Life Sciences Commons^™

A Review On Bioremediation Of Heavy Metals And Hydrocarbons Through Plant Growth-Promoting Bacteria And Composting, Hafsa Saleem, Hadia Farooq, Rimsha Mazhar, Sadaf Shakil, Sahar Fazal

Journal of Bioresource Management

Soil is an important ecosystem component; it serves as a habitat for many microscopic organisms that play an important role in the ecosystem's maintenance and crops grown in soil. Unfortunately, human activities have adversely affected not only the environment but also the soil health. Soil has been contaminated with heavy metals hydrocarbons and hydrocarbon-based products that are affecting soil health and the growth of plants. In this review two methods of heavy metal remediation are discussed; one is the role of plant growth-promoting bacteria (PGPB) in the enhancement of phytoremediation and the other is composting. PGPB help plants to grow …

Microbes Mediated Keratin Extraction From Poultry Waste And Assessment Of Its Efficacy In Growth Promotion Of Zea Mays And Triticum Aestivum, Imran Ahmad, Aisha Waheed Qurashi, Roheela Yasmeen

Journal of Bioresource Management

Feathers are a waste product of the poultry industry and are considered a major pollutant of the environment. Soil associated with poultry farms is a rich source of indigenous bacteria that are involved in biodegradation of keratin of the feathers of Gallus gallus domesticus. The present study was designed to understand the role of microbes in the degradation of a feather’s keratin and its possible utilization as a bio-protein for plants. In our studies, Keratinolytic activity was high at 37-45 ℃ and at 7.5-9.5 pH. Pseudomonas aeruginosa was identified by 16S rRNA sequencing as the most active keratinolytic strain. …

Isolation And Characterization Of Microbes From Agricultural Fields And Their Evaluation For The Control Of Plant Pests, Hira Liaqat Ali, Sumaira Mazhar, Afeefa Chaudhry

Journal of Bioresource Management

Plant diseases due to pests cause huge loss to crop fields every year. For the control of plant pest, pesticides are under use. Fusarium wilt is caused by phytopathogen Fusarium oxysporum. More than 100 species were affected due to this disease caused by this virus. Fungi also affect onion plant crop yield every year, when chemical pesticides are applied to onion crop field. It will increase the cost of onion production and it is also dangerous for the environment and living organism other than the targeted pests. A number of microorganisms like fungi, bacteria and nematode are currently being …

A Review On Validation Of Enzyme Linked Immunosorbent Assay (Elisa) Techniques For Detection And Quantification Of Different Contaminant In Aquatic Environment, Sanwal Aslam, Zhen Zhang, Ali Muhammad

Journal of Bioresource Management

In world, contaminants and microbes are considered as serious problem in house water supplies as well as in aquatic environment for life. As a result, different countries are working to set level of these contaminants and microbes in domestic supply of water and in aquatic environment. In addition to these scientists are also working on reliable sampling and then their suitable analysis method for accuracy. It is also very important to improve and give justification for analytical techniques used in laboratories, which enable us to monitor level of various contaminants in aquatic environment. In this review, we have focused on …

Ochrobactrum, Bacillus And Enterobacter Isolates Of Hot Water Spring Augment The Growth Of Zea Mays Seedlings, Noor-E-Saba Naz, Rida Batool, Nazia Jamil

Journal of Bioresource Management

Hot springs situated at high altitudes is a kind of remarkable ecosystem for the exploration of microbial flora. It was hypothesized that hot springs can harbor bacteria with plant growth-promoting and exopolysaccharides (EPS) producing ability that can favour the growth of plants. For the investigation of this hypothesis, seven EPS producing bacterial isolates were isolated from a water sample of hot water spring of Tattapani, Azad Kashmir and characterized morphologically and biochemically. Three out of seven isolates (BE1, BN1 and BN3) showed significant production of EPS (14-15 mg / 100 ml). Growth kinetics study revealed that optimum EPS production was …

Sero-Epidemiology Of Rotavirus Gastroenteritis In Children In Ilorin, Kwara State, Omosigho Omoruyi Pius, Izevbuwa Osazee Ekundayo, Saheed Ibrahim Damilare

Journal of Bioresource Management

Rotavirus is responsible for the most severe dehydrating diarrhea among young children due to gastroenteritis. In this study, we aimed to ascertain the occurrence of childhood gastroenteritis caused by Rotavirus among infants and young children who are younger than 5 years of age in Ilorin, Kwara State and determined the risk factors posing the challenges to be susceptible to diarrhea associated with rotavirus in Ilorin, Kwara State. Diarrhea stool samples were collected from children who passed watery stools, who met predetermined inclusion criteria and who presented at the study hospitals Viz: General Hospital and Specialist Hospital Alagbado and Children Specialist …

Endophytic Potential Of Entomopathogenic Fungi For The Remediation Of Wastewater, Huda Bilal, Hasnain Raza, Sana Sarfaraz, Danyal Haider Khan

Journal of Bioresource Management

Phytoremediation has the potential to significantly reduce water contamination caused by excessive harmful chemicals. The degradative properties of fungi are used in fungal phytoremediation to eliminate or neutralise the hazardous pollutants present in water. The goal of the current study was to endophytize water lettuce with the two entomopathogenic fungus Metarhizium anisopilae and Trichoderma harazium. The plant is inoculated with the fungus using the root-dipping procedure. There were two main treatments and a control all with five replications. The analysis of plant and wastewater were analyzed initially like frequency of fungus remained in plant weight, root length and for …

A Review On Antibiotic Resistance And The Use Of Medicinal Plants In The Management Of Uropathogenic Bacteria, Hubza Ruatt Khan, Mehvish Javeed, Iqra Batool, Rabeea Anwar, Asma Ashraf, Sara Janiad

Journal of Bioresource Management

UTIs are the most prevalent infections and are caused by uropathogenicmicrobes such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus spp. Antibiotic resistance has hampered the management of UTIs over the years, with direct repercussions on the treatment cost, the infection severity, and the duration of hospitalization. This review discussed the route of infections, risk factors connected to UTIs, antibiotic resistance issues as well as an alternative therapy to overcome the problem of antibiotic resistance. The medicinal plants which have been utilized for thousands of years to cure a variety of ailmentsrepresent a significant antibiotic …

In Vitro Evaluation Of Antibacterial And Anticoagulant Activities Of Harmala Alkaloids Roots, Amel Benbott, Sabah Boukeria, Hakima Beldi, Kanza Kadi, Abdelouahab Yahia

Journal of Bioresource Management

Peganum harmala L. is a wild herbal plant rich in active compounds, and is among the most used plants in local medicine. The purpose of this study is to contribute to the evaluation of some biological activities of alkaloids extract of P. harmala roots. Using the classical method of extraction of β-carboline alkaloids, the yield of alkaloids in the roots was estimated at 2.34 %. The thin layer chromatography (TLC) method identified the two components Harmine and Hrmaline in the total alkaloid extract of the roots. We evaluated the antibacterial activity of harmala alkaloids in roots against three referenced bacterial …

Assessment Of Antimicrobial Competence Of Epiphytes And Endophytes From Osmium Basilicum And Trigonella Foenum Graecum, Iram Asim, Atia Iqbal, Muhammad Ikrama Tanveer

Journal of Bioresource Management

Plant-associated bacteria are an unexplored group of microorganisms that has enormous potential. These bacteria are the source of finding new antimicrobial substances. The present study aimed to isolate and characterize the epiphytes and endophytes from Osmium basilicum and Trigonella foenum graecum plants and to determine their antimicrobial potential against pathogenic bacteria from Nishtar Hospital Multan, Pakistan. The sum of 18 stems and roots along leaf specimens were assembled as of the plant's Osmium basilicum plus Trigonella foenum graecum as of the distinct locales of the Multan city. Overall 73 bacterial strains were isolated and their colony morphology, gram staining, spore …

Prevalence And Evaluation Of Multidrug Resistance Pattern Of Pseudomonas Aeruginosa Among Critical And Non-Critical Areas At A Tertiary Care Hospital Of Multan, Romah Ishfaq, Hubza Ruatt Khan, Mehvish Javeed, Muhammad Ikrama Tanveer, Asma Ashraf

Journal of Bioresource Management

Pseudomonas aeruginosa is an extremely wide spread microorganism linked to nosocomial illnesses. Effective inspection of variations in antimicrobial resistance patterns of P. aeruginosa is vital for selecting suitable antimicrobial drugs for pragmatic treatment. The current research has been performed for assessing antimicrobial sensitivity profile of P. aeruginosa isolated as of a variety of medical specimens collected from critical and non-critical admitted patients of Nishtar Hospital, Multan. The isolates were detected utilizing standard lab practices, as well as the sensitivity was examined employing Kirby-Bauer disk diffusion method corresponding to Clinical and Laboratory Standard Institute (CLSI) recommendations 2019. Out of 373 samples, …

Evaluation Of Antibacterial Potential Of Artemisinin Extracts Of Artemisia Annua In Vivo And In Vitro, Arsalan Khan, Akhtar Ali, Imtiaz Ali Shah, Umm-E- Aimen, Waseem Ullah, Imtiaz Khan

Journal of Bioresource Management

To ensure universal health care, the World Health Organization recognized the significance of complementary and alternative medicines (CAM) and recommended the use of natural herbs and plants bearing therapeutic potential and fewer adverse effects. Therefore, Artemisia annua herb was evaluated for its antibacterial potential and therapeutic efficacy against Staphylococcus aureus, Streptococcus and Escherichia coli both in vitro and in vivo. Artemisinin was extracted from Artemisia annua by chemical treatment. Subsequently, the culture sensitivity tests were performed on MHA by disk diffusion method to determine the antibacterial potential of the Artemisinin extracts against the test bacteria (in vitro phase). …

Presence Of Blaper-1 And Blaveb-1 Beta-Lactamase Genes Among Isolates Of Pseudomonas Aeruginosa From Burn And Trauma Hospital Peshawar, Pakistan, Suleman Khan, Samiyah Tasleem, Alfarah Rehmat Ullah,, Sarwat Moon, Saad Alghamdi, Raina Saad Suliman, Muhammad Ateeq, Muhmmad Salman, Anas S. Dablool,, Banan Atwah, Farkad Bantun

Journal of Bioresource Management

Pseudomonas aeruginosa spp are the most prevalent bacteria that cause nosocomial infections in hospitals. Most antibiotics, including novel new β-lactams, are already resistant to them, and they can become resistant during treatment, which can make the treatment fail. P. aeruginosa isolates from ICU patients who had Per-1 and VEB-1 were the main focus of this study. These two ESBLs are the two most common in ICU patients who had them. 50 isolates were gathered from Peshawar's LRH ICU facilities in the year 2021. The antibiotic susceptibility test was conducted in accordance with the Clinical and Laboratory Standards Institute's standards (CLSI). …

Microbiological Contamination Associated With The Proximity Of A Refuse Dumpsite To A River Situated In Okada, Edo State, Nigeria, Ebere J. Okafor-Elenwo, Odaro S. Imade, Osazee E. Izevbuwa

Journal of Bioresource Management

This study was performed to evaluate the potential contamination of the Okponha river situated near a dumpsite in Okada, Edo State, Nigeria. Water samples were collected and analyzed for bacteriological and parasitological quality using standard procedures. Isolation and enumeration of bacterial colonies were performed by pour plate technique and the isolated bacteria were identified by standard phenotypic tests. Helminths and protozoa were screened by the direct smear technique. The values of HPC (3.79 ± 0.12 log₁₀ CFU/ml) and TCC (2.20 ± 0.14 log₁₀ CFU/ml) obtained from the river water samples exceeded WHO and NAFDAC recommended limits (≤ 2 …

Amelioration Of Cr (Vi) Into Cr (Iii) By Some Heavy Metal Resistant Bacterial Strains Isolated From Naran Valley, Ifrah Fayyaz, Asma Kalsoom, Rida Batool

Journal of Bioresource Management

Total thirty-two strains were isolated from both water and soil samples collected from Naran Valley which is surrounded by the rugged mountains, located in the province of Khyber Pakhtunkhwa, Pakistan. Streak plate method was used to screen the bacterial strains for resistance against selected heavy metals like chromium (Cr), cobalt (Co), copper (Cu), nickel (Ni) and zinc (Zn). Resistance against multiple heavy metals was shown by all the strains, but S15 and W15 were significant as they exhibited a maximum resistance to chromium and copper (1200 µg/ml). These strains also presented multiple antibiotic (ampicillin, tetracycline, chloramphenicol, and erythromycin) resistance patterns. …

Current Epidemiological Status And Antibiotic Resistance Profile Of Urinary Tract Infection, Muddasir Khan, Syed Hussain Shah, M. Abdullah, Muhammad Salman, Fawad Hayat, Raheela Sarwar, Neelma Hassan, Sohail Ahmad Jan, M. Saddam, Ziaur Rahman, Imran Qazi

Journal of Bioresource Management

With a longitudinal (2020–2021) completion, a set of 300 urine samples of individuals suspected of UTI was characterized based on the biochemical analysis and Kirby Bauer's disc diffusion method. Throughout, a total of 167 samples revealed UTI positivity by obtaining bacterial growth. Reporting that E. coli (69.4 %) was the most dominant when compared with Klebsiella (14.3 %), Proteus (5.9 %), S. aureus (4.7 %), P. aeruginosa (3.5 %), and Enterobacter (1.7 %), respectively. Gender-wise discrimination showed that E. coli was highly distributed among female (50.2 %) patients. A prominent percentage of E. coli (41.9 %) was found in the …

Expression Of Sars Cov2 Receptors Influenced Upon Cytokine Polarizations (Il-4 And Ifnγ) In Hemangioendothelioma Cells, Chandra Lekha Koopari

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Endothelial cells are distinct multifunctional cells with essential metabolic and synthetic roles along with their ability to function as selective permeability barrier. Endothelial cells (ECs), the major component of blood vessels, essentially interact directly yet differently with inflammatory cytokines. ECs are well recognized to be polarized cells, but little is known about the potential function of inflammatory mediators. Covid 19 may have long-term health effects on par with chronic illnesses. Vascular inflammatory disease and coagulopathy linked to COVID-19 are exacerbated by endothelial cell (EC) dysfunction. SARS-CoV-2 enters the host cell through the Angiotensin Converting Enzyme 2 (ACE2) receptor and primes …

Ifn-Γ Increases The Expression Of Sars-Cov-2 Receptors On Vero E6 Cells, Bindu Madhavi Madabattula

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Respiratory epithelial cells are the initial target for SARS-CoV-2 infection. IFN-γ is known to increase the expression of ACE-2, an initial receptor for SARS-CoV-2, on epithelial cells. This study focuses on examining the effect of IFN-γ for ACE-2, TMPRSS2, and neuropilin-1 on Vero E6 cells using two immunofluorescence methods, namely, direct (membrane) fluorescence method and Cytation5 method. Direct fluorescence was determined using an Accu-Scope and ImageJ analysis. Using this method, significance (p<0.023) was observed only for ACE-2 when Vero E6 cells were treated with IFN-γ. Cytation5 fluorescence was determined using a Bio-tek Cytation5 plate reader. The results showed that IFN-γ significantly increased (p<0.001) the expression of ACE-2, neuropilin-1, and TMPRSS2. These results indicate Cytation5 is a more sensitive method for determining the expression of receptors on Vero E6 cells. The elevated levels of SARS-CoV-2 receptors expression resulting from IFN-γ treatment makes the epithelial cells more susceptible targets of SARS-CoV-2 infection. IFN-γ is most likely provided by innate immune cells in the initial COVID-19 infection, consequently contributing to the severity of disease.

Factors Affecting The Epizootics Of Entomopathogenic Fungi-A Review, Mirza Abdul Qayyum, Huda Bilal, Unsar Naeem Ullah:, Habib Ali, Hasnain Raza, Muhammad Wajid

Journal of Bioresource Management

Entomopathogenic fungi (EPF) specifically infect and kill insects can serve as a potential biological control agent. Several biotic and abiotic factors affect their occurrence, persistence, and epizootics caused by them. In biotic factors, fungi characters (host range, latency, spore density and dispersal, mainly host-pathogen compatibility), insect host factors (behavioral, morphological, and physiological), plant-mediated effects (plant architecture, surface chemistry, and leaf topology) are included supports or causes the barrier to mycobiopesticide. Abiotic factors which affect the EPF field persistence are mainly environmental (temperature, sunlight, humidity, rainfall) physical and chemical soil properties (soil texture, pH, E.c, moisture, C/N content, and organic matter) …

Prevalence And Multidrug Resistance Pattern Of E. Coli Among Urinary Tract Infection Patients In Tertiary Care Hospital Of Multan, Hubza Ruatt Khan, Mehvish Javeed, Asghar Javed, Nisma Farooq

Journal of Bioresource Management

Urinary Tract Infection is alarming problem worldwide due to the intensity of antimicrobial resistance. Escherichia coli is the most predominant organism in UTI. This study was planned to evaluate demographic parameters, the prevalence of E. coli, and antimicrobial resistance patterns among E. coli isolates from UTI patients in Nishtar Hospital of Multan from January to June 2018. A total of 350 mid-stream urine samples were collected from different patients having age group from 25 to 60 years and processed by standard laboratory procedures. Out of 350 samples, 100 samples were observed as critical bacteremia. Escherichia coli and Klebsiella …

Plastic Biodegradation Through Insects And Their Symbionts Microbes: A Review, Huda Bilal, Hasnain Raza, Haseena Bibi, Tehmina Bibi

Journal of Bioresource Management

Plastic waste has recently been identified as one of the most serious environmental issues, affecting all life forms, natural habitats, and the economy, and is one of the most serious global environmental problems, second only to climate change. Seeking alternative environmentally sustainable options, such as biodegradation instead of conventional disposal, is critical in the face of this challenge. However, there is currently a lack of information about the mechanisms and efficacy of plastic biodegradation. From this perspective, this study aims to illustrate the negative environmental impacts of the plastic waste. It also addresses the role of insects and gut microbiota …

A Review Of Novel Coronavirus: Cross-Disciplinal Perspective, Iram Asim, Hafsa Rehman, Rabeea Anwar, Humaira Yasmeen

Journal of Bioresource Management

The whole world is under the influence of coronavirus after its first report from Wuhan, China in December 2019. The virus is from coronaviridae family which has zoonotic viruses that can spread from animals to humans. The coronavirus like other viruses of this family produces mild flu-like symptoms within 2-14 days in the human host which progresses to death in severe cases. Unlikely coronavirus spread fast among humans-infectious diseases. Up till now (27/04/2020) around 2.97M cases and 207K deaths have been reported. The presence of a virus in respiratory secretions is diagnosed through molecular methods and chest scans. For this …

Psychological Impact Of Novel Coronavirus Covid-19 Across The Globe-A Review, Sadaf Shaheen, Iram Asim, Rida Zainab, Humaira Yasmeen

Journal of Bioresource Management

The recent outbreaks of novel coronavirus disease have unprecedent impact on mental health of patients, front-line healthcare workers and local population. However, the impact is not fully documented. This review explores stress-driven factors, stress-vulnerable groups and stress management interventions. Repetitive exposure to mass media and inappropriate health protective measures has heightened stress responses. Fear of not getting recovered from COVID-19 and disaffection has profound impact of infected individuals and their families. They may experience fear, anxiety, anger, sleep deprives and anorexia which may weakens their immune system thus making them vulnerable to COVID-19. The COVID-19 pandemic has underscored potential gaps …

Profile Of Antimicrobial Susceptibility From Cattles’S Milk Isolates Suffering From Mastitis In District Lahore, Muhammad Imran, Iqra Rehman, Abdul Qayyum Khan Sulehria, Yasser Mustafa Butt, Abdul Majid Khan, Azizah Ziauddin

Journal of Bioresource Management

Mastitis is highly prevalent infection in cattle causing cost-effective loss in dairy milk production. Escherichia coli is the most frequently isolated bacteria causing mastitis worldwide. The current study was performed to investigate the mastitis prevalence and effect of different antibiotics against pathogens causing it. In sum, 216 milk samples were collected randomly including 108 each from both cows and buffaloes. These samples were subjected to Surf Field Mastitis Test for prevalence of mastitis that found 18.50% and 23.14% in buffaloes and cows respectively. Mastitis was more prevalent both in buffaloes and cows at the age of 9-10 years that was …

Investigating Streptococcus Pneumoniae And Adenovirus Co-Infections Of Lung Epithelial Cells, Mark Nicholas Calabro

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Co-infection is common among viruses and bacteria in the human respiratory system. Adenovirus (AdV) and Streptococcus pneumoniae are clinically relevant respiratory pathogens that cause morbidity and mortality in a variety of patient populations with the highest morbidity occurring among immunocompromised individuals, but also prevalent in infants and the elderly. Acute respiratory distress syndrome may become severe in healthy individuals when co-infection with S. pneumoniae and AdV occurs due to synergistic effects of the pathogens on the host. I hypothesized that S. pneumoniae infection decreases AdV transduction of airway epithelia. To test this hypothesis, we utilized the polarized immortalized airway epithelial …

Il-10 And Tgf-Beta Increase Connexin-43 Expression And Membrane Potential Of Hl-1 Cardiomyocytes Coupled With Raw 264.7 Macrophages, Cora B. Cox

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Cardiomyocytes and macrophages have been found to interact via connexin-43 hemichannels. The role of connexin-43, however, is not fully understood. This study shows that these interactions aid in increasing the membrane potential of cardiomyocytes allowing contraction of the cells. HL-1 cardiomyocytes and RAW 264.7 macrophages in coculture increased expression of connexin-43 compared to cardiomyocytes alone. Co-cultures also increased the fluorescence of Di-8-ANEPPS potentiometric dye indicating an increase in cardiomyocyte membrane potential. Treatment with IL-10 and TGF-beta further increased connexin-43 expression and membrane potential. Treatment with SOCS3 inhibited the effects of TGF-beta and IL-10 while having no effect on its own. …

Investigating Streptococcus Pneumoniae And Adenovirus Co-Infections Of Lung Epithelial Cells, Mark Nicholas Calabro

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Co-infection is common among viruses and bacteria in the human respiratory system. Adenovirus (AdV) and Streptococcus pneumoniae are clinically relevant respiratory pathogens that cause morbidity and mortality in a variety of patient populations with the highest morbidity occurring among immunocompromised individuals, but also prevalent in infants and the elderly. Acute respiratory distress syndrome may become severe in healthy individuals when co-infection with S. pneumoniae and AdV occurs due to synergistic effects of the pathogens on the host. I hypothesized that S. pneumoniae infection decreases AdV transduction of airway epithelia. To test this hypothesis, we utilized the polarized immortalized airway epithelial …

Microbial Diversity In The Sawmill Environment: Implications On The Health Of Sawmill Workers And Merchants, Nigeria, Ebere J. Okafor-Elenwo Ej, Odaro S. Imade Os, Osazee E. Izevbuwa Eo

Journal of Bioresource Management

There is an increasingly high suspicion of occupational health diseases amongst workers in sawmill factories. Hence this study aimed to determine the microbial diversity in some sawmill factories in Okada, Edo State, Nigeria to evaluate potential implications of the factory’s processing area on the health of the workers and wood merchants. Bacteria and fungi count in the bioaerosols within the processing area of each factory was performed with the passive air sampling technique. While in the sawdust samples, microbial counts were carried out with the pour plate technique. Identification of the microbes was performed with macroscopic and microscopic examinations as …

Seroprevalence, Biochemical Investigation And Risk Factor Assessment For Hbv & Hcv Infection In Hospital Based Patients Of Islamabad, Pakistan, Narmeen Adnan Rana, Bushra Munir, Nazeer Hussain, Nazia Imtiaz, Muhammad Asif Gondal Dr., Fahed Parvaiz Dr.

Journal of Bioresource Management

Viral hepatitis poses a serious threat to mankind. Hepatitis B and C Virus are blood borne pathogens that affect millions of individually globally. This study was conducted on a hospital-based population in Islamabad, Pakistan over a period of 4 months, utilizing ELISA as the diagnostic technique which suggested a higher seroprevalence rate for both HBV and HCV i.e. 2.07% and 8.24% respectively. A correlational analysis of the biochemical parameters of these individuals with HBV and HCV infection was carried out and the results indicated a positive correlation of HBV with Alkaline Phosphatase (ALP), HCV with Total Bilirubin (TBil) and both …

Comment: Probably A Good News: Covid-19 Is Not Spreading Fast In Tropical Region Like In Temperate Region, Madeeha Manzoor

Journal of Bioresource Management

My understanding of Covid-19 is that, there are some climatic factors involved in seed spread of this virus. As it started from the area of China which falls under temperate zone, its second victim was Iran’s city Qom which also falls under temperate zone and third is Italy which is completely in temperate zone.

Data from two countries from Temperate zone and three countries from Tropical zone was compared. The days counted from the 100 cases each country reached as day one and further days studied on equal basis.