Extended-spectrum b-lactamases (ESBLs) are enzymes that can hydrolyze extended- spectrum cephalosporins and monobactams. ESBL-producing Klebsiella pneumoniae are responsible for serious morbidity and mortality among paediatric patients. This study aimed to determine the frequency of ESBL-producing K. pneumoniae, phenotypic characterization techniques and antimicrobial resistance pattern. The study was also established to determine the molecular characterization of blaSHV, blaTEM, blaCTX-M genes which are responsible for ESBL-mediated antibiotic resistance. The study was conducted at The Children’s Hospital & Institute of Child Health, Lahore, Pakistan during May 2010 to February 2012. The molecular studies of blaSHV, blaTEM, blaCTX-M and integron genes were performed during October 2012 to April 2013 at the Microbiology and Immunology Department, The University of Melbourne, Australia. Various clinical samples were collected and studied from paediatric patients, including blood, central venous pressure line, cerebrospinal fluid, ear swab, endotracheal tube, peritoneal dialysis catheter, pleural fluid, pus, tracheal secretion, urine and wound swab. The organisms were identified using various biochemical tests and the API 20E system. ESBL production was determined using double disk synergy test (DDST) and Clinical and Laboratory Standards Institute (CLSI) confirmatory test. The antimicrobial resistance pattern of ESBL-producing K. pneumoniae was determined using Kirby-Bauer disc diffusion method with various antibiotic groups. The target genes were amplified and DNA sequencing was performed for blaSHV and blaTEM genes to find out the mutations responsible for ESBL genotype. Screening of 710 K. pneumoniae isolates showed 214 (30.1%) were ESBL screen positive K. pneumoniae. The CLSI confirmatory test showed significantly greater sensitivity (p<0.0001) compared to DDST. There were 82 (38.3%) neonates infected with ESBL K. pneumoniae and 152 (71.0%) of the total cases were males. The most common sources of ESBL K. pneumoniae were blood (117; 54.7%) and urine (46; 21.5%). Of the 214 cases, 92 (43.0%) cases were isolated from Neonatal Nursery Unit and (47; 22.0%) Nephrology. Patients presented with various symptoms such as fever (125 cases; 58.4%) and respiratory distress (104 cases; 48.6%). Important interventions given to the patients included intravenous line (209 cases; 97.7%), urinary catheters (46; 21.5%) and endotracheal tube (18; 8.4%). The outcome of the patients showed the successful discharge of 127 (59.0%) patients after treatment while there were 56 (26.0%) cases of mortality and 31 (15.0%) left against medical advice (LAMA). There was no significant correlation (p=0.1396) found between length of stay and mortality of the patient. Neonates infected with K. pneumoniae had a significantly higher chance of mortality than the older age groups (p=0.0140), while there was no association of outcome (p=1.0000) between the two genders. A higher mortality rate (p=0.0005) was seen among the septicemic patients. The mortality rate was significantly higher (p=0.0013) in patients who presented with respiratory distress symptoms. An antibiotic resistance profile of ESBL-producing K. pneumoniae was performed against 18 antibiotics. All ESBL K. pneumoniae isolates were resistant to ceftazidime, ceftriaxone, cefotaxime and cefuroxime. The antibiotics that K. pneumoniae were most resistant to, include co-amoxiclav (212; 99.1%), cefpodoxime (210; 98.1%), co-trimoxazole (207; 96.7%), gentamicin (201; 93.9%), tobramycin (199; 93.0%), aztreonam (192; 89.7%), cefepime (171; 79.9%) and amikacin (164; 76.6%). Only 41 (19.2%) isolates were resistant to cefoxitin and 96 (44.9%) showed medium level resistance to ciprofloxacin. Only one (0.5%) isolate showed resistance to imipenem and meropenem. The number of isolates displaying resistance to sulbactam-cefoperazone and piperacillin-tazobactam were 13 (6.1%) and 7 (3.3%), respectively. The number of antibiotics to which K. pneumoniae were resistant in each patient were compared in patients with (n=67) or without (n=147) history of antibiotic use in the last three months. No significant difference (p=0.5298) found between the two groups. Amplification and analysis of bla genes showed the majority of K. pneumoniae isolates carry the blaSHV (99.5%), blaTEM (93.0%) and blaCTX-M (99.0%) genes. All of the TEM genes isolated in this study were wild type TEM-1 β-lactamases. The ESBL type SHV detected in the present study were SHV-28 (19.2%), SHV-12 (5.2%) and SHV-110 (0.5%), while non-ESBL type SHV were SHV-1 (20.2%), SHV-11 (31.5%), SHV-42 (1.9%) and SHV-27 (1.4%). The CTX-M-1 group β-lactamases was identified in 99% of the strains. K. pneumoniae isolates in the present study were also studied for the presence of an integrase gene and it was found that 94.9% of isolates had a class 1 integrase, while the class 2 and 3 integrase genes were identified in 1.4% and 0.9% of isolates, respectively. This is the first study conducted on clinical isolates of ESBL-producing K. pneumoniae among paediatric patients from a tertiary care paediatric hospital of Pakistan. The high prevalence of ESBL-producing K. pneumoniae among paediatric patients is responsible for prolonged hospital stay and an increased financial burden on parents and the government. Cephalosporins, monobactams, aminoglycosides and sulfonamide drugs do not prove to be a good choice for the treatment of ESBL-producing K. pneumoniae infections to high rates of resistance to these antibiotics. This study recommends the use of carbapenems, sulbactam-cefoperazone and piperacillin-tazobactam for the treatment of ESBL K. pneumoniae infections but they should be used as a last resort following culture and susceptibility testing. It is being recommended that a stricter infection control policy should be implemented to control the horizontal transfer of blaSHV, blaTEM, blaCTX-M genes and integrons in clinical isolates of K. pneumoniae and other bacteria.
سید شاہ محمد محی الدین پھلواروی افسوس ہے پچھلے مہینہ اپریل کی ۲۲؍ تاریخ کوصوبۂ بہار کے امیر شریعت اورپھلواری شریف کی خانقاہِ مجیبیہ کے سجادہ نشین مولانا الحاج السید شاہ محمد محی الدین صاحب نے صبح کے وقت تقریباً آٹھ بجے وفات پائی۔مولانا علم وعمل، تقوی وطہارت اوراخلاق وعادات کے اعتبار سے علمائے سلف کانمونہ تھے۔ آپ کاروحانی اوراخلاقی فیض واثر بہت وسیع تھا اور آپ کی ذات لاکھوں مسلمانوں کی عقیدت وارادت کامرکز تھی۔اب جبکہ وقت آرہاتھا کہ امارتِ شریعت کاادارہ سیاسی محکومیت کی بندشوں سے آزاد ہوکر اپنے فرائض وواجبات صحیح طور پرانجام دے سکے۔آپ ایسے بزرگ کا رحلت کرجانا مسلمانوں کے لیے جس قدر بھی افسوس اور رنج والم کاباعث ہوکم ہے۔ بہرحال مشیتِ ایزدی میں کسی کوکیا مجالِ دم زدن ہے۔ اﷲ تعالیٰ حضرت مرحوم کوجنت الفردوس میں مقامِ جلیل عطافرمائے اور بیش از پیش نعمتوں اورنوازشوں سے سرفراز فرمائے ۔آمین۔ اسی شمارہ میں مولانا مرحوم کے حالات وسوانح پرایک مختصر مقالہ بھی شریکِ اشاعت ہے۔ [مئی ۱۹۴۷ء]
The Holy Quran is that last scripture of Allah Almighty that has been revealed for the guidance of the human beings. Prior of its revelation the mankind was indulged in the darkness of illiteracy, ignorance, barbarism, lawlessness and idolatry. But the Quran revealed them the wealth of knowledge and in the time the followers of this Great Book became mentors of the whole Ummah. In Arabia, mankind had lost their dignity before the Quran was sent. They lacked national unity. They considered it to be a binding of themselves to Quarrel with one another. The past religious teaching had been altered. The life of the poor had been made tough by the local Arab Tribal leaders. Allah almighty Showered by sending the Holy Quran in such circumstances. The Quran is such a code of life which is a sure Source of success if lead our lives according to its Teachings. The Root Cause of our Problems is our lack of knowledge about the Quranic Teachings. It is the need of Hour that the Teachings of the Quran should be extended so that the Muslim Youth should be able to build their character according to the Quranic principles.
Prostate adenocarcinoma (PCa) is one among the most common global disorders affecting men and is a leading cause of cancer related mortality. In the present study, TP53, PTEN and AR genes in 680 histologically confirmed prostate cancer patients and 500 controls from Pakistan were investigated to unravel their role in prostate cancer. Exon specific primers were designed for all the exons of the three genes along with flanking intronic and UTR regions. Sequence changes were initially screened by PCR-SSCP and then confirmed through automated sequencing. Our data showed that none of the samples had a complete deletion of any of the three genes. A total of twenty six novel exonic mutations and eight novel intronic variants were detected in the target genes along with the four previously reported exonic alterations. The intronic variants were observed both in cases and controls and thus were further investigated for their possible association with the disease through un-conditional logistic regression under different genetic models. The g.7675016T>A variant of TP53 showed significant association with increased PCa risk under allelic contrast (OR=1.84, 95%CI=1.29-2.63, p-value=0.001), heterozygous model (OR=2.25, 95%CI=1.37-3.69, p-value=0.001) and dominant model (OR=2.13, 95%CI=1.38-3.28, p-value=0.001) while its g.7674991T>A variant showed positive association in all genetic models except TT vs TC and recessive model. In our studied population, we observed non-significant association for g.87891382G>A variant of PTEN in all models except allelic contrast (OR=1.87, 95%CI=1.36-2.58, p-value=0.000), while the other three variants of PTEN showed positive correlation with increased PCa risk. Similarly, both the intronic variants of AR gene were also found associated with increased PCa risk. The AA genotypes of g.67637091T>A variant and CC of g.67724021T>C variant are more common in cases. Homozygous individuals for A allele has 3.68 times higher risk of prostate cancer. Majority of the mutations are novel and thus establishing their pathogenicity is of prime diagnostic importance. A comprehensive structural and functional annotation was therefore reported in the present study for the observed mutations through the application of bioinformatics approaches. It was noticed that 5/8 of TP53 mutations (P152A, T170M, E171R, H179Q and V203G), 7/9 of PTEN mutations (A86P, E91K, H93Q, Q97H, E99X, H272F and E288F), while 11/13 of the AR gene mutations (I870fs, I870_splice, D880Y, E884X, I900V, S909C, K906N, L908P, V912G, I915F and Y916S) were predicted to endorse changes in their respective protein structure and thus are functionally damaging. Phenotypic data was collected in integration with the genotypic data from all the study participants and was statistically evaluated for their correlation and impact on individual’s survival. Age, smoking CVDs, BMI, dairy products consumption, physical activity, family history and hypertension was observed responsible for increasing prostate cancer risk under uni-variate and multi-variate models. The diabetic patients were observed at lower risk of having PCa while others risk factors were found non-associated. Overall survival analysis showed a median survival time of 21 months for the PCa patients (95%CI= 18-26). Intra-group differences in patient’s survival after adjusting for confounder age were confirmed through a proportional hazard model. It was observed that age, BMI, smoking, prostectomy and physical activities are associated with survival probability while the rest of risk factors have no effect on survival. It was further noticed that diabetic patients has better survival length as compared to the non-diabetic counterparts. Our results elucidate the significance of combining molecular and in silico approaches to fully distinguish pathogenic mutations from benign which will have profound effects on the patient management. We also have developed an integrated platform, pakprostate.com during the course of study to provide users the facility to search literature, genes, drugs, to have access to download verified dataset, can upload data, can apply basic stats and visualize data just on click of a button. The repository pakprostate is freely available online at the URL www.pakprostate.com. Case-control samples were collected from all the four provinces of Pakistan including Capital territory, Gilgit-baltistan and Kashmir. It is thus hoped that, this study is a good representation of Pakistani population.