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Controlling the hazard of sclerotia produced by the Sclerotinia sclerotiorum is very complex, and it is urgent to adopt an effective method that is harmonious environmentally to control the disease. Among the six isolates isolated from the rhizosphere of lettuce, the isolate HZA84 demonstrated a high activity in its antagonism towards Sclerotinia sclerotiorum in vitro, and produces siderophore. By amplification of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II subunit (RPB2) genes, the isolate HZA84 was identified as Trichoderma asperellum, which was confirmed by analysis of phylogenetic tree. The Scanning electron microscope monitoring detected that the isolate HZA84 spread over the sclerotial surface, thus, damaging, decomposing, and distorting the globular cells of the outer cortex of the sclerotia. The Real-time polymerase chain reaction (RT-qPCR) analysis disclosed the overexpression of two genes (chit33 and chit37) encoding the endochitinase in addition to one gene (prb1) encoding the proteinase during 4 and 8 days of the parasitism behavior of isolate HZA84 on the sclerotia surface. These enzymes aligned together in the sclerotia destruction by hyperparasitism. On the other hand, the pots trial revealed that spraying of isolate HZA84 reduced the drop disease symptoms of lettuce. The disease severity was decreased by 19.33 and the biocontrol efficiency was increased by 80.67% within the fourth week of inoculation. These findings magnify the unique role of Trichoderma in disrupting the development of plant diseases in sustainable ways., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

One of the most concerning global environmental issues is the pollution of agricultural soils by heavy metals (HMs), especially cadmium, which not only affects human health through Cd-containing foods but also impacts the quality of rice. The soil's nitrification and denitrification processes, coupled with the release of volatile organic compounds by plants, raise substantial concerns. In this review, we summarize the recent literature related to the deleterious effects of Cd on both soil processes related to the N cycle and rice quality, particularly aroma, in different water management practices. Under both continuous flooding (CF) and alternate wetting and drying (AWD) conditions, cadmium has been observed to reduce both the nitrification and denitrification processes. The adverse effects are more pronounced in alternate wetting and drying (AWD) as compared to continuous flooding (CF). Similarly, the alteration in rice aroma is more significant in AWD than in CF. The precise modulation of volatile organic compounds (VOCs) by Cd remains unclear based on the available literature. Nevertheless, HM accumulation is higher in AWD conditions compared to CF, leading to a detrimental impact on volatile organic compounds (VOCs). The literature concludes that AWD practices should be avoided in Cd-contaminated fields to decrease accumulation and maintain the quality of the rice. In the future, rhizospheric engineering and plant biotechnology can be used to decrease the transport of HMs from the soil to the plant's edible parts.

Competing Interests: Authors declare no conflicts of interest.

Verticillium dahliae is a soilborne fungal pathogen that causes vascular wilt diseases in a wide range of economically important crops, including eggplant. Trichoderma spp. are effective biological control agents that suppress a wide range of plant pathogens through a variety of mechanisms, including mycoparasitism. However, the molecular mechanisms of mycoparasitism of Trichoderma spp. in the degradation of microsclerotia of V. dahliae are not yet fully understood. In this study, the ability of 15 isolates of Trichoderma to degrade microsclerotia of V. dahliae was evaluated using a dual culture method. After 15 days, isolate HZA14 showed the greatest potential for microsclerotial degradation. The culture filtrate of isolate HZA14 also significantly inhibited the mycelial growth and conidia germination of V. dahliae at different dilutions. Moreover, this study showed that T. virens produced siderophores and indole-3-acetic acid (IAA). In disease control tests, T. virens HZA14 reduced disease severity in eggplant seedlings by up to 2.77%, resulting in a control efficacy of 96.59% at 30 days after inoculation. Additionally, inoculation with an HZA14 isolate increased stem and root length and fresh and dry weight, demonstrating plant growth promotion efficacy. To further investigate the mycoparasitism mechanism of T. virens HZA14, transcriptomics sequencing and real-time fluorescence quantitative PCR (RT-qPCR) were used to identify the differentially expressed genes (DEGs) of T. virens HZA14 at 3, 6, 9, 12, and 15 days of the interaction with microsclerotia of V. dahliae . In contrast to the control group, the mycoparasitic process of T. virens HZA14 exhibited differential gene expression, with 1197, 1758, 1936, and 1914 genes being up-regulated and 1191, 1963, 2050, and 2114 genes being down-regulated, respectively. Among these genes, enzymes associated with the degradation of microsclerotia, such as endochitinase A1, endochitinase 3, endo-1,3-beta-glucanase, alpha-N-acetylglucosaminidase, laccase-1, and peroxidase were predicted based on bioinformatics analysis. The RT-qPCR results confirmed the RNA-sequencing data, showing that the expression trend of the genes was consistent. These results provide important information for understanding molecular mechanisms of microsclerotial degradation and integrated management of Verticillium wilt in eggplant and other crops.

Background Thorough and precise operative notes play a vital role in patient care, facilitating communication among healthcare teams and serving as essential documents for legal purposes. Poor documentation can jeopardize patient safety and the quality of care provided. The use of standardized guidelines, such as those endorsed by recognized surgical organizations, is crucial to ensure consistent and detailed record-keeping. This study aims to assess the alignment of postoperative notes with established guidelines, with the goal of enhancing documentation practices in the healthcare setting. Objectives This study aimed to evaluate the quality and comprehensiveness of postoperative surgical notes and assess their alignment with established guidelines for surgical documentation, specifically focusing on adherence to recognized standards in surgical practice. Methods This cross-sectional audit assessed 150 operative notes (79 pre-implementation and 71 post-implementation of the Royal College of Surgeons (RCS) guidelines) in the General Surgery Unit at Khyber Teaching Hospital Peshawar, Pakistan. Data included peri-operative findings, operative diagnosis, team information, operational details, complications, procedures, prosthesis, closure, DVT prophylaxis, time out, postoperative orders, and signatures. Results Post-implementation, peri-operative findings were noted in 68 (95.7%) notes, compared to 56 (70.8%) pre-implementation. Operative diagnosis consistently increased from 65 (82.3%) to 69 (97.2%). Post-implementation, operation type, date, and time were consistently included in 67 (94.4%) notes. Complications, additional procedures, and tissue alterations surged to 66 (92.9%), 64 (90.1%), and 60 (84.5%), respectively. Prosthesis and closure techniques were recorded in 65 (91.5%) and 66 (92.9%). Deep vein thrombosis (DVT) prophylaxis and "time out" were documented in 68 (95.8%) notes. Postoperative orders and signatures improved to 70 (98.6%) and 69 (97.2%), respectively. Conclusion Our study revealed the significant positive impact of RCS guideline implementation on operative note documentation. Improvements were noted in essential components such as peri-operative findings, diagnosis, team details, complications, procedures, and more. These enhancements have far-reaching implications, bolstering patient care and ensuring clear communication among healthcare providers, all while serving a vital role in medico-legal matters. By adopting the RCS guidelines, healthcare institutions commit to a higher documentation standard, ultimately supporting good clinical governance., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Toru et al.)

Background: Anal cancer, mainly attributed to human papillomavirus (HPV) infection, is rising in prevalence among the general population in Pakistan. This study aimed to examine the knowledge, attitudes, and practices (KAP) towards anal cancer screening and HPV of the general population in Pakistan., Method: We surveyed anal cancer KAP using social media and snowball sampling from December 2022 to May 2023. The questionnaire had 16 knowledge, 12 attitudes, 6 practice questions, and socio-demographic variables. We applied validity criteria for inclusion and exclusion and used cutoffs ≥50% for each KAP category. We analyzed data in R with Guttman's λ2 for reliability, did univariate and bivariate analysis, and reported frequencies, percentages, p-values, coefficients, odds ratios, and 95% confidence intervals., Results: We surveyed 1620 people and discovered low awareness of HPV and anal cancer causes prevention, and screening (11%-24%), high stigma and embarrassment for screening (54%-70%), strong moral beliefs (89%), condom nonuse (91%), and low engagement in health services and programs (9.1%-14%). Knowledge (75.23%, OR = 1.0984, p = 0.05) was shaped by socio-demographic factors, attitude, and practice, with higher education enhancing knowledge (OR = 1.0984, p = 0.05). Attitude (78.45%, OR = 6.6052, p< 0.001) was influenced by socio-demographic factors, practice, and knowledge as well. Younger females, single, unemployed, students, living with more family members, earning more income, and residing in Islamabad had a more positive attitude (ORs from 1.0115 to 6.6052, p< 0.05), while religion did not affect attitude (p = 0.51). Practice (9.16%, OR = 0.1820, p< 0.001) was determined by socio-demographic factors, knowledge, and attitude. Older males, employed teachers, living with more family members, earning less income, and residing in Islamabad had better practice (ORs from 0.1323 to 3.8431, p< 0.05), but marital status and religion did not influence practice (p > 0.05)., Conclusion: Pakistani young adults need more education, awareness, health services, and programs on HPV and anal cancer, as they have low awareness, high stigma, and socio-cultural challenges. In addition, it is recommended for more research and policy initiatives are needed to address socio-cultural factors and increase anal Pap to overcome anal cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Awan, Naeem, Khattak, Mahmood, Kamran, Khan, Guo, Yongjing, Liu and Nasir.)

Bacterial brown stripe disease caused by Acidovorax oryzae is a major threat to crop yields, and the current reliance on pesticides for control is unsustainable due to environmental pollution and resistance. To address this, bacterial-based ligands have been explored as a potential treatment solution. In this study, we developed a protein-protein interaction (PPI) network for A. oryzae by utilizing shared differentially expressed genes (DEGs) and the STRING database. Using a maximal clique centrality (MCC) approach through CytoHubba and Network Analyzer, we identified hub genes within the PPI network. We then analyzed the genomic data of the top 10 proteins, and further narrowed them down to 2 proteins by utilizing betweenness, closeness, degree, and eigenvector studies. Finally, we used molecular docking to screen 100 compounds against the final two proteins (guaA and metG), and Enfumafungin was selected as a potential treatment for bacterial resistance caused by A. oryzae based on their binding affinity and interaction energy. Our approach demonstrates the potential of utilizing bioinformatics and molecular docking to identify novel drug candidates for precision treatment of bacterial brown stripe disease caused by A. oryzae , paving the way for more targeted and sustainable control strategies. The efficacy of Enfumafungin in inhibiting the growth of A. oryzae strain RS-1 was investigated through both computational and wet lab methods. The models of the protein were built using the Swiss model, and their accuracy was confirmed via a Ramachandran plot. Additionally, Enfumafungin demonstrated potent inhibitory action against the bacterial strain, with an MIC of 100 µg/mL, reducing OD 600 values by up to 91%. The effectiveness of Enfumafungin was further evidenced through agar well diffusion assays, which exhibited the highest zone of inhibition at 1.42 cm when the concentration of Enfumafungin was at 100 µg/mL. Moreover, Enfumafungin was also able to effectively reduce the biofilm of A. oryzae RS-1 in a concentration-dependent manner. The swarming motility of A. oryzae RS-1 was also found to be significantly inhibited by Enfumafungin. Further validation through TEM observation revealed that bacterial cells exposed to Enfumafungin displayed mostly red fluorescence, indicating destruction of the bacterial cell membrane., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Khattak, Qian, Xu, Tomah, Ibrahim, Khan, Ahmed, Hatamleh, Al-Dosary, Ali and Li.)

The environmental impact of industrial development has been well-documented. The use of physical and chemical methods in industrial development has negative consequences for the environment, raising concerns about the sustainability of this approach. There is a growing need for advanced technologies that are compatible with preserving the environment. The use of fungi products for nanoparticle (NP) synthesis is a promising approach that has the potential to meet this need. The genus Trichoderma is a non-pathogenic filamentous fungus with a high degree of genetic diversity. Different strains of this genus have a variety of important environmental, agricultural, and industrial applications. Species of Trichoderma can be used to synthesize metallic NPs using a biological method that is environmentally friendly, low cost, energy saving, and non-toxic. In this review, we provide an overview of the role of Trichoderma metabolism in the synthesis of metallic NPs. We discuss the different metabolic pathways involved in NP synthesis, as well as the role of metabolic metabolites in stabilizing NPs and promoting their synergistic effects. In addition, the future perspective of NPs synthesized by extracts of Trichoderma is discussed, as well as their potential applications in biomedicine, agriculture, and environmental health.

Dengue virus (DENV) has caused about 12 large outbreaks in Pakistan, resulting in 286,262 morbidities and 1108 deaths. The most affected province is Khyber Pakhtunkhwa (KP). This study was conducted to determine the average DENV prevalence in different areas of the Haripur endemic district of KP and the causing factors of DENV., Methods: This work was a cross-sectional study that was performed in the DENV endemic district Haripur. A total of 761 individuals were included in this study. The data were categorized according to sex, age and symptoms (like fever, body aches, bleeding, and skin rash). For data analysis, SPSS 23 version was applied. ArcGIS version 10.8 was used to map the study area., Results: In this study, there were 716 confirmed cases of DENV fever, including 421 males (58.8%) and 295 females (41.2%). The most affected age range, 16-30 years, reported by 301 (42.0%), was followed by 31-45 years, 184 (25.7%), above 46 years, 132 (18.4%), and 0-15 years, 99 (13.8%). The positive IgG cases were 581(81.0%). Those whose age ranges from 1 to 15 years 82 (8.7%) cases, 16-30 years 244 (34.1%), 31-45 years 156 (21.8%), above 46-year age 99 (13.8%) cases. In addition, this suggests that those between the ages of 16 and 30 are at the highest risk for DENV infection. However, this might be the fact that individuals in this age range are more likely to be out in the environment, making them more vulnerable to the virus., Conclusion: Over the past ten years, DENV fever has become increasingly prevalent in Pakistan. The risk is substantially higher for males. Dengue outbreaks hit those between the ages of 16 and 30 the hardest. The proper monitoring and assessment of DENV are necessary for prevention and controlling the disease. Disease surveillance includes identification and molecular characterization of infected persons and monitoring mosquito populations in high-risk locations for the purpose of vector surveillance. In order to assess the community's willingness to participate in DENV preventive efforts, behavioral impact surveillance is also necessary., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Some endophyte bacteria can improve plant growth and suppress plant diseases. However, little is known about the potential of endophytes bacteria to promote wheat growth and suppress the Fusarium seedling blight pathogen Fusarium graminearum . This study was conducted to isolate and identify endophytic bacteria and evaluate their efficacy for the plant growth promotion and disease suppression of Fusarium seedling blight (FSB) in wheat. The Pseudomonas poae strain CO showed strong antifungal activity in vitro and under greenhouse conditions against F. graminearum strain PH-1. The cell-free supernatants (CFSs) of P. poae strain CO were able to inhibit the mycelium growth, the number of colonies forming, spore germination, germ tube length, and the mycotoxin production of FSB with an inhibition rate of 87.00, 62.25, 51.33, 69.29, and 71.08%, respectively, with the highest concentration of CFSs. The results indicated that P. poae exhibited multifarious antifungal properties, such as the production of hydrolytic enzymes, siderophores, and lipopeptides. In addition, compared to untreated seeds, wheat plants treated with the strain showed significant growth rates, where root and shoot length increased by about 33% and the weight of fresh roots, fresh shoots, dry roots, and dry shoots by 50%. In addition, the strain produced high levels of indole-3-acetic acid, phosphate solubilization, and nitrogen fixation. Finally, the strain demonstrated strong antagonistic properties as well as a variety of plant growth-promoting properties. Thus, this result suggest that this strain could be used as an alternate to synthetic chemicals, which can serve as an effective method of protecting wheat from fungal infection.

Malaria is one of the major public health issues globally. Malaria infection spreads through mosquito bites from infected female Anopheles mosquitoes. This study aims to conduct a systematic review and meta-analysis on malaria prevalence in Pakistan from 2006 to 2021. We searched PubMed, Science Direct, EMBASE, EMCare, and Google Scholar to acquire data on the prevalence of malaria infections. We performed a meta-analysis with a random-effects model to obtain the pooled prevalence of malaria, Plasmodium vivax, and Plasmodium falciparum. Meta-analysis was computed using R 4.1.2 Version statistical software. I 2 and time series analysis were performed to identify a possible source of heterogeneity across studies. A funnel plot and the Freeman-Tukey Double Arcsine Transformed Proportion were used to evaluate the presence of publication bias. Out of the 315 studies collected, only 45 full-text articles were screened and included in the final measurable meta-analysis. Pooled malaria prevalence in Pakistan was 23.3%, with Plasmodium vivax , Plasmodium falciparum , and mixed infection rates of 79.13%, 16.29%, and 3.98%, respectively. Similarly, the analysis revealed that the maximum malaria prevalence was 99.79% in Karachi and the minimum was 1.68% in the Larkana district. Amazingly, this systematic review and meta-analysis detected a wide variation in malaria prevalence in Pakistan. Pakistan's public health department and other competent authorities should pay close attention to the large decrease in mosquito populations to curb the infection rate., Competing Interests: The authors declare no conflict of interest., (© 2023 Published by Elsevier Ltd.)

Competing Interests: We declare no competing interests. UAA and XG are joint first authors.

Aims: Cancer is a disease that takes lives of thousands of people each year. There are more than 100 different types of cancers known to man. This fatal disease is one of the leading causes of death today., Background: Astrocyte elevated gene-1(AEG-1)/Metadherin (MTDH) activates multiple oncogenic signaling pathways and leads to different types of cancers. MTDH interacting with staphylococcal nuclease domain containing 1(SND1) supports the survival and growth of mammary epithelial cells under oncogenic conditions., Objective: Silencing MTDH or SND1 individually or disrupting their interaction compromises the tumorigenic potential of tumor-initiating cells. The aim of our present study was to investigate novel interactions of staphylococcal nuclease domain containing 1 (SND1) binding domain of AEG-1/MTDH with different lead compounds through molecular docking approach using MOE software., Methods: Molecular docking was done by docking the ChemBridge database against important residues of MTDH involved in interaction with SND1. After docking the whole ChemBridge database, the top 200 interactive compounds were selected based on docking scores. After applying Lipinski's rule, all the remaining chosen compounds were studied on the basis of binding affinity, binding energy, docking score and protein-ligand interactions. Finally, 10 compounds showing multiple interactions with different amino acid residues were selected as the top interacting compounds., Results: Three compounds were selected for simulation studies after testing these compounds using topkat toxicity and ADMET studies. The simulation study indicated that compound 32538601 is a lead compound for inhibiting MTDH-SND1 complex formation., Conclusion: These novels, potent inhibitors of MTDH-SND1 complex can ultimately help us in controlling cancer up to some extent., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Helicobacter pylori (HP) is a vital element in the etiology of peptic ulcers and gastric cancer. This research aimed to determine the frequency, distribution, and determinants of HP infection in adults and adolescents with gastric symptoms in district Haripur, Khyber Pakhtunkhwa, Pakistan. This cross-sectional study was performed from June 2018 to June 2020 at the Medical Laboratory Technology Department, The University of Haripur, Pakistan. Presence of HP was a research variable, while sex, age groups, education status, overcrowding, dining habits, milk intake, drinking water source and animal contact were grouping variables. Immuno-chromatographic technique (ICT) was used to for serological detection of HP antibodies. All variables were represented by frequency and percentage with 95%CI. Prevalence of HP and its distribution by eight socio-demographic variables was testified by the chi-square goodness-of-fit test while association was testified by chi-square test of association. Out of total 1160 cases, 557 (48%) were positive for HP. Population prevalence was higher in men, in the age group 20-40 years, illiterate, family size ≤ 10 persons, taking restaurant food, using tetra pack, using municipal water, and having animal contact. The observed prevalence of HP was similar to its expected prevalence in the population. The observed distribution of HP in the sample was different from its expected distribution in population by eight socio-demographic variables. Presence of HP was associated with all eight socio-demographic variables besides age groups.

Chikungunya virus (CHIKV) is one of the emerging viruses around the globe. It belongs to the family Togaviridae and genus Alphavirus and is an arthropod borne virus that transmits by the bite of an infected mosquito, mainly through Aedes aegypti and Aedes albopcitus. It is a spherical, enveloped virus with positive single stranded RNA genome. It was first discovered during 1952-53 in Tanganyika, after which outbreaks were documented in many regions of the world. CHIKV has two transmission cycles; an enzootic sylvatic cycle and an urban cycle. CHIKV genome contains 11,900 nucleotides and two open reading frames and shows great sequence variability. Molecular mechanisms of virus host-cell interactions and the pathogenesis of disease are not fully understood. The disease involves three phases; acute, post-acute and chronic with symptoms including high-grade fever, arthralgia, macupapular rashes and headache. There is no licensed vaccine or specific treatment for CHIKV infection. This lack of specific interventions combined with difficulties in making a precise diagnosis together make the disease difficult to manage. In this review we aim to present the current knowledge of global epidemiology, transmission, structure, various aspects of diagnosis as well as highlight potential antiviral drugs and vaccines against CHIKV., (© 2021 John Wiley & Sons Ltd.)

Introduction: Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria-endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan., Methods: Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016-December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation., Results: Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. Plasmodium vivax was found in 623, Plasmodium falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively., Conclusion: The conventional microscopy method has low sensitivity to detect the mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR make this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Background: Malaria among pregnant women is one of the major causes of maternal and infant mortality and morbidity, especially in high-risk areas. Therefore, our study identified the burden of malaria for pregnant women, non-pregnant women, and children under 5 years of age, and malaria service health facilities in Bannu district, Khyber Pakhtunkhwa, Pakistan. Methods: A cross-sectional study was conducted. In this survey, 15,650 individuals were surveyed, and 1,283 were malaria-positive detected. The data were collected from 80 different healthcare centers. SPSS version 23 was used for data analysis. ArcGIS version 10.8 was used for study area mapping. Results: Malaria was detected in 23.3% of children under five, 4.4% of pregnant women, and 72.3% of non-pregnant women, respectively. Moreover, P. falciparum, P. vivax , and mixed infection had a prevalence of 2.1, 96.8, and 1.1%. The most often used and effective medications to treat malaria were chloroquine (29.7%) and primaquine (69.4%). Conclusion: This study's findings depict that malaria's prevalence in the non-pregnant women's group was high. Additionally, P. vivax infection was found to be more prevalent than other types of malaria infection. Due to the scarcity of healthcare facilities in this endemic region, special attention should be directed to strengthening the malaria surveillance and eradication programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qureshi, Khan, Ahmad, Awan, Khattak, Khan and Sun.)

Hematological and hematopoietic cells malignancies of the genes and hematopoietic cells are associated with the genetic mutation, often at the chromosomal level. The standard cytogenetic study is widely accepted as one of the main diagnostics and prognostic determinants in patients. Therefore, the current descriptive and cross-sectional study sought to determine the cytogenetic analysis of frequent hematological malignancies in Pakistan. A total of 202 peripheral bone marrow or blood samples from patients with benign and malignant hematological malignancy were taken using a conventional G-banding technique. Among enrolled patients, the mean age was 21.5 years ± 23.4, and gender-wise distribution showed a marked predominance of the male 147 (73%) population compared to the female 55 (27%). Patients in the age group (2-10 years) had the highest frequency, 48 (24%), of hematological neoplasms, followed by age (11-20 years) with 40 (20%). Normal karyotypes (46, XX/46, XY) was found in 51% (n=103) patients. Furthermore, the frequency of complex karyotype was 30 (15%), while normal was seen in 171 (85%) patients. Pre-B Acute Lymphoblastic Leukemia (Pre-B ALL) was the most prevalent malignancy of 66 (33%), followed by Chronic Myelogenous Leukemia (CML) of 41 (20%) and Acute Lymphocytic Leukemia of 29 (14%). Translocation was the most prevalent 50 (25%), followed by hypotriploidy 14 (7%) and monosomy 8 (4%) on chromosome aberration analysis. In addition, t(9:22) translocation was found to be 20 (10%) in CML, with the majority in the age group (31-40 years). This study recommends that karyotyping should be tested frequently in hematological conditions because it may provide insight into the relative chromosomal changes associated with particular malignancies.

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan's war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Objective: To isolate and characterise multidrug resistant strains of Staphylococcus aureus from healthcare workers who are at potential risk of nosocomial infections., Methods: The observational, cross-sectional study was conducted from November 2014 to April 2015 at different hospitals of Haripur and Abbottabad, Pakistan, and comprised ward and operation theatre staff. The isolates were identified on the basis of microbiological and biochemical tests and further confirmed by polymerase chain reaction. Disc diffusion method was used for antibiotic sensitivity testing, and panton valentine leukocidin and methicillin resistance mecA genes were detected using polymerase chain reaction., Results: Of 208 isolates, 108(52%) were from the ward staff and 100(48%) were from the operation theatre staff. Overall, 167(80.3%) isolates were positive for Staphylococcus aureus, and 75(36%) were methicillin-resistant Staphylococcus aureus. The number of antibiotic-resistant isolates was 75(45%) cefoxitin, 60(36%) ofloxacin, 152(91%) erythromycin, 52(31%) doxycycline, 127(76%) lincomycin, 53(32%) amoxicillin-clavulanate, 67(40%) ciprofloxacin, and 89(53%) ceftriaxone., Conclusions: A high number of hospital staff, including those working in operation theatres, were found to be carrying methicillin-resistant Staphylococcus aureus and multidrug resistant strains in their nasal passage that may be a source of infection to patients.

Malaria is the second highest reported disease from public health sector which affects about 4 million people each year in Pakistan. The study sought to evaluate the haematological changes in malarial patients in order to find any relation of these changes with malarial pathogenesis. This cross sectional descriptive study was designed at the pathology department of Gomal Medical College, Dera Ismail Khan during March 2015 to February 2017. Blood samples were collected from 400 malaria microscopy positive in EDTA tubes for the analysis of haematological parameters like Total leucocyte count, Haemoglobin, Platelets, Mean Cell Volume, Mean Cell Haemoglobin and Mean Cell Haemoglobin Concentration. Out of the 400 cases of malaria, 366 (91.5%) were Plasmodium vivax, 24 (6.0%) were Plasmodium falciparum and 10 (2.5%) were co-infection of Plasmodium vivax and Plasmodium falciparum infection. Anaemia was seen in 148 (37%) of cases; thrombocytopenia was 316(79%) and 60 (15%) cases had leucopenia. This study shows that malarial patients exhibited important changes in haematological parameters like thrombocytopenia; anaemia and leucopenia are significant predictors of malaria infection. When used in combination with other clinical and microscopy methods, these parameters could improve the diagnosis and treatment of malaria.

Background: The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether-lumefantrine as first-line treatment in 2017., Methods: Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. Plasmodium falciparum infections were confirmed by rapid diagnostic test or microscopy. Plasmodium falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism., Results: No artemisinin resistance associated SNPs were identified in the pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections., Conclusion: This study provides an update on the current prevalence of molecular markers associated with reduced P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.

After publication of the original article [1], we were notified that an author's name has been erroneously spelled. Aamir Ali Khattak should be replaced with Aamer Ali Khattak.

Background: Malaria is the fifth leading cause of death worldwide. Pakistan is considered as a moderate malaria-endemic country but still, 177 million individuals are at risk of malaria. Roughly 60% of Pakistan's population, live in malaria-endemic regions. The present study is based upon the survey of various health care centers in 10 major cities of Northern and Southern Punjab to find out the malarial infection patterns in 2015. The diagnosis, seasonal variations, age and gender-wise distribution of Plasmodium spp. circulating in the study area were also included in the objectives., Methods: The malaria-suspected patients '16075' were enrolled for malaria diagnosis using microscopy, out of which 925 were malaria positive which were processed for molecular analysis using nested PCR. The 18S rRNA genes of Plasmodium species were amplified, sequenced, blast and the phylogenetic tree was constructed based on sequences using online integrated tool MEGA7., Results: The 364 cases recruited from Northern Punjab with the highest incidence in Rawalpindi (25.5%) and lowest in Chakwal (15.9%). From Southern Punjab 561 cases were enlisted Rajanpur (21.4%) maximum and lowest from Multan and Rahim Yar Khan (18%). The slide positivity rate, annual parasite incidence, and annual blood examination rates were 5.7 per 1000 population, 0.1, and 0.2% respectively. The only P. vivax (66.7%), P. falciparum (23.7%) and mixed infection by these two species (9.6%) were diagnosed. The same trend (P. vivax > P. falciparum > mixed infection) in species identification %age was confirmed from molecular analysis. However, the occurrence of malaria was higher in Southern Punjab (5.5%) as compared to the Northern Punjab (4.0%). The overall malaria percentage occurrence of treatment-seeking patients in all recruited cities of Punjab was 4.9%. The age-group of 1-20 and males among genders were more affected by malaria. The comparison of different seasons showed that the malaria infection was at a peak in Summer and post-monsoon., Conclusion: The incidence of malaria was high in the flood infected rural areas of Southern Punjab, Summer, and post-monsoon. The age group (1-20) and gender-wise males were more affected by malaria.

Background: In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan., Methods: A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing., Results: The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance., Conclusion: The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

Objective: To check the epidemiology of malaria along Pak-Afghan bordering area.., Methods: The prospective study was conducted at Hatmi Medical Centre, Barmal district, Afghanistan, from March 2014 to February 2016, and comprised patients living in bordering areas between Pakistan's North Waziristan Agency and Afghanistan's Paktika province. Blood samples were collected from subjects with symptoms of malaria for plasmodium microscopy in Giemsa-stained thick and thin film. Slides were stored in a secure slide box and were reconfirmed by microscopy expert at the Department of Pathology, Gomal Medical College, Dera Ismail Khan, Pakistan. SPSS 19 was used for data analysis., Results: Of the total 3,840 patients, 2,655(69.1%) tested negative for malaria while the remaining 1,185(30.9%) tested positive - of whom 788(66.5%) were males and 397(33.5%) were females. Of the positive cases, positive, 1,074(90.6%) were Plasmodium vivax, 76(6.4%) Plasmodium falciparum and 35(3%) were mixed species infections with no case of Plasmodium ovale and Plasmodium malariae., Conclusions: Strict malaria-control measures are needed in both countries.