Effectiveness Of Pcr Pool Testing For Screening Of Covid-19 Infection In Pakistan.

BACKGROUND: We tested the utility of mini-pool PCR testing for the rational use of PCR consumables in screening for CoViD-19. METHODS: After pilot experiments, 3-samples pool size was selected. One step RT-PCR was performed. The samples in the mini-pool having COVID gene amplification were tested individually. RESULTS: 1548 samples tested in 516 mini-pools resulted 396 mini-pools as negative and 120 as positive. Upon individual testing, 110 samples tested positive and 9 were inconclusive. 876 PCR reactions were performed to test 1548 samples, saving 43% PCR reagents. Centres with low prevalence resulted in most saving on reagents (50%), while centres with high prevalence resulted in more test reactions. Testing of individual samples resulted in delays in reporting. CONCLUSIONS: Pooling can increase lab capacity, however, pooling delays results and cause degradation of samples.

Antidiabetic and pancreas-protective potential of Parthenium Hysterophorous (Carrot grass) in Alloxan induced diabetic rabbits.

Parthenium hysterophorus has been used to cure cancer, fever, malaria, diarrhea, dysentery, and neurologic disorders. This study evaluates the anti-diabetic effects of methanolic extract of P. hysterophorus (MEPH) in alloxan-induced diabetic rabbits. Twenty-five rabbits were divided into 5groups (N=5). Group-I served as a negative control. Groups II to V were injected with freshly prepared alloxan solution 150 mg/kg intraperitoneally to induce diabetes. Group II till V received following treatments orally: Group II: Alloxan 150 mg/kg alone; group III: Alloxan + MEPH (50 mg/kg); group IV: Alloxan + MEPH (100 mg/kg); group V: Alloxan +Glucophage (62.5 mg/kg), respectively for 10 days. The body weight of all animals was recorded on the 1st, 4th, 7th and 10th days. Short-term (1st, 3rd, 5th and 7th hour) and long-term (4th, 7th and 10th day) hypoglycemic effects were also recorded. All animals were sacrificed on the 10th day to isolate the pancreas for histopathological examination. The results showed that MPEH reduced the blood glucose levels in all the groups of alloxan-induced diabetic rabbits. The histopathological studies depicted that 100 mg/kg of MEPH most effectively repaired alloxan-induced pancreatic damage. The study showed that the MPEH is useful for developing effective phytomedicine to treat diabetes mellitus.

Nephrocurative Effect of Parthenium hysterophorus (Carrot Grass) in Paracetamol Induced Nephrotoxicity in Rabbits.

Parthenium hysterophorus is conventionally used to treat urinary tract infections, joint pain, and hyperglycaemia. This study evaluates the reno-curative effects of methanolic extract of P. hysterophorus (MEPH) in paracetamol-induced nephrotoxic rabbits. Thirty male rabbits were divided into V groups: Group I served as the negative control. Group-II to V were treated with 2 g/kg of paracetamol to induce nephrotoxicity. Group II served as paracetamol (PCM) intoxicated control. Group III till V were fed orally with the following treatments: III paracetamol (PCM) 40 mg/kg MEPH; IV PCM+80 mg/kg MEPH; V PCM+Cystone (5 ml/kg), respectively, for 14 days. The body weight of all animals was recorded on days 1, 7 and 14. All the animals were dissected on the 14th day and blood, urine and kidneys were collected. The results showed that P. hysterophorus had no effect on body weight but lowered urea and creatinine levels and brought urine parameters back to the normal range in experimental groups of PCM-induced nephrotoxic rabbits. The 80 mg/kg dose of MEPH reduced urea and creatinine levels and normalized urine parameters more effectively compared to low doses of MEPH and the standard drug, i.e., Cystone. Kidney histopathological studies exhibited that 80 mg/kg of MEPH repaired paracetamol-induced renal damage, whereas Cystone only provided reno protection as no repair in damaged tissue was investigated in histopathology of Cystone treated animals. The results suggested that P. hysterophorus exhibited significant reno-curative activity on paracetamol-induced nephrotoxic rabbits.

Haploinsufficiency of EXT1 and Heparan Sulphate Deficiency Associated with Hereditary Multiple Exostoses in a Pakistani Family.

Background and Objectives: Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes (EXT1 and EXT2) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods: Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results: The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion (c.247Cdel) in exon 1 of EXT1, segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion: Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis.

Detection of blaNDM-1 gene in ESBL producing Escherichia coli and Klebsiella pneumoniae isolated from urine samples.

INTRODUCTION: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. METHODOLOGY: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumß-lactamase (ESBL), carbapenemase and metallo ß-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. RESULTS: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene. CONCLUSIONS: The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population.

Variations in Mineral/heavy Metals Profiling and Preventive Role of Trichomes in Peach Fruits Treated with CaC2.

BACKGROUND: Phytonutrients in peach fruits have health-promoting antioxidants against various chronic diseases. However, there is no extensive data to show the nutritional values of Local peach cultivars after post-harvest treatments. OBJECTIVE: Mainly this study was objective to determine the effect of calcium carbide on nutritional value and quality of fruits of Pakistani peach cultivars. METHODS: The peach fruits were collected from three different peach orchids of KPK and the fruits were divided into 4 groups while 5th group was collected from a local fruit shop. Each experimental group was treated with different concentrations of calcium carbide whereas control group was not treated. The peel and pulp samples were oven dried and ground to fine powder separately. The elemental compositions were determined using Particle Induced X-ray emission and Pelletron Tandem Accelerator. RESULTS: Sixteen elements were identified in peach fruits and the elements were Al, P, S, Cl, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, and Se. In peel, the concentration of some elements increased or decreased after treatment with CaC2 while in pulp the conc. of nearly all detected elements was increased in treated samples. We found a significantly higher amount of heavy metals traces, including As, Se, Co, Si, and P in peach fruits treated with CaC2 Interestingly, the presence of trichomes in peach skin prevents the transfer of these heavy metals deep into the pulp which was also verified by the elemental profiling of nectarines. CONCLUSION: Conclusively, the artificial ripening with CaC2 changed the nutritional value of peach fruits that has higher health risks if consume with the peel. According to our best knowledge, this is the first report that highlights the effects of CaC2 which deteriorate the nutritional value of peach fruits in Pakistan.

Detection of MCR-1 Gene in Multiple Drug Resistant Escherichia coli and Klebsiella pneumoniae in Human Clinical Samples from Peshawar, Pakistan.

BACKGROUND: The presence of plasmid mediated mcr-1 gene in multidrug resistant Gram-negative bacteria poses a serious public health concern in today's world. OBJECTIVE: The present study was aimed to detect the presence of plasmid mediated mcr-1 encoding resistance to colistin in multiple drug resistant (MDR) E. coli and K. pneumoniae isolates. METHODS: A total of 180 clinical isolates of E. coli (n=120) and K. pneumoniae (n=60) were isolated from different clinical specimens, i.e., urine, blood, stool and pus, from diagnostic labs of two major public sector tertiary care hospitals in Peshawar, Pakistan. MDR profile of these isolates was assessed through Kirby-Baur disc diffusion method. All isolates were screened for colistin resistance by dilution methods. Colistin resistant isolates were subjected to PCR for mcr-1 detection and confirmation was done by Sanger sequencing method. RESULTS: Overall, 83.3% (100/120) E. coli and 93.3% (56/60) K. pneumoniae were detected as MDR. Colistin resistance was found in 23.3% (28/120) E. coli and 40% (24/60) K. pneumoniae isolates, whereas mcr-1 gene was detected in 10 out of 52 colistin resistant isolates, including six E. coli and four K. pneumoniae isolates. Minimum inhibitory concentrations (MICs) of colistin in these ten mcr-1 positive isolates ranged from 4µg/ml to 16µg/ml. All mcr-1 positive isolates showed 99% sequence similarity when compared with other present sequences in GenBank. CONCLUSION: Hence, our study confirms the presence of mcr-1 mediated colistin resistance in the studied area. Therefore, urgently larger scale surveillance studies are recommended to investigate prevalence of mcr-1 mediated colistin resistance and to prevent its further spread in the area.

Plasmid-mediated mcr-1 gene in Acinetobacter baumannii and Pseudomonas aeruginosa: first report from Pakistan.

INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.

Assessment of the knowledge of fathers, uptake of routine immunization, and its associated factors in a rural community of North West Nigeria.

Introduction: Father's involvement is essential for the successful immunization of the child, as man is the head of the family and he takes responsibility for all decisions including health and financial issues. This study aimed to assess the knowledge of fathers, uptake of routine immunization (RI), and its associated factors in a rural community of North West Nigeria. Materials and Methods: The study was a community-based cross-sectional study conducted among the male heads of households residing in a rural community of Sokoto state. Systematic sampling was used to recruit 276 respondents. Data were obtained using a structured interviewer-administered questionnaire. Data obtained was entered into the IBM Software package and subsequently analyzed. Level of significance was set at 5%. Results: Only 2.5% and 1.4% of the respondents knew the age measles and yellow fever vaccines were given, respectively. Majority (75.4%) of the respondents' last-born child did not receive bacillus Calmette-Guérin at birth. Only (7.6%) of their last-born child were completely immunized for age. Majority of the respondents that had poor knowledge of RI had no formal education (P = 0.043). Conclusion: The study reported the knowledge of RI among fathers was poor. Having formal education and perception that children should be allowed to receive RI were correlates of good knowledge and uptake of RI. Parents, fathers, in particular, should be educated on the schedule of RI.

RésuméIntroduction: L'implication du père est essentielle à la réussite de la vaccination de l'enfant que l'homme est le chef de famille et il assume la responsabilité de toutes les décisions, y compris les questions de santé et financiers. Cette étude visait à évaluer les connaissances des pères, l'absorption de la vaccination de routine et de ses facteurs associés dans une communauté rurale du nord-ouest du Nigeria. matériaux et méthodes: L'étude était une communauté étude transversale basée menée entre les chefs de famille résidant dans une communauté rurale de l'Etat de Sokoto. L'échantillonnage systématique a été utilisé pour recruter 276 répondants. Les données ont été obtenues à l'aide d'un enquêteur structuré questionnaires. Les données obtenues ont été saisies dans progiciel IBM et ensuite analysés. Le niveau de signification a été fixé à 5%. Résultats: Seulement 2,5% et 1,4% des personnes interrogées connaissaient la rougeole d'âge et les vaccins contre la fièvre jaune ont reçu respectivement. La majorité (75,4%) des répondants de l'enfant dernier-né n'a pas reçu le BCG à la naissance. Seulement (7,6%) de leur dernier enfant ont été complètement vaccinés pour l'âge. La majorité des répondants qui avaient une mauvaise connaissance du RI avait pas d'éducation formelle (p = 0,043). Conclusion: L'étude des connaissances déclarée de vaccination de routine chez les pères était pauvre. Avoir l'éducation formelle et de la perception que les enfants devraient être autorisés à recevoir RI étaient corrélats de la bonne connaissance et l'absorption de la vaccination systématique. Les parents, les pères en particulier, doivent être éduqués sur le calendrier du RI.

Plasmid-mediated mcr-1 gene in acinetobacter baumannii and pseudomonas aeruginosa: first report from pakistan

Abstract INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.