Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Exome sequencing identifies homozygous variants in MBOAT7 associated with neurodevelopmental disorder.

Intellectual disability (ID) is a large group of neurodevelopmental disorders characterized by a congenital limitation in intellectual functioning (reasoning, learning, and problem solving), adaptive behavior (conceptual, social, and practical skills), originated at birth and manifested before the age of 18. By whole exome sequencing of five consanguineous Pakistani families presenting hallmark features of ID, global developmental delay, aggressive and self-injurious behaviors, microcephaly, febrile seizures and facial dysmorphic features, we identified three novel homozygous missense variants (NM_024298.5: c.588G > T; p.Trp196Cys, c.736 T > C; p.Tyr246His and c.524A > C; p. Asp175Ala) and one rare homozygous in-frame deletion variant (c.758_778del;p.Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7) gene previously associated with autosomal recessive neurodevelopmental disorder. The segregation of the variants was validated by Sanger sequencing in all family members. In silico homology modeling of wild-type and mutated proteins revealed substantial changes in the structure of both proteins, indicating a possible effect on function. The identification and validation of new pathogenic MBOAT7 variants in five cases of autosomal recessive ID further highlight the importance of this genes in proper brain function and development.

Cystic echinococcosis: an emerging zoonosis in southern regions of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cystic echinococcosis (CE) is one of the principal causes of economic loss to the livestock industry because of its morbidity and mortality of food-producing animals and condemnation of important visceral organs. Pakistan being an agricultural country having an extensive livestock sector, is mostly practiced by poor people, which has a fundamental role in the economy. The present study was aimed to conduct a cross-sectional survey and PCR based confirmation of Echinococcus granulosus in sheep, goats, cows, and buffaloes from southern regions (three districts: Lakki Marwat, Bannu, and Karak) of Khyber Pakhtunkhwa, Pakistan. During the study, a total of 2833 animals were examined randomly including; sheep (n = 529), goats (n = 428), cows (n = 1693), and buffaloes (n = 183). Hydatid cysts were collected and examined for the presence of protoscoleces using microscopy. Detection of DNA was performed by using PCR and two mitochondrial genetic markers namely; NAD-1 and COX-1 were amplified. RESULTS: The overall prevalence of CE was found to be (9%) among the examined animals. The hydatid cyst infection was highly prevalent in buffaloes (12%), followed by sheep (10%), cows (9%), and goats (5.1%). Cystic echinococcosis was more prevalent (10%; 96/992) in district Lakki Marwat followed by district Bannu (9%; 112/1246) and Karak (7%; 39/595). Female animals were more likely to be infected with CE (11.6%) than male animals (5.3%) (p = 0.001). Similarly, the infection was higher in the older group of animals as compared to younger (p = 0.001). Mostly (52.2%; n = 129) of hydatid cysts were found in the liver, while (64.4%; n = 159) cysts of the infected animals were infertile. PCR based identification confirmed the presence of E. granulosus sensu stricto (s.s) in the study area. CONCLUSION: Cystic echinococcosis was found to be highly prevalent in southern regions of Khyber Pakhtunkhwa and could be a potential threat to human health. Moreover, molecular sequencing and phylogenetic analyses should be carried out in future to identify the prevailing genotype (s) of E. granulosus s.s.

Glycyrrhiza glabra HPLC fractions: identification of Aldehydo Isoophiopogonone and Liquirtigenin having activity against multidrug resistant bacteria.

BACKGROUND: Medicinal plants have been founded as traditional herbal medicine worldwide. Most of the plant's therapeutic properties are due to the presence of secondary metabolites such as alkaloids, glycosides, tannins and volatile oil. METHODS: The present investigation analyzed the High-Pressure Liquid Chromatography (HPLC) fractions of Glycyrrhiza glabra (Aqueous, Chloroform, Ethanol and Hexane) against multidrug resistant human bacterial pathogens (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa). All the fractions showed antibacterial activity, were subjected to LC MS/MS analysis for identification of bioactive compounds. RESULTS: Among total HPLC fractions of G. glabra (n = 20), three HPLC fractions showed potential activity against multidrug resistant (MDR) bacterial isolates. Fraction 1 (F1) of aqueous extracts, showed activity against A. baumannii (15 ± 0.5 mm). F4 from hexane extract of G. glabra showed activity against S. aureus (10 ± 0.2 mm). However, F2 from ethanol extract exhibited activity against S. aureus (10 ± 0.3 mm). These active fractions were further processed by LC MS/MS analysis for the identification of compounds. Ellagic acid was identified in the F1 of aqueous extract while 6-aldehydo-isoophiopogonone was present in F4 of hexane extract. Similarly, Liquirtigenin was identified in F2 of ethanol. CONCLUSIONS: Glycyrrhiza glabra extracts HPLC fractions showed anti-MDR activity. Three bioactive compounds were identified in the study. 6-aldehydo-isoophiopogonone and Liquirtigenin were for the first time reported in G. glabra. Further characterization of the identified compounds will be helpful for possible therapeutic uses against infectious diseases caused by multidrug resistant bacteria.

Baseline characteristics of infected foot ulcers in patients with diabetes at a tertiary care hospital in Pakistan.

OBJECTIVE:: Studies on diabetic foot ulcers (DFU) involving a representative sample of patients in Pakistan are scarce. This study aimed to determine baseline characteristics of infected DFUs in patients hospitalised at the Pakistan Institute of Medical Sciences Islamabad. METHOD:: In this cross-sectional study, carried out during May 2015 and June 2016, foot ulcer characteristics of patients with DFUs were investigated and documented. From infected DFUs, aerobic bacterial pathogens were isolated, identified and evaluated for antimicrobial susceptibility. RESULTS:: A total of 214 patients were recruited to the study, 62.6% of which were male, 90.2% were aged ≥40 years, 76.2% had type 1 diabetes and 78.5% had poor glycaemic control at time of presentation to hospital. Most patients had grade 3/moderate ulceration (based on the Wagner and International Working Group on the Diabetic Foot/Infectious Diseases Society of America criteria). Over half of the DFUs (57.9%) were of ≤3 months' duration and 70.1% were ≥3 cm2. Of the patients with deep infection grade ulcers, 26.6% underwent amputation, accounting for their prolonged hospital stay (≥20 days). Significant differences were observed between patients with type 1 and type 2 diabetes with DFUs in relation to gender (p≤0.0001), ulcer size (p=0.0421) and duration of hospital stay (p=0.0253). The most significant predictors for lower extremity amputation were osteomyelitis (p=0.0114), retinopathy (p=0.0001) and neuropathy (p=0.0001. Piperacillin/tazobactam was found to be an effective antibiotic against the most commonly isolated Staphylococcus non-aureus (35.48%), Pseudomonas aeruginosa (22.26%), and Staphylococcus aureus (20.96%) species indentified in the DFU infections. CONCLUSION:: The findings of this study may be helpful in the optimal management and appropriate treatment of patients with infected DFUs.

Review: Ethnomedicinal, phytochemical and antibacterial activities of medicinal flora of Pakistan used against Pseudomonas aeruginosa-A Review.

Medicinal plants have been used from ancient time against different infectious diseases caused by microorganisms across the globe. The present review represents different medicinal plants of Pakistan used traditionally for the treatment of variety of ailments caused by Pseudomonas aeruginosa, their in-vitro activities against P. aeruginosa and phytochemistry. These plants were extracted with different solvents that showed good in-vitro activities against P. aeruginosa, due to the presence of active phytoconstituents including alkaloids, terpenoids etc. Among all the solvents used for extraction process, alcoholic extracts were mostly preferred in Pakistan. However, non-alcoholic solvents like ethyl acetate and chloroform also showed good anti-P. aeruginosa activities. Statistically, increase in concentration (mg/ml) of ethyl acetate and chloroform extracts significantly increase (p=0.000 and p= 0.046) inhibitory activity against P. aeruginosa. This review provides scientific validation of the traditional knowledge in using medicinal plants for the treatment of different diseases caused by this bacterium. Reported Pakistani medicinal plants contain variety of phytochemical compounds that could be very useful in the production of new drugs with fewer side effects on living system compared to some allopathic drugs. This review also provides baseline information for future research studies on the phytochemistry of unexplored plants. Further research studies should be carried out on non-alcoholic extracts that could be helpful in the extraction new compounds, which could lead to the development of some novel drugs in the pharmaceutical industries of Pakistan.

Molecular characterization and growth optimization of halo-tolerant protease producing Bacillus Subtilis Strain BLK-1.5 isolated from salt mines of Karak, Pakistan.

Microbial proteolytic enzyme is one of the most important industrial enzymes that hydrolyze proteins. The applications of proteases under harsh industrial conditions like alkalinity, salinity, and temperature make them inactive and unstable. This suggests need for search for novel microbial sources for protease production having diverse properties. For this purpose, 54 bacterial strains were isolated from different salt mines of Karak, Pakistan and were investigated for their proteolytic activity on skim milk agar plates. The strain which showed maximum protease activity was characterized by 16S rRNA gene sequence analysis. Furthermore, growth and protease production was optimized for the characterized bacteria under different physical factors, i.e., pH, temperature and salinity. The isolate BLK-1.5 exhibited strong protease production and was identified as Bacillus subtilis based on biochemical characteristics and 16S rRNA gene sequence analysis. Maximum production of protease was recorded at pH 10, 37 °C and 7 % (w/v) NaCl. Molecular weight of proteases was estimated 38 kDa and its optimum activity was observed at pH 10, 50 °C and 2 % (w/v) NaCl. In conclusion, the protease produced by halo-tolerant Bacillus subtilis strain BLK-1.5 has diverse characteristics and could be useful in various industrial applications.

PCR/RFLP-based analysis of genetically distinct Plasmodium vivax population of Pvmsp-3α and Pvmsp-3ß genes in Pakistan.

BACKGROUND: Plasmodium vivax is one of the widespread human malarial parasites accounting for 75% of malaria epidemics. However, there is no baseline information about the status and nature of genetic variation of Plasmodium species circulating in various parts of Pakistan. The present study was aimed at observing the molecular epidemiology and genetic variation of Plasmodium vivax by analysing its merozoite surface protein-3α (msp-3α) and merozoite surface protein-3ß (msp-3ß) genes, by using suballele, species-specific, combined nested PCR/RFLP detection techniques. METHODS: A total of 230 blood samples from suspected subjects tested slide positive for vivax malaria were collected from Punjab, Sindh, Khyber Pakhtunkhwa, and Balochistan during the period May 2012 to December 2013. Combined nested PCR/RFLP technique was conducted using Pvmsp-3α and Pvmsp-3ß genetic markers to detect extent of genetic variation in clinical isolates of P. vivax in the studied areas of Pakistan. RESULTS: By PCR, P. vivax, 202/230 (87.82%), was found to be widely distributed in the studied areas. PCR/RFLP analysis showed a high range of allelic variations for both msp-3α and msp-3ß genetic markers of P. vivax, i.e., 21 alleles for msp-3α and 19 for msp-3ß. Statistically a significant difference (p ≤ 0.05) was observed in the genetic diversity of the suballelic variants of msp-3α and msp-3ß genes of P. vivax. CONCLUSION: It is concluded that P. vivax populations are highly polymorphic and diverse allelic variants of Pvmsp-3α and Pvmsp-3ß are present in Pakistan.

Incidence of Helminthic and Viral Coinfections in Malaria Patients in the Tertiary Care Hospital Setup.

Introduction: This study determines the incidence of common viral and helminth coinfections with malaria in the tertiary care hospital set up in southern Khyber Pakhtunkhwa, Pakistan. Materials and Methods: The multidimensional research included malaria patients admitted to different hospitals of district Kohat during January and December 2021. Stool samples and blood were assembled from the patients. Giemsa-stained microscopy-positive samples were processed by the immunochromatography technique (ICT) to identify Plasmodium species. Common viral infections such as viral hepatitis (A, B, and C), HIV, and dengue (DENV) were analyzed by ICT kits while SARS-CoV-2 was confirmed through real-time PCR. Furthermore, the intestinal helminths were identified using the Kato-Katz thick smear method. Results: Among 1278 patients, 548 were diagnosed with malaria, 412 (75.2%) were positive for P. vivax infection, 115 (21%) for P. falciparum, and 21 (3.8%) for mixed malaria infection (P. vivax/P. falciparum), with a higher incidence among males (65.2%) than females (34.8%). Coinfection with helminths was positive in 215 (39.3%) malaria patients. The most common infections were caused by the Ascaris lumbricoides species (42.6%) followed by Enterobius vermicularis (31.7%) and hookworm. A total of 24.6% of malaria-positive cases were also coinfected with different viruses with higher frequencies of confection for HAV (8.2%) and DENV (6.2%), respectively. The patients revealed higher incidence of coinfections with P. falciparum (57%) as compared with P. vivax (39.2%) and mixed infections (3.7%). Conclusion: This study demonstrated that the study population exhibited a significant incidence of coinfections with intestinal helminth and viral malaria.

Consanguineous marriages increase the incidence of recurrent tuberculosis: Evidence from whole exome sequencing.

BACKGROUND: In this study, we have identified multiple mutations in the IL-12R1 gene among Pakistani patients who have inherited them through consanguineous marriages. These patients have experienced severe Bacille-Calmette-Guérin (BCG) infection as well as recurrent tuberculosis. We will demonstrate the pivotal role of interleukin (IL)-12/interferon (IFN)-γ axis in the regulation of mycobacterial diseases. METHODOLOGY: First, we checked the patients' medical records, and then afterward, we assessed interferon-gamma (IFN-γ) production through ELISA. Following that, DNA was extracted to investigate IL-12/IFN- abnormalities. Whole exome sequencing was conducted through Sanger sequencing. Secretory cytokine levels were compared from healthy control of the same age groups and they were found to be considerably less in the disease cohort. To evaluate the probable functional impact of these alterations, an in silico study was performed. RESULTS: The study found that the patients' PBMCs produced considerably less IFN-γ than expected. Analysis using flow cytometry showed that activated T cells lacked surface expression of IL-12Rß1. Exon 7 of the IL-12Rß1 gene, which encodes a portion of the cytokine binding region (CBR), and exon 10, which encodes the fibronectin-type III (FNIII) domain, were found to have the mutations c.641 A > G; p.Q214R and c.1094 T > C; p.M365T, respectively. In silico analysis showed that these mutations likely to have a deleterious effect on protein function. CONCLUSION: Our findings indicate the significant contribution of the IL-12/IFN-γ is in combating infections due to mycobacterium. Among Pakistani patients born to consanguineous marriages, the identified mutations in the IL-12Rß-1 gene provide insights into the genetic basis of severe BCG infections and recurrent tuberculosis. The study highlights the potential utility of newborn screening in regions with mandatory BCG vaccination, enabling early detection and intervention for primary immunodeficiencies associated with mycobacterial infections. Moreover, the study suggests at the potential role of other related genes such as IL-23Rß1, TYK2, or JAK2 in IFN-γ production, warranting further investigation.

Biallelic variants in CSMD1 are implicated in a neurodevelopmental disorder with intellectual disability and variable cortical malformations.

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade, an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders, the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing, we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay, intellectual disability, microcephaly, and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary, we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder.

Novel mutations in CYBB Gene Cause X-linked chronic Granulomatous Disease in Pakistani patients.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a primary immunodeficiency that causes susceptibility to recurrent fungal and bacterial infections. The CYBB gene encodes gp91phox component of the Phagocytic Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and specifically, X-linked CGD is caused by mutations in the CYBB gene, located on the X chromosome. The aim of the study was to characterize functional and genetic mutations in X-linked CGD. METHODS: Functional analysis was conducted on the whole blood of seventeen male individuals who were suspected to have X-linked chronic granulomatous disease (CGD). Flow cytometry was employed to assess the capacity of NADPH oxidase, measuring both H2O2 production and gp91phox protein expression in neutrophils. Additionally, DNA Sanger sequencing was performed for genetic analysis. The pathogenicity of novel mutations was assessed by pathogenicity prediction tools. RESULT: Among the seventeen patients evaluated, five patients (P1, P2, P3, P4, and P5) displayed impaired H2O2 production by their neutrophils upon stimulation with Phorbol myristate acetate (PMA), accompanied by abnormal gp91phox expression. DNA sequencing of the CYBB gene identified specific mutations in each patient. In P1 and P2 (previously reported cases), a hemizygous missense mutation, c.925G > A/p.E309K was identified. In P3 and P4 (novel cases), hemizygous nonsense mutations, c.216T > A/p.C72X were found. Lastly, in P5 (also a novel case), a hemizygous missense mutation, c.732T > G/p.C244W was detected. These mutations reside in exons 9,3 and 7 of the CYBB gene, respectively. CONCLUSIONS: The current study contributes to the understanding of the clinical and genetic spectrum associated with X-linked chronic granulomatous disease (CGD). It highlights the significance of early diagnosis in CGD and emphasizes the importance of lifelong prophylaxis to prevent severe infections.

Aurora kinase-C-T191D is constitutively active mutant.

BACKGROUND: Aurora kinases (Aurora-A, B and C) belong to a family of conserved serine/threonine kinases which are key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved in cell cycle regulation while aurora-C is meiotic chromosome passenger protein. As Aurora kinase C is rarely expressed in normal somatic cells and has been found over expressed in many cancer lines. It is suggested that Aurora-C-T191D is not hyperactive mutant. RESULT: Aurora-C-T191D variant form was investigated and compared with wild type. The overexpression of Aurora-C-T191D was observed that it behaves like Aurora-C wild type (aurC-WT). Both Aurora-C-T191D and aurC-WT induce abnormal cell division resulting in centrosome amplification and multinucleation in transiently transfected cells as well as in stable cell lines. Similarly, Aurora-C-T191D and aurC-WT formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3 T3 stable cell lines overexpressing Aurora-C-T191D and its wild type partner induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumour aggressiveness was positively correlated with the rate of kinase activity, making Aurora-C a potential anti-cancer therapeutic target. CONCLUSION: These findings proved that Aurora C-T191D is not hyperactive but is constitutively active mutant.

Knowledge, attitude, and practices towards cutaneous leishmaniasis in referral cases with cutaneous lesions: A cross-sectional survey in remote districts of southern Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cutaneous leishmaniasis is a neglected tropical disease caused by Leishmania spp. and transmitted by female sandflies. Terrorism and counter-insurgency military operations in Federally Administered Tribal Areas (FATA) lead to a large-scale migration of internally displaced persons (IDPs) in Khyber Pakhtunkhwa and thus, new outbreaks of several infectious diseases such as cutaneous leishmaniasis occurred. This study intended to find the prevalence of cutaneous leishmaniasis in people with cutaneous lesions suspected of having cutaneous leishmaniasis in four remote districts of Khyber Pakhtunkhwa and to assess the participant's knowledge, attitude, and practices about the infection and its control. METHODS: A cross-sectional study was carried out in four remote districts of Khyber Pakhtunkhwa including Karak, Lakki Marwat, Tank, and Dera Ismail Khan (D. I. Khan) and a total of 1,674 participants were recruited using a convenience sampling technique. RESULTS: The prevalence of cutaneous leishmaniasis among the participants with cutaneous lesions was 50.4% and the infection was comparatively more prevalent in district Karak. Among participants, 56.8% were male and mostly, 53.8% were under the age of 16 years with 52.8% living in kutcha houses and were from rural areas. Multiple skin lesions were more common, and the face was frequently affected body part. The ratio of participants with lesions older than a month was higher and the majority confronted infections with blood protozoan parasites for the first time. Most participants were unaware of the signs/symptoms of the disease, basic knowledge of the vectors, anthroponotic spread, preventive measures, secondary infections, and reservoir hosts. The use of wood/animal dung as fuel, closeness with reservoir animals, and no use of insect repellents were some of the notable risk factors. CONCLUSION: Cutaneous leishmaniasis is highly prevalent in the study area and a very low level of awareness was reported among the participants. This study necessitates the planning and execution of regulations and preventive programs, public health education, awareness campaigns, and disease management practices to overcome future incidence of cutaneous leishmaniasis.

Analysis of associated risk factors among recurrent cutaneous leishmaniasis patients: A cross-sectional study in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Leishmaniasis is the second and fourth highest cause of mortality and morbidity respectively among all tropical diseases. Recurrence in the onset of leishmaniasis is a major problem that needs to be addressed to reduce the case fatality rate and ensure timely clinical intervention. Here we are investigating the association of risk factors with recurrent cutaneous leishmaniasis to address this issue. MATERIAL AND METHODS: Patients received by Nasser Ullah Khan Babar Hospital in Peshawar, Pakistan from March 2019 to July 2020 were enrolled in this study. Those patients who developed symptoms after completion of treatment were included in Group-A while those who had atypical scars like leishmaniasis but were negative for cutaneous leishmaniasis were included in the comparison group tagged as Group B. All those individuals who had completed six weeks of treatment for CL but had normal complete blood counts (CBC) were included to avoid other underlying immunological pathologies, while we excluded those participants who had co-morbidities like diabetes, liver disease, cardiac disease, and pregnant and lactating women through their history Association was tested between Group-A and Group-B with other explanatory variables through chi-square test. The regression model was proposed to determine the predictors. RESULT: A total of 48 participants of both sexes were included in the study with a mean age of 32.2 ± 15.10. The data suggest that females are overrepresented among the patients with recurrent leishmaniasis [21(53.8 %,); p = 0.07]. Compared to patients; healthy participants had a higher proportion of adults (19-59 years) versus adolescents (13-18 years) [26(66.7 %) vs 07(17.9), p = 0.004]. Multivariate logistic regression analysis shows that females are 2.1 times more prone to infections among cases as compared to healthy individuals [unadjusted OR 2.20, 95 % confidence interval (CI) 1.5-10.6, p = 0.02; adjusted OR 2.1, 95 % CI 1.50-10.69, p = 0.02]. We propose that patients receiving intradermal were less likely to be infected as compared to those receiving intralesional injections [unadjusted OR 0.07.0, 95 % confidence interval (CI) 1.18-3.37, p = 0.03; adjusted OR 0.06, 95 % CI 1.18-3.38, p = 0.03]. CONCLUSION: Old age (adults) and sex (females) were the strongest predictors to be associated with recurrent leishmaniasis. Similarly, the choice of intradermal as compared to intralesional injection and the prolonged treatment duration were strongly associated with greater chances of recurrence.

Phyto-Extract-Mediated Synthesis of Silver Nanoparticles (AgNPs) and Their Biological Activities.

Background: Nanotechnology finds broad applications in the field of nanomedicine, an emerging new field used for diagnosis, treatment, prevention of diseases, and improvement of health. Objectives: To synthesize silver nanoparticles (AgNPs) from Withania somnifera and Fagonia indica and to carry out their antimicrobial, insecticidal, and phytotoxic activities, a step toward the new range of nanomedicines. Methods: Silver nanoparticles were synthesized from Withania somnifera and Fagonia indica by chemical reduction method, and further biological activities of these nanoparticles were compared with crude methanolic extract, prepared through cold maceration process, at the concentration of 50 mg/ml. Results: Among all tested bacterial pathogens, crude extract of W. somnifera showed a statistically high significant inhibition zone in millimeter against Pseudomonas aeruginosa (21; p < 0.01). AgNPs showed highly significant result against Streptococcus pneumonia (14; p < 0.01). In comparison with crude extracts, AgNPs showed statistically significant (p < 0.01) results against S. pneumonia (AgNPs, 14; crude, 8.33 mm). Crude extract showed significant inhibition zone against two bacterial strains, P. aeruginosa (crude, 21; AgNPs, 11.67 mm) and Klebsiella pneumoniae (crude, 11.33; AgNPs, 8 mm). Crude extracts of F. indica showed the significant activity against Vibrio cholera (p < 0.01; 11.33 mm). Silver nanoparticles of F. indica exhibited the highest significant activity against Aspergillus flavus and Fusarium oxysporum while AgNPs of W. somnifera were active only against A. flavus. Extracts of W. somnifera and F. indica showed increasing phytotoxic activity with increasing concentrations. The highest significant inhibition was obtained for crude extract (46.7) and AgNPs (45.7) of F. indica at 1000 µg/ml. Insecticidal activity of crude and AgNPs of both plants showed significant inhibition against all tested insects with increasing time intervals, and the highest significant result was obtained at 72 hours with a value of p < 0.01 except T. castaneum. Conclusions: Both crude and AgNPs showed potent activity; however, in comparison, silver nanoparticles showed slightly enhanced activity. Crude and AgNPs of both plants showed good phytotoxic and insecticidal inhibition. Antimicrobial studies of AgNPs on diseases causing pathogens open a door for new antimicrobial agents and could be the answer to antibiotic resistance after further analysis.

SPTBN5, Encoding the ßV-Spectrin Protein, Leads to a Syndrome of Intellectual Disability, Developmental Delay, and Seizures.

Whole exome sequencing has provided significant opportunities to discover novel candidate genes for intellectual disability and autism spectrum disorders. Variants in the spectrin genes SPTAN1, SPTBN1, SPTBN2, and SPTBN4 have been associated with neurological disorders; however, SPTBN5 gene-variants have not been associated with any human disorder. This is the first report that associates SPTBN5 gene variants (ENSG00000137877: c.266A>C; p.His89Pro, c.9784G>A; p.Glu3262Lys, c.933C>G; p.Tyr311Ter, and c.8809A>T; p.Asn2937Tyr) causing neurodevelopmental phenotypes in four different families. The SPTBN5-associated clinical traits in our patients include intellectual disability (mild to severe), aggressive tendencies, accompanied by variable features such as craniofacial and physical dysmorphisms, autistic behavior, and gastroesophageal reflux. We also provide a review of the existing literature related to other spectrin genes, which highlights clinical features partially overlapping with SPTBN5.

Prevalence of HBV and HBV vaccination coverage in health care workers of tertiary hospitals of Peshawar, Pakistan.

BACKGROUND: Hepatitis B Virus (HBV) may progress to serious consequences and increase dramatically beyond endemic dimensions that transmits to or from health care workers (HCWs) during routine investigation in their work places. Basic aim of this study was to canvass the safety of HCWs and determine the prevalence of HBV and its possible association with occupational and non-occupational risk factors. Hepatitis B vaccination coverage level and main barriers to vaccination were also taken in account. RESULTS: A total of 824 health care workers were randomly selected from three major hospitals of Peshawar, Khyber Pakhtunkhwa. Blood samples were analyzed in Department of Zoology, Kohat University of Science and Technology Kohat, and relevant information was obtained by means of preset questionnaire. HCWs in the studied hospitals showed 2.18% prevalence of positive HBV. Nurses and technicians were more prone to occupational exposure and to HBV infection. There was significant difference between vaccinated and non-vaccinated HCWs as well as between the doctors and all other categories. Barriers to complete vaccination, in spite of good knowledge of subjects in this regard were work pressure (39.8%), negligence (38.8%) un-affordability (20.9%), and unavailability (0.5%). CONCLUSIONS: Special preventive measures (universal precaution and vaccination), which are fundamental way to protect HCW against HBV infection should be adopted.

Prevalence of hepatitis B virus genotypes in HBsAg positive individuals of Afghanistan.

BACKGROUND: The structural and functional differences between hepatitis B virus (HBV) genotypes are the mainstay to severity, complications, treatment and possibly vaccination against the virus. This study was conducted to determine the HBV genotypes in HBsAg positive patients of Afghanistan as no such large scale data available previously. METHODS: Two hundred and fourteen HBsAg-positive patients were included in this study. All patients were anti-HCV and anti-HIV negative. All the samples were confirmed for HBV DNA with nested PCR while HBV DNA positive samples were subjected to type specific PCR for HBV genotyping (A-F). RESULTS: Of the total samples, 168 (78.5%) were males and 46 (21.49%) females, aged ranged between 18 to 71 years. This study demonstrated that genotype D (35.67%) is the predominant genotype circulating in Afghani's population. Genotype C was observed in 32.16% followed by genotype A (19.30%), and genotype B (7.02%) while 6.07% of the individuals were not typed. CONCLUSION: This study has shown a heterogeneous distribution of HBV genotypes. Further more, extensive studies are required to investigate genetic and geographical divergence and characteristics of the virus in the country, as no such large sample sized study has been carried out so far in this country.

Rising burden of Hepatitis C Virus in hemodialysis patients.

AIM: High prevalence of Hepatitis C virus (HCV) has been reported among the dialysis patients throughout the world. No serious efforts were taken to investigate HCV in patients undergoing hemodialysis (HD) treatment who are at great increased risk to HCV. HCV genotypes are important in the study of epidemiology, pathogenesis and reaction to antiviral therapy. This study was performed to investigate the prevalence of active HCV infection, HCV genotypes and to assess risk factors associated with HCV genotype infection in HD patients of Khyber Pakhtunkhwa as well as comparing this prevalence data with past studies in Pakistan. METHODS: Polymerase chain reaction was performed for HCV RNA detection and genotyping in 384 HD patients. The data obtained was compared with available past studies from Pakistan. RESULTS: Anti HCV antibodies were observed in 112 (29.2%), of whom 90 (80.4%) were HCV RNA positive. In rest of the anti HCV negative patients, HCV RNA was detected in 16 (5.9%) patients. The dominant HCV genotypes in HCV infected HD patients were found to be 3a (n = 36), 3b (n = 20), 1a (n = 16), 2a (n = 10), 2b (n = 2), 1b (n = 4), 4a (n = 2), untypeable (n = 10) and mixed (n = 12) genotype. CONCLUSION: This study suggesting that i) the prevalence of HCV does not differentiate between past and present infection and continued to be elevated ii) HD patients may be a risk for HCV due to the involvement of multiple routes of infections especially poor blood screening of transfused blood and low standard of dialysis procedures in Pakistan and iii) need to apply infection control practice.