B-511*T and IL-1RN*2 alleles (OR 19.064; 95% CI 2.319-156.7; p = 0.001) in H. pylori infected individuals had markedly increased risk of GC development. In Pakistani population, an increased risk of GC development is associated with the carriage of IL-1B-511*T and IL-1RN*2 alleles. Synergistic effect of H. pylori infection and IL-1B-511*T/IL-1RN*2 genotypes was also observed in association with significantly higher risk of developing GC. Further prospective and large scale studies are needed to establish the clinical impact of these findings.

Species Identification and Antibiotic Susceptibilities of Coagulase- Negative Staphylococci Isolated from Urinary Tract Infection Specimens.

OBJECTIVE: To determine the incidence of Coagulase- negative S. aureusin urinary tract infections and sensitivities of these isolates to antimicrobial agents. STUDY DESIGN: Cohort study. PLACE AND DURATION OF STUDY: Dr. Essa Laboratory and Immunology and Infectious Disease Research Laboratory (IIDRL), Microbiology Department, University of Karachi, from January 2009 to January 2010. METHODOLOGY: Urine specimens, suggestive of urinary tract infection (UTI), were identified. Speciation of isolates was done using API-20 Staph.system. Screening of extracellular products was done using SDS-PAGE electrophoresis and Hemolysin on blood-agar plates. Minimum inhibitory concentration (MICs) of antibiotics was estimated by microtiter well plate method. Frequency and percentages were determined and chi-square test was used for comparing proportions with significance at p < 0.05. RESULTS: Coagulase - negative S. aureus(CONS) were the cause of urinary tract infection in 56 out of 1866 outpatient (3%) and 164 of 1261 inpatient (13%), urinary tract infections (p < 0.001). Two hundred and twenty CONS isolates were identified. The most common CONS identified was S. saprophyticus (31%, 68 strains). The relative frequency of Coagulase - negative S. aureuswas 6% (13 strains). All isolates were sensitive to Vancomycin and Linezolid. Resistance was 69% to Ampicillin, 53% to Methicillin, and 37.5% to Ciprofloxacin. CONCLUSION: CONS are a potential uropathogens, with capability of slime production and resistance to common empirical prescriptions. This also warrants formulation of an appropriate antibiotic policy that covers CONS.

Causal Assessment of Serum Urate Levels in Cardiometabolic Diseases Through a Mendelian Randomization Study.

BACKGROUND: Although epidemiological studies have reported positive associations between circulating urate levels and cardiometabolic diseases, causality remains uncertain. OBJECTIVES: Through a Mendelian randomization approach, we assessed whether serum urate levels are causally relevant in type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), ischemic stroke, and heart failure (HF). METHODS: This study investigated 28 single nucleotide polymorphisms known to regulate serum urate levels in association with various vascular and nonvascular risk factors to assess pleiotropy. To limit genetic confounding, 14 single nucleotide polymorphisms exclusively associated with serum urate levels were used in a genetic risk score to assess associations with the following cardiometabolic diseases (cases/controls): T2DM (26,488/83,964), CHD (54,501/68,275), ischemic stroke (14,779/67,312), and HF (4,526/18,400). As a positive control, this study also investigated our genetic instrument in 3,151 gout cases and 68,350 controls. RESULTS: Serum urate levels, increased by 1 SD due to the genetic score, were not associated with T2DM, CHD, ischemic stroke, or HF. These results were in contrast with previous prospective studies that did observe increased risks of these 4 cardiometabolic diseases for an equivalent increase in circulating urate levels. However, a 1 SD increase in serum urate levels due to the genetic score was associated with increased risk of gout (odds ratio: 5.84; 95% confidence interval: 4.56 to 7.49), which was directionally consistent with previous observations. CONCLUSIONS: Evidence from this study does not support a causal role of circulating serum urate levels in T2DM, CHD, ischemic stroke, or HF. Decreasing serum urate levels may not translate into risk reductions for cardiometabolic conditions.

Camellia sinensis Mediated Enhancement of Humoral Immunity to Particulate and Non-particulate Antigens.

The most common drinking beverage in large portion of the world is Camellia sinensis (green tea). In the present study, we evaluated the adjuvant effect of green tea and tea polyphenols to particulate and non-particulate antigens. BALB/c mice were immunized with particulate and non-particulate antigens. Modulation of immunoglobulin-secreting splenocytes, IgG-mediated and IgM-mediated immunity, was evaluated by hemolytic plaque assay and enzyme-linked immunosorbent assay, respectively. Dose-dependent response of tea polyphenols was also assayed. Phenolic content was measured in crude preparations of green tea. We observed a stimulatory effect of green tea preparations on humoral immune response mediated by the increased number of antibody-secreted cells in spleen. A significant increase in IgM-mediated and IgG-mediated immune response to non-particulate antigen was also observed in green tea-treated animals. A dose-dependent adjuvant effect was seen in the case of tea polyphenols for a longer period of time compared with crude tea preparations. This study indicates polyphenols as major constituents responsible for the enhanced and sustained adjuvant activity of green tea. We suggest that tea polyphenols might be considered for real-life evaluation during adjuvant-mediated vaccination trial programs.

Physical activity, smoking, and genetic predisposition to obesity in people from Pakistan: the PROMIS study.

BACKGROUND: Multiple genetic variants have been reliably associated with obesity-related traits in Europeans, but little is known about their associations and interactions with lifestyle factors in South Asians. METHODS: In 16,157 Pakistani adults (8232 controls; 7925 diagnosed with myocardial infarction [MI]) enrolled in the PROMIS Study, we tested whether: a) BMI-associated loci, individually or in aggregate (as a genetic risk score--GRS), are associated with BMI; b) physical activity and smoking modify the association of these loci with BMI. Analyses were adjusted for age, age(2), sex, MI (yes/no), and population substructure. RESULTS: Of 95 SNPs studied here, 73 showed directionally consistent effects on BMI as reported in Europeans. Each additional BMI-raising allele of the GRS was associated with 0.04 (SE = 0.01) kg/m(2) higher BMI (P = 4.5 × 10(-14)). We observed nominal evidence of interactions of CLIP1 rs11583200 (P(interaction) = 0.014), CADM2 rs13078960 (P(interaction) = 0.037) and GALNT10 rs7715256 (P(interaction) = 0.048) with physical activity, and PTBP2 rs11165643 (P(interaction) = 0.045), HIP1 rs1167827 (P(interaction) = 0.015), C6orf106 rs205262 (P(interaction) = 0.032) and GRID1 rs7899106 (P(interaction) = 0.043) with smoking on BMI. CONCLUSIONS: Most BMI-associated loci have directionally consistent effects on BMI in Pakistanis and Europeans. There were suggestive interactions of established BMI-related SNPs with smoking or physical activity.

Synergy between antibiotics and natural agents results in increased antimicrobial activity against Staphylococcus epidermidis.

INTRODUCTION: Staphylococcus epidermidis is one of the most frequent causes of biofilm-associated infections on indwelling medical devices. With the emergence of methicillin-resistant S. epidermidis (MRSE), there is an urgent need to discover novel active agents against a range of Gram-positive pathogens. We screened the clinical isolates of S. epidermidis for susceptibility/resistance against commonly prescribed antibiotics. Furthermore, we tested some natural agents alone and in combination with antibiotics to find possible synergistic antimicrobial effects. METHODOLOGY: S. epidermidis clinical isolates were screened for susceptibility/resistance against vancomycin, erythromycin, tetracycline, chloramphenicol, ampicillin, ofloxacin, cephalexin, and gentamicin using the Kirby-Bauer disk diffusion method. The antimicrobial potential of Camellia sinensis, Juglans regia, and Hippophae rhamnoides alone and in combination with antibiotics were examined using the disk diffusion method, where the antimicrobial potential activity was measured in terms of formation of zones of inhibition. RESULTS: Most S. epidermidis isolates were found to be resistant to one or more antibiotics. Gentamycin and ofloxacin were found to be the most effective antibiotics against S. epidermidis isolates. Extracts of Hippophae rhamnoides, Juglans regia, and Camellia sinensis were found to be equally effective against S. epidermidis isolates. In combination with antibiotics, these extracts exhibited appreciable synergistic activity; the highest synergistic activity was observed with erythromycin and cephalexin. In the case of cephalexin, a reversion in resistance was observed. CONCLUSIONS: The plant extracts used in the study exhibited additive and synergistic antibacterial activity against S. epidermidis, hence providing an effective alternative to deal with the problem of multidrug resistance.

Cervical cancer screening and HPV genotype distribution among asymptomatic patients of Karachi Pakistan.

BACKGROUND: Cervical cancer is the second most prevalent cancer in females worldwide. Human papillomavirus (HPV) infection is a sexually transmitted infection. However, in addition to HPV infection, other factors exist that influence the risk of developing cervical cancer. In Pakistan most women who developed cervical cancer have been infrequently or never screened. OBJECTIVE: To determine the prevalence of HPV infection and its subtype profile among asymptomatic patients with pre cancerous cervical intraepithelial lesion. METHODS: In this hospital-based descriptive study, 160 asymptomatic females attending gynecology clinics were subjected to HPV screening after obtaining informed consent. Cervical Scrapings were examined by cytopathology and colposcopic directed biopsies taken. High-grade intraepithelial lesion (HSIL) CIN-2, and Low-grade intraepithelial lesion (LSIL) CIN-1 were selected. Samples were analyzed for the presence of HPV-DNA general and type specific genotype 16 and 18. HPV- DNA was extracted by QIA amp DNA kit protocol and amplification was done by polymerase chain reaction (PCR) and genotyped by type specific primers. RESULTS: Out of 160, 17 Pap smear tests were positive, 6 (35.3%) with abnormal results (HSIL) CIN-2 were HPV-DNA positive. Among them, 5 (83.3%) had subtype 16 and in 1 (16.7%) case the genotype was undetectable. The remaining 11(6.9%) with pre cancer minimal abnormal (LSIL) CIN-1 presented. Out of them 3 (27.3%) were HPV-DNA positive with subtype 16. Five (45.4%) were followed by repeated pap smear every six months for two years, and the rest of 3 (27.3%) patients refused for the test. CONCLUSION: A high incidence of Human papillomavirus (HPV) infection is found in women with pre cancerous lesion of cervix in Pakistani women.

Correlation of ABH blood group antigens secretion with Helicobacter pylori infection in Pakistani patients.

OBJECTIVES: A and B blood group antigens are fucosylated carbohydrate present on human erythrocytes and body secretions. Their presence in body secretions depends on the expression of a dominant allele of secretor gene FUT2 and is correlated with susceptibility to various infectious and non-infectious diseases. We investigated the correlation of blood group and ABH antigen secretion with Helicobacter pylori infection and gastroduodenal symptoms and analysed the distribution of babA gene among ABH secretors and non-secretors. METHODS: Two hundred and ninety patients who underwent gastroduodenal endoscopy during 2011 to 2012 participated. Gastric biopsy, saliva and blood samples were obtained from every patient. Gastric biopsies were subjected to rapid urease test and PCR for the detection of H. pylori and babA gene. Blood grouping and ABH antigens secretions were determined by Lewis blood group phenotyping and haemagglutination inhibition test. RESULTS: 50.34% of patients were ABH antigen secretors and 45.51% non-secretors. Distribution analysis of blood group revealed that 40 blood group B, 67 blood group A 20 blood group O and 19 blood group AB patients secreted ABH antigens in saliva. Fifty-six blood group O, 19 blood group B, 32 blood group A and 17 blood group AB patients were non-secretors. Gastroduodenal complaints were common among non-secretors. Sixty-two percent of patients with a combination of duodenal ulcer and gastro-oesophageal reflux and 54% of patients with gastritis were non-secretors. Of 290 samples, 31.02% were positive for H. pylori. Thirty percent of these tested positive for babA gene; the majority belonged to non-secretor blood group O. CONCLUSIONS: Our results suggest that the infection of H. pylori is correlated with ABO blood groups and blood group antigens secretion in body fluids.

Prevalence and Susceptibility Pattern of Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa in Karachi.

OBJECTIVE: To determine the frequency and susceptibility pattern of multi-drug resistant (MDR) Pseudomonas aeruginosa isolated from clinical specimens in Karachi. METHODS: This cross sectional study was conducted in Microbiology Department, University of Karachi, from January 2012 to January 2013. Clinical specimens were collected from different hospitals of Karachi. Clinical isolates were identified by standard and specific microbiological methods. The antibiotic susceptibility pattern was determined by Kirby Bauer Disc diffusion method. Clinical and Laboratory Standards Institute (CLSI) guidelines were used to determine the results. RESULTS: The frequency of MDR P. aeruginosa isolated from different clinical specimens was found to be 30%. Amikacin was found to be the most effective antibiotic, followed by Co-trimaxazole and Quinolones. CONCLUSION: Antibiotic resistant P. aeruginosa are emerging as a critical human health issue. There is an urgent need to resolve the issue by taking some preventive measures. Combined efforts of health care professionals and researchers are required to educate people about the proper use of antibiotics and other infection control measures.

Oxidative DNA damage as a potential early biomarker of Helicobacter pylori associated carcinogenesis.

Helicobacter pylori infection is an established risk factor for gastritis, gastric ulcer, peptic ulcer and gastric cancer. CagA +ve H. pylori has been associated with oxidative DNA damage of gastric mucosa but their combined role in the development of gastric cancer is still unknown. Here we compare the combined expression of cagA and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in normal, gastritis and gastric cancer tissues. Two hundred gastric biopsies from patients with dyspeptic symptoms, 70 gastric cancer tissue samples and 30 gastric biopsies from non-dyspeptic individuals (controls) were included in this study and 8-OHdG was detected by immunohistochemistry (IHC). Histological features and the presence of H. pylori infection were demonstrated by Hematoxylin and Eosin (HE), Giemsa and alcian blue-periodic acid-Schiff ± diastase (AB-PAS ± D) staining. DNA was extracted from tissues and polymerase chain reaction (PCR) performed to determine the presence of ureaseA and cagA genes of H. pylori. The results showed the presence of H. pylori in 106 (53 %) gastric biopsies out of 200 dyspeptic patients, including 70 (66 %) cases of cagA + ve H. pylori. The presence of cagA gene and high expression of 8-OHdG was highly correlated with severe gastric inflammation and gastric cancer particularly, in cases with infiltration of chronic inflammatory cells (36.8 % cagA + ve, 18 %), neutrophilic activity (47.2 %, 25.5 %), intestinal metaplasia (77.7 %, 35.7 %) and intestinal type gastric cancer (95 %, 95.4 %) (p ≤ 0.01). In conclusion, H. Pylori cagA gene expression and the detection of 8-OHdG adducts in gastric epithelium can serve as potential early biomarkers of H. Pylori-associated gastric carcinogenesis.