Jawad Ahmed

ANTIMICROBIAL SUSCEPTIBILITY AND ESBL PREVALENCE IN PSEUDOMONAS AERUGINOSA ISOLATED FROM BURN PATIENTS IN THE NORTH WEST OF PAKISTAN

Pseudomonas aeruginosa is one of the most prevalent pathogen in burn infections. Infections with P. aeruginosa are associated with higher mortality rate and antibiotic costs in hospitalized patients. These bacteria also produce enzymes called Expanded Spectrum Beta-Lactamases (ESBL) which render penicillins and cephalosporins inactive. The aim of this study was to assess the antimicrobial susceptibility pattern and prevalence of ESBL in P. aeruginosa in Peshawar, North West of Pakistan. During 2005-2006, one hundred and six P. aeruginosa isolates were collected from burn patients at a tertiary care hospital. Antibiotic susceptibility testing and ESBL detection were carried out according to Clinical Laboratory and Standards Institute (CLSI) criteria. Eighteen antibiotics were tested in this study. A total of 38 (35.85%) isolates were found to be ESBL producers. Thirty one (29.24%) isolates were resistant to 3 or more antibiotics (multidrug resistance). Meropenem and imipenem showed high potency with 99% and 96% isolates being susceptible respectively. Susceptibility to amikacin was 70%; gentamicin 25%; ciprofloxacin 49%; enoxacin 47%; gatifloxacin 42%; doxycycline 21% and to co-trimoxazole only 16%. This study reveals that P. aeruginosa isolated from burns in this region are multidrug resistant and produce ESBL in large proportions.

Neuroprotective and Anti-Aging Potentials of Essential Oils from Aromatic and Medicinal Plants

The use of essential oils (EOs) and their components is known since long in traditional medicine and aromatherapy for the management of various diseases, and is further increased in the recent times. The neuroprotective and anti-aging potentials of EOs and their possible mechanism of actions were evaluated by numerous researchers around the globe. Several clinically important EOs and their components from Nigella sativa, Acorus gramineus, Lavandula angustifolia, Eucalyptus globulus, Mentha piperita, Rosmarinus officinalis, Jasminum sambac, Piper nigrum and so many other plants are reported for neuroprotective effects. This review article was aimed to summarize the current finding on EOs tested against neurodegenerative disorders like Alzheimer disease (AD) and dementia. The effects of EOs on pathological targets of AD and dementia including amyloid deposition (Aβ), neurofibrillary tangles (NFTs), cholinergic hypofunction, oxidative stress and glutamatergic abnormalities were focused. Furthermore, effects of EOs on other neurological disorders including anxiety, depression, cognitive hypofunction epilepsy and convulsions were also evaluated in detail. In conclusion, EOs were effective on several pathological targets and have improved cognitive performance in animal models and human subjects. Thus, EOs can be developed as multi-potent agents against neurological disorders with better efficacy, safety and cost effectiveness.

Frequency of cryptosporidium infection in children under five years of age having diarrhea in the North West of Pakistan.

Cryptosporidium species are minute, coccidian protozoan parasites that have been associated with enterocolitis. It has worldwide distribution and has emerged as an important cause of diarrhea, particularly in children less than 5 years of age and in immunocompromised individuals. Waterborne transmission is particularly troublesome because Cryptosporidium parvum oocysts are not eliminated by chlorination or domestic disinfectants. In the present study, single stool specimens from young children (< 5 years) presented with diarrhea were collected in Khyber teaching hospital, Peshawar, Pakistan. Wet mount preparation and modified Ziehl-Neelsen staining were used for identification of oocysts in stool specimens. Cryptosporidium oocysts were found in 18 (9.0%) out of 200 children suffering from diarrhea. Infection was common in children between 1 - 24 months of age and associated with abdominal cramps (50%), vomiting (61.1%) and prolonged duration of diarrhea (88.9%). Direct and indirect contact with animals was present in most of C. parvum infected children (83.3%). Most of C. parvum infected children were consumers of well water (77.8%). Cryptosporidium spp. are highly infectious causes of diarrheal illness around the world. It is an important cause of diarrhea in children. Clinician and laboratories should be encouraged to include C. parvum diagnostic techniques while dealing with diarrheal stool samples of young children.

Deletion mutation in BSCL2 gene underlies congenital generalized lipodystrophy in a Pakistani family.

BackgroundCongenital generalized lipodystrophy (CGL) also known as Berardinelli-Seip Congenital Lipodystrophy (BSCL) is a genetically heterogeneous disorder characterized by loss of adipose tissues, Acanthosis nigricans, diabetes mellitus, muscular hypertrophy, hepatomegaly and hypertriglyceridemia. There are four subclinical phenotypes of CGL (CGL1-4) and mutations in four genes AGPAT2, BSCL2, CAV1 and PTRF have been assigned to each type.MethodsThe study included clinical and molecular investigations of CGL disease in a consanguineous Pakistani family. For mutation screening all the coding exons including splice junctions of AGPAT2, BSCL2, CAV1 and PTRF genes were PCR amplified and sequenced directly using an automated DNA sequencer ABI3730.ResultsSequence analysis revealed a single base pair deletion mutation (c.636delC; p.Tyr213ThrfsX20) in exon 5 of BSCL2 gene causing a frame shift and premature termination codon.ConclusionMutation identified here in BSCL2 gene causing congenital generalized lipodystrophy is the first report in Pakistani population. The patients exhibited characteristic features of generalized lipodystrophy, Acanthosis nigricans, diabetes mellitus and hypertrophic cardiomyopathy.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1913913076864247.

Cellular efflux transporters and the potential role of natural products in combating efflux mediated drug resistance

Efflux mediated multidrug resistance (MDR) is a major problem in the treatment of bacterial, fungal and protozoal infections in addition to cancer chemotherapy. Among other well known mechanisms, efflux pumps are significant contributors to chemo-resistance. Efflux mediated resistance generally occurs through up-regulation of genes responsible for the expression of transporter proteins extruding drugs from the cell to create intracellular sub-therapeutic concentrations leading to resistance. The rapid expansion of MDR pathogens necessitates the discovery of resistance modifying drugs, which in combination with antimicrobial or chemotherapeutic agents would tend to reinstate the action of these drugs and avert the emergence of acquired resistance. This review describes the existence of efflux pumps in prokaryotes and eukaryotes as well as their role in chemo-resistance with a special focus on natural product-derived efflux pump inhibitors.

New patterns of HCV subtypes distribution in the Khyber Pakhtunkhwa province of Pakistan

Hepatitis C virus (HCV) is the leading cause of liver diseases including cirrhosis and hepatocellular carcinoma and it affects an estimated 3% of the world’s population which corresponds to over 130 million HCV positive patients worldwide.1 HCV is an enveloped virus with a positive single stranded RNA genome in the family Flaviviridae. Due to its RNA genome, HCV shows substantial nucleotide sequence variability with nucleotide substitution rate of 1.44 × 10−3 per site/year resulting in evolution into seven major genotypes including multiple subtypes and quasispecies.2 RNA viruses evolve rapidly2 and changes in epidemiological patterns have been reported from different regions of the world. Several studies have reported HCV genotypes distribution in Khyber Pakhtunkhwa (KP), Pakistan,1,3,4 while none has so far reported the changing epidemiological pattern of HCV genotypes in the province. The aim of this study was to find out the frequency and existing pattern of HCV genotypes distribution in KP by a modified genotyping assay and sequencing. A total of 510 HCV infected patients who were registered at various healthcare units at Peshawar city for free treatment or diagnosis under the prime minister’s Hepatitis C control program were included in this study as per their consent and ethical approval of the Institutional committee. Qualitative detection of HCV and HCV genotyping was carried out by a modified reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the core gene was used for genotype authentication. Out of the total, active HCV infection was detected in 422 (82.75%) patients. HCV 3a (45.5%) was found to be the most abundant subtype followed by a high proportion of mixed genotypes infection (22.99%). HCV 1b accounted for infection among 11.61% of the patients while 3b was detected in 5.21% of the cases. Rare genotypes or subtypes encountered were 2a, 2b, and 1a. Among mixed genotypes, 3a/1b was the most prevalent (49%) followed by an equal distribution of 3a/3b and 1a/1b (15%). Similarly, 1b/3b, 1b/2a, and 1a/3b mixed genotypes were detected in 9%, 6%, and 4% of the patients, respectively. Previous studies have reported a much higher prevalence of HCV 3a (ranging from 70 to 90%) both in KP3,4 and Pakistan,5 while in this study only 45.5% of individuals were infected by 3.79% 3.32%

Antifungal susceptibility testing of vulvovaginal Candida species among women attending antenatal clinic in tertiary care hospitals of Peshawar

Background Vulvovaginal candidiasis (VVC) is considered as a pervasive gynecological problem among women worldwide. Owing to this fact, in the current study, we aimed at assessing the prevalence rate of Candida spp. causing VVC in symptomatic pregnant women and their antifungal susceptibility pattern. Methods This study was carried out in the tertiary care hospitals of Peshawar during the period of July 1, 2016 to December 31, 2016. The study group included 450 pregnant women in the age group of 17–44 years with symptoms of excessive vaginal discharge, pain and pruritis. In all, 108 pregnant women were culture positive for Candida. Antimicrobial susceptibility testing (AST) was conducted on specimens against various azoles and polyene F group of antifungals. Results Out of 108 Candida spp. isolated from vaginal swabs, there were 45 (41.7%) Candida albicans, 18 (16.7%) Candida tropicalis, 18 (16.7%) Candida krusei, 16 (14.8%) Candida glabrata and 11 (10.2%) Candida dubliniensis. According to age distribution, 27 years was the mean age. Pregnancy trimester distribution among patients was as follows: 21 (19.4%) patients were in their first trimester, 65 (60.2%) patients were in their second trimester and 22 (20.4%) patients were in the third trimester. Susceptibility of fluconazole was determined as follows: 33.3% of the Candida isolates were sensitive, 4.6% were susceptible dose dependent (SDD) and 62% were resistant. Susceptibility of Candida spp. with respect to nystatin in patients with VVC was as follows: 25% were sensitive, 16.7% were SDD and 58.3% were resistant. Susceptibility of clotrimazole was analyzed, and it was sensitive in 21.3% of patients, SDD in 19.4% of patients and resistant in 59.3% of patients. Voriconazole susceptibility was recorded to be sensitive in 85.2% of patients, SDD in 4.6% of patients and resistant in 10.2% of patients suffering from VVC. Susceptibility results for itraconazole in patients with VVC were as follows: 42.6% of patients were sensitive, 16.7% of patients were SDD, and 40.7% of patients were resistant. Conclusion In this study, frequency of VVC was noted to be high in the second trimester of pregnancy, with the highest frequency of C. albicans isolated, followed by C. tropicalis and C. krusei. Antifungal susceptibility testing revealed that fluconazole was exceedingly resistant against Candida species (62%), followed by clotrimazole (59.3%) and nystatin (58.3%). On the contrary, voriconazole had the highest antimicrobial activity against Candida species (85.2%).

Biallelic variants in LINGO1 are associated with autosomal recessive intellectual disability, microcephaly, speech and motor delay

PurposeTo elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability.MethodsA combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California–San Francisco Chimera software.ResultsAll five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein.ConclusionLINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.

Whole Genome Sequencing instead of Whole Exome Sequencing is required to identify the Genetic Causes of Polycystic Ovary Syndrome in Pakistani families

Background & Objective: Polycystic Ovary Syndrome (PCOS) is the major cause of infertility in females. PCOS is a complex and multifactorial disease, genetic and environmental factors being important predisposing factors. Diagnosis of PCOS is difficult due to the complexity of this disease; hence, better diagnostic tests are required to improve its management. Aim of the study was to elucidate the genetic causes of PCOS in three Pakistani families. Methods: Three Pakistani families segregating PCOS in an apparently autosomal recessive mode were recruited. Whole genome Single Nucleotide Polymorphism (SNP) genotyping and Whole Exome Sequencing (WES) were carried out to identify the candidate genes. Results: SNP genotypes data analyses identified multiple regions of homozygosity on different chromosomes. WES was performed in affected members of the family. Screening for pathogenic mutations in homozygous regions failed to detect any mutation/variant of interest. Conclusion: PCOS is multifactorial and complex disease so variants in the coding as well as in non-coding regions may be the genetic causes of the disease. To elucidate the genetic cause(s) of the PCOS, Whole Genome Sequencing (WGS) is recommended to cover both coding and non-coding regions of the genome.

Expression analysis of cyclooxygenase-2 in patients suffering from esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the aggressive malignancies and mechanisms underlying its pathogenesis remain unclear. Cyclooxygenase-2 (COX-2) enzyme system plays a crucial role in many gastrointestinal malignancies and is an important regulator of cell growth, proliferation, apoptosis, differentiation and transformation. More precise outcome of COX-2 in ESCC is less investigated. In this study we investigated the risk factors of ESCC and expression of COX-2 in Carcinoma in situ (CIS) and ESCC compared to normal esophageal mucosa. ESCC relationship to clinico-pathological parameters using immunohistochemistry was also part of this investigation. Current study was conducted in the Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. A total of 69 diagnosed patients of ESCC, both Pakistanis and Afghans were enrolled. Various risk factors associated with ESCC were recorded. Mean age at the time of diagnosis was 55 years. Out of 69 patients of ESCC 46 (67%) were users of dipping tobacco (Naswar). Expression of COX-2 was determined in normal esophageal mucosa, CIS and invasive ESCC using Immunohistochemistry (IHC). Differences of mean were computed using ANOVA followed by applying Post Hoc test. Patients were categorized as positive with high expression or negative with low to nil expression. ANOVA showed large differences in expression of COX-2 in normal healthy mucosa compared with CIS and ESCC with the mean difference of -9.529 and -7.370 respectively, p-value being <.05 at 95% confidence interval (CI). No significant difference was noticed in the expression of COX-2 in CIS compared with ESCC with p-value >.05 at 95% CI. Our complete cohort (23–85 years) showed statistically significant difference in the expression of COX-2 gene in ESCC and CIS tissue samples compared with normal healthy mucosa. Results of this study indicate that over-expression of COX-2 is positively associated with ESCC.