The Role of Plasmapheresis in Severe Leptospirosis Refractory to Standard Therapy: A Case Report and a Comprehensive Literature Review.

Leptospirosis, a zoonotic infection prevalent in Pakistan, presents diverse clinical manifestations ranging from mild flu-like symptoms to severe multiorgan failure known as Weil's disease. This case study reports on a 24-year-old woman with leptospirosis complicated by acute kidney injury and hyperbilirubinemia, unresponsive to standard therapies. Despite initial treatment with antibiotics and hemodialysis, her condition deteriorated. Following a single session of plasmapheresis, marked clinical and laboratory improvements were observed. Notably, plasma exchange effectively reduced bilirubin levels, underscoring its potential benefit in severe leptospirosis. This case highlights the role of plasmapheresis as rescue therapy in critically ill patients, demonstrating significant outcomes in cases resistant to conventional management. Further research is warranted to refine guidelines on the optimal timing and frequency of plasma exchange in such settings.

Assessment of knowledge, attitude, and practice related to brucellosis among livestock farmers and meat handlers in Saudi Arabia.

Background: Brucellosis is a bacterial zoonotic infection that is endemic in Saudi Arabia and associated with clinical and economic impacts. Several studies from countries endemic for brucellosis evaluated the knowledge and attitude of livestock farmers regarding brucellosis. However, no such study was conducted in Saudi Arabia. This study aimed to evaluate the knowledge, attitude, and practice of livestock farmers and meat handlers in Saudi Arabia. Methods: This was a cross-sectional questionnaire-based study, where participants were interviewed in-person in Arabic in livestock markets between September-December 2023. Convenient sampling was utilized. The questionnaire included basic demographics and questions to assess the knowledge, attitude, and practice toward personal protection and protection of the animals from brucellosis. The questionnaire was adapted from a previously validated survey and included 59 questions. Providing at least one correct answer to a certain question indicated a good knowledge about this item or a safe practice. The participants were divided into: farmers (shepherds working for the animal owners), commercial animal owners (those who rent a stockyard in the livestock market and employ farmers to sell their animals), and private animal owners (owners of private farms from which they sell their animals). Results: 545 participants were interviewed (n = 291 farmers, n = 118 commercial animal owners, n = 113 private animal owners, and n = 23 animal slaughterhouse workers). >90% have heard of brucellosis. Lack of education and short experience (<5 years) of working with livestock were negatively associated with good knowledge of brucellosis symptoms and transmission (OR, 0.30; 95%CI, 0.10-0.94; p = 0.038 and OR, 0.23; 95%CI, 0.08-0.62; p = 0.004, respectively). Taking sick animals to the veterinarian was reported by 61.2%, whereas 36.4% follow safe practices when disposing of aborted fetuses. While 34% consume raw milk, only 10% consume rare/medium-rare meat. 51.2% acknowledged the need for more information on brucellosis. Conclusion: This study revealed the need to augment the knowledge of people working with animals, particularly those with no school education and those with short work experience, via providing educational visits or materials or through veterinarians. This should help them identify human and animal brucellosis symptoms and increase the knowledge on how to protect oneself and animals from this disease.

Staphylococcus pseudintermedius and Pseudomonas aeruginosa Lubbock Chronic Wound Biofilm (LCWB): a suitable dual-species model for in vitro studies.

Antimicrobial treatment of methicillin-resistant Staphylococcus pseudintermedius associated with canine wounds represents an important challenge. The aim of this study was to create a canine wound infection model, Lubbock Chronic Wound Biofilm (LCWB), with a focus on S. pseudintermedius, drawing inspiration from the established human model involving Staphylococcus aureus. Methicillin-resistant S. pseudintermedius 115 (MRSP) and Pseudomonas aeruginosa 700 strains, isolated from dog wounds, were used to set up the LCWB at 24, 48 and 72 h. The LCWBs were evaluated in terms of volume, weight, and microbial CFU/mg. The microbial spatial distribution in the LCWBs was assessed by SEM and CLSM imaging. The best incubation time for the LCWB production in terms of volume (3.38 cm3 ± 0.13), weight (0.86 gr ± 0.02) and CFU/mg (up to 7.05 × 106 CFU/mg ± 2.89 × 105) was 48 h. The SEM and CLSM images showed a major viable microbial colonization at 48 h with non-mixed bacteria with a prevalence of MRSP on the surface and P. aeruginosa 700 in the depth of the wound. The obtained findings demonstrate the capability of S. pseudintermedius to grow together P. aeruginosa in the LCWB model, representing the suitable model to reproduce the animal chronic wound in vitro.

Monitoring Influenza C and D Viruses in Patients With Respiratory Diseases in Japan, January 2018 to March 2023.

BACKGROUND: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long-term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown. METHODS: We established a duplex real-time RT-PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap-dependent endonuclease inhibitor baloxavir marboxil. RESULTS: We detected three influenza C viruses belonging to the C/Kanagawa- or C/Sao Paulo-lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa-lineage, showed similar antigenicity to the reference C/Kanagawa-lineage strain and was susceptible to baloxavir. CONCLUSIONS: Our duplex real-time RT-PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus.

First detection of tick-borne encephalitis virus (TBEV) in raw milk samples in North-Western Iran.

Tick-borne encephalitis virus (TBEV) is a significant cause of flaviviral infections affecting the human central nervous system, primarily transmitted through tick bites and the consumption of unpasteurized milk. This study aimed to assess the prevalence of TBEV and identify new natural foci of TBEV in livestock milk. In this cross-sectional study, unpasteurized milk samples were collected from livestock reared on farms and analysed for the presence and subtyping of TBEV using nested reverse transcription-polymerase chain reaction , alongside the detection of anti-TBEV total IgG antibodies using ELISA. The findings revealed that the highest prevalence of TBEV was observed in goat and sheep milk combined, whereas no TBEV was detected in cow milk samples. All identified strains were of the Siberian subtype. Moreover, the highest prevalence of anti-TBEV antibodies was detected in sheep milk. These results uncover new foci of TBEV in Iran, underscoring the importance of thermal processing (pasteurization) of milk prior to consumption to mitigate the risk of TBEV infection.

Prevalence of Acute Hepatitis E Virus Infections in Swiss Blood Donors 2018-2020.

INTRODUCTION: Hepatitis E virus (HEV) genotype 3 is the major cause of acute viral hepatitis in several European countries. It is acquired mainly by ingesting contaminated pork, but has also been reported to be transmitted through blood transfusion. Although most HEV infections, including those via blood products, are usually self-limiting, they may become chronic in immunocompromised persons. It is thus essential to identify HEV-infected blood donations to prevent transmission to vulnerable recipients. AIMS: Prior to the decision whether to introduce HEV RNA screening for all Swiss blood donations, a 2-year nationwide prevalence study was conducted. METHODS: All blood donations were screened in pools of 12-24 samples at five regional blood donation services, and HEV RNA-positive pools were subsequently resolved to the individual donation index donation (X). The viral load, HEV IgG and IgM serology, and HEV genotype were determined. Follow-up investigations were conducted on future control donations (X + 1) and previous archived donations of the donor (X - 1) where available. RESULTS: Between October 2018 and September 2020, 541,349 blood donations were screened and 125 confirmed positive donations were identified (prevalence 1:4331 donations). At the time of blood donation, the HEV RNA-positive individuals were symptom-free. The median viral load was 554 IU/mL (range: 2.01-2,500,000 IU/mL). Men (88; 70%) were more frequently infected than women (37; 30%), as compared with the sex distribution in the Swiss donor population (57% male/43% female, p < 0.01). Of the 106 genotyped cases (85%), all belonged to genotype 3. Two HEV sub-genotypes predominated; 3h3 (formerly 3s) and 3c. The remaining sub-genotypes are all known to circulate in Europe. Five 3ra genotypes were identified, this being a variant associated with rabbits. In total, 85 (68%) X donations were negative for HEV IgM and IgG. The remaining 40 (32%) were positive for HEV IgG and/or IgM, and consistent with an active infection. We found no markers of previous HEV in 87 of the 89 available and analyzed archive samples (X - 1). Two donors were HEV IgG-positive in the X - 1 donation suggesting insufficient immunity to prevent HEV reinfection. Time of collection of the 90 (72%) analyzed X + 1 donations varied between 2.9 and 101.9 weeks (median of 35 weeks) after X donation. As expected, none of those tested were positive for HEV RNA. Most donors (89; 99%) were positive for anti-HEV lgG/lgM (i.e., seroconversion). HEV lgM-positivity (23; 26%) indicates an often-long persistence of lgM antibodies post-HEV infection. CONCLUSION: The data collected during the first year of the study provided the basis for the decision to establish mandatory HEV RNA universal screening of all Swiss blood donations in minipools, a vital step in providing safer blood for all recipients, especially those who are immunosuppressed.

Chagas Disease: A Silent Threat for Dogs and Humans.

Chagas disease (CD) is a vector-borne Neglected Zoonotic Disease (NZD) caused by a flagellate protozoan, Trypanosoma cruzi, that affects various mammalian species across America, including humans and domestic animals. However, due to an increase in population movements and new routes of transmission, T. cruzi infection is presently considered a worldwide health concern, no longer restricted to endemic countries. Dogs play a major role in the domestic cycle by acting very efficiently as reservoirs and allowing the perpetuation of parasite transmission in endemic areas. Despite the significant progress made in recent years, still there is no vaccine against human and animal disease, there are few drugs available for the treatment of human CD, and there is no standard protocol for the treatment of canine CD. In this review, we highlight human and canine Chagas Disease in its different dimensions and interconnections. Dogs, which are considered to be the most important peridomestic reservoir and sentinel for the transmission of T. cruzi infection in a community, develop CD that is clinically similar to human CD. Therefore, an integrative approach, based on the One Health concept, bringing together the advances in genomics, immunology, and epidemiology can lead to the effective development of vaccines, new treatments, and innovative control strategies to tackle CD.

Clinical features of pasteurellosis without an animal bite or scratch in comparison with bite/scratch pasteurellosis.

Pasteurellosis is a common zoonotic infection that occurs after an animal bite or scratch (B/S). We compared the clinical features of six patients with non-B/S pasteurellosis with those of 14 patients with B/S infections. Pasteurella multocida was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in all six non-B/S infections, whereas 13 of the 14 B/S infections were identified with diagnostic kits. The non-B/S infections were pneumonia (n = 3), skin and soft tissue infections (n = 2), and bacteremia (n = 1). Pneumonia occurred in two patients with underlying pulmonary disease, whereas ventilator-associated pneumonia developed in one patient with cerebral infarction. Pasteurella multocida was isolated from a blood specimen and nasal swab from a patient with liver cirrhosis (Child-Pugh class C) and diabetes. Cellulitis developed in one patient with diabetes and normal-pressure hydrocephalus, who had an open wound following a fall, and in one patient with diabetes and a foot ulcer. Three patients with non-B/S infections had no pet and no episode of recent animal contact. The rate of moderate-to-severe comorbidities was significantly higher in patients with non-B/S infections than in those with B/S infections (100% and 14.3%, respectively, p < 0.001). In conclusion, non-B/S infections can develop in patients with chronic pulmonary disease, invasive mechanical ventilation, or open wounds, or who are immunocompromised, irrespective of obvious animal exposure. In contrast to B/S infections, non-B/S pasteurellosis should be considered opportunistic.

An In-silico Approach to Design and Validate siRNA against Monkeypox Virus.

INTRODUCTION: The monkeypox virus has emerged as an uncommon zoonotic infection. The recent outbreak of MPXV in Europe and abroad in 2022 presented a major threat to individuals at risk. At present, no specific MPXV vaccinations or medications are available. METHODS: In this study, we predicted the most effective siRNA against the conserved region of the MPXV and validated the activity by performing molecular docking studies. RESULTS: Ultimately, the most efficient siRNA molecule was shortlisted against the envelope protein gene (B6R) based on its toxicity, effectivity, thermodynamic stability, molecular interaction, and molecular dynamics simulations (MD) with the Human Argonaute 2 protein. CONCLUSION: Thus, the strategy may offer a platform for the development of potential antiviral RNA therapeutics that target MPXV at the genomic level.

Synthetic Frog-Derived-like Peptides: A New Weapon against Emerging and Potential Zoonotic Viruses.

Given the emergence of the coronavirus disease 2019 (COVID-19), zoonoses have raised in the spotlight of the scientific community. Animals have a pivotal role not only for this infection, but also for many other recent emerging and re-emerging viral diseases, where they may represent both intermediate hosts and/or vectors for zoonoses diffusion. Today, roughly two-thirds of human infections are derived from animal origins; therefore, the search for new broad-spectrum antiviral molecules is mandatory to prevent, control and eradicate future epidemic outbreaks. Host defense peptides, derived from skin secretions of amphibians, appear as the right alternative to common antimicrobial drugs. They are cationic peptides with an amphipathic nature widely described as antibacterial agents, but less is reported about their antiviral potential. In the present study, we evaluated the activity of five amphibian peptides, namely RV-23, AR-23, Hylin-a1, Deserticolin-1 and Hylaseptin-P1, against a wide panel of enveloped animal viruses. A strong virucidal effect was observed for RV-23, AR-23 and Hylin-a1 against bovine and caprine herpesviruses, canine distemper virus, bovine viral diarrhea virus, and Schmallenberg virus. Our results identified these three peptides as potential antiviral-led candidates with a putative therapeutic effect against several animal viruses.

Public Knowledge, Attitudes, and Practices Regarding Brucellosis in Taif City, Saudi Arabia.

BACKGROUND: Brucellosis is a serious zoonotic infectious disease. Humans contract the disease by coming into contact with infected animals or their products. In Saudi Arabia, brucellosis is considered to be an endemic disease, with an annual incidence of 15.34 per 100,000 population from 2003 to 2018. Because of the devastating consequences for human health, raising awareness is an essential component in preventing brucellosis. Our study aims to assess the knowledge, awareness, and attitudes regarding brucellosis among the residents of Taif City, Saudi Arabia. METHODS: A descriptive, cross-sectional survey conducted in June-October 2022 targeted the population of Taif City, Saudi Arabia. The data were collected by an online questionnaire, which included questions on sociodemographic characteristics, awareness regarding brucellosis, behavior and attitude toward animals, and consuming animal-based products. RESULTS: A total of 743 participants were included. The participants were 18-70 years old, 63.4% were females, and 79.4% had a university education. Only 450 participants answered yes to the first question: "Do you know about brucellosis or have you heard about it?" Therefore, they were asked to answer knowledge questions. It was found that out of 450 participants, 46.9% demonstrated a "poor" knowledge level. Participants aged 26-55 years old demonstrated significantly more "good" knowledge than the other age groups (p = 0.001). Males demonstrated significantly more "good" knowledge (30.6%) than females (14.9%) (p < 0.001). The practices and attitudes of animal breeder participants (16.2%) were satisfactory because more than half of them did not participate in the birth of animals (53.4%), 50.7% did not participate in the birth with abortion, and approximately 61% used gloves when taking care of animals. The practices were unsatisfactory because 53.4% of the participants reported that they "always" eat the meat of animals they keep, and 64.4% reported that they personally slaughter sheep or cows from the herd. CONCLUSION: Our study showed that most of the participants were aware of brucellosis; however, at the same time, the knowledge level regarding brucellosis was not satisfactory.

Abdominal Calcifications in a Patient From the Congo Basin with History of Snake Meat Consumption: Radiographic Evidence of Prior Armillifer Infection.

We report a case of a 60-year-old asymptomatic male with history of consumption of uncooked snake meat while living in the Congo basin and prior imaging showing multiple abdominal calcifications. Patient had multiple subepithelial colonic lesions identified during screening colonoscopy and microscopic examination of the lesions demonstrated a calcified nodule in the submucosa with overlying normal mucosa. However, no parasite was identified within the calcified nodule. Given the history of consumption of uncooked snake meat and the typical radiographic feature of multiple abdominal calcifications, it is very likely that the patient's radiographic abnormalities are due to prior Armillifer armillatus infection, a parasitic infection acquired from consumption of uncooked snake meat. Patient was asymptomatic at the time of evaluation and was not given anti-parasitic treatment.

An investigation of household dogs as the source in a case of human bacteraemia caused by Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a commensal and an opportunistic pathogen in dogs, and is also an opportunistic pathogen in humans. Here we report about a case of bacteraemia with a fatal outcome in a 77-year-old co-morbid male likely caused by a S. pseudintermedius and the investigation into the possible transmission from the two dogs in the patient's household. The two dogs carried the same S. pseudintermedius strain, but this dog strain was unrelated to the strain from the patient. In contrast to the patient strain, the dog strain showed reduced susceptibility to several antibiotics and both dogs had received antibiotic treatment prior to sampling. So, it is conceivable that these treatments can have eliminated the patient's strain between the transmission event and the dog sampling. It is also worth noting that the patient strain was positive for the expA gene, which encodes an exfoliative toxin closely related to the S. aureus exfoliative toxin B. This toxin has been linked to canine pyoderma, but its effect on humans remains unknown. Transmission of S. pseudintermedius was confirmed in the household between the dogs. However, we could not verify that the dogs were the source for the S. pseudintermedius in the patient.

Leptospirosis Manifesting as HELLP (Hemolysis, Elevated Liver Enzymes, and Low Platelets) Syndrome: A Rare Case of Leptospirosis During Pregnancy.

Pregnancy is characterized by a reduced immune response, making pregnant women more susceptible to infections. We present a case of a 24-year-old woman in her second pregnancy who arrived at the hospital at 36 weeks gestation in active labor. The patient had received regular antenatal care including routine prenatal check-ups, screenings, and appropriate vaccinations. She complained of abdominal pain for 5-6 hours, sudden onset of hematuria, and a history of low-grade fever for two days. Physical examination revealed paleness, grade three pedal edema, and elevated blood pressure. Diagnostic tests showed mild anemia, thrombocytopenia, proteinuria, elevated liver enzymes, and kidney dysfunction. The patient was admitted to the labor ward, and a tentative diagnosis of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome was made. Shortly after arrival, she spontaneously delivered a healthy baby. However, post-delivery, her fever profile indicated the presence of leptospira IgM antibodies, leading to a diagnosis of leptospirosis mimicking HELLP syndrome. Immediate medical treatment resulted in symptom resolution within two weeks and normal biochemical values within a month. Leptospirosis, caused by the gram-negative spirochete bacteria leptospira, is a zoonotic infection rarely observed during pregnancy and can be misdiagnosed due to its atypical presentation. It can mimic other pregnancy-related conditions such as viral hepatitis, obstetric cholestasis, HELLP syndrome, and acute fatty liver of pregnancy. Early detection and treatment are crucial as this disease can have serious consequences for both the mother and fetus. Therefore, leptospirosis should be considered a potential differential diagnosis, particularly in endemic areas.

Toxocara canis seropositivity in different exposure groups in the Khyber Pakhtunkhwa province of Northwest Pakistan.

Human toxocariasis is a highly prevalent zoonosis worldwide but is underreported in most countries. This study was conducted to evaluate Toxocara canis seropositivity in different exposure sub-groups located in the Mardan, Swabi, and Nowshera districts of the Khyber Pakhtunkhwa province of Northwest Pakistan. A total of 400 blood samples were collected from males 15 years of age and older with no animals, with livestock, with dogs and/or cats living in the house, and from butchers and veterinarians or para-veterinarians. Serum was tested using a commercial ELISA kit for detection of IgG antibodies against T. canis. Proportion seropositive was presented for each group and differences between groups were evaluated using the chi-square or Fisher's exact test as appropriate. Possible risk factors obtained through administration of a questionnaire were also evaluated for each sub-population. Overall T. canis seroprevalence was 14.2%, with a significant difference found between the seroprevalence of individuals with no animals (5.0%; 5/100), individuals with dogs and/or cats living in the household (8.0%; 8/100), individuals with livestock (18.0%; 18/100), veterinarians or para-veterinarians (24.0%; 12/50), and butchers (28.0%; 14/50) (p < 0.001). Significant differences in seropositivity by income bracket, education level, and working in the fields were found for some sub-groups. Study findings demonstrate that certain sub-populations, in Northwest Pakistan, may be at greater risk of T. canis infection. Development and implementation of targeted preventive strategies may, therefore, be needed.

Diagnostic and management strategies for chronic hepatitis E infection.

INTRODUCTION: Hepatitis E Virus (HEV) was initially thought to cause only acute infections, but the discovery of chronic hepatitis E in immunocompromised patients has profoundly changed our understanding of the virus. AREAS COVERED: We describe the physiopathology, diagnosis, and clinical management of chronic HEV infection. The virus can persist in nearly two-thirds of immunosuppressed patients. Reducing immunosuppression is the first immunomodulatory strategy to cure chronic hepatitis E. But this may not always be feasible or effective. Ribavirin monotherapy for 3 months has been recommended as first-line treatment for chronically infected patients. Ribavirin is around 80% effective at eradicating HEV in retrospective studies. Apart from ribavirin, interferon has been successfully used in liver transplants recipients, but if the patient does not respond, no other alternative drug is available. The vaccine available to prevent HEV infection is one available only in China. EXPERT OPINION: HEV infection is a major concern in immunocompromised patients. But the therapeutic arsenal is limited to ribavirin and interferon. Both produce several side effects and new drugs are urgently needed. Moreover, preventive strategies to limit HEV transmission and/or evolution to a chronic infection are also required.

Post-Operative Wound Infection by Multiple Zoonotic Organisms.

Rhodococcus hoagii (formerly Rhodococcus equi), originally isolated from equine species, is primarily transmitted through inhalation of contaminated aerosols such as dust and even breath of infected livestock. Pasteurella multocida is a common cause of cellulitis secondary to canine and feline bites. These are 2 zoonotic microbes that can present with potentially life-threatening, systematic illness in immunocompromised patients.In this brief report, we delineate the clinical course of a 42-year-old post-operative patient (status post-elective exploratory laparotomy, extensive adhesiolysis, and excision of enterocutaneous fistula with creation of an end ileostomy) who developed multiple infections involving these two zoonotic microbes. The patient is an enthusiast of exotic animals with an extensive occupational history as a zookeeper. She was determined to be HIV negative but has a significant history of Crohn's disease managed with systemic immunosuppressive therapy. This case highlights her unusual constellation of post-operative infections and her subsequent medical management.

Assessment of Geographical Distribution of Emerging Zoonotic Toxoplasma gondii Infection in Women Patients Using Geographical Information System (GIS) in Various Regions of Khyber Pakhtunkhwa (KP) Province, Pakistan.

Toxoplasmosis is a zoonotic parasitic disease caused by T. gondii, an obligate intracellular apcomplexan zoonotic parasite that is geographically worldwide in distribution. The parasite infects humans and all warm-blooded animals and is highly prevalent in various geographical regions of the world, including Pakistan. The current study addressee prevalence of Toxoplasma infection in women in various geographical regions, mapping of endemic division and t district of Khyber Pakhtunkhwa province through geographical information system (GIS) in order to locate endemic regions, monitor seasonal and annual increase in prevalence of infection in women patients. Setting: Tertiary hospitals and basic health care centers located in 7 divisions and 24 districts of Khyber Pakhtunkhwa (KP) province of Pakistan. During the current study, 3586 women patients from 7 divisions and 24 districts were clinically examined and screened for prevalence of T. gondii infection. Participants were screened for Toxoplasma infection using ICT and latex agglutination test (LAT) as initial screening assay, while iELISA (IgM, IgG) was used as confirmatory assay. Mapping of the studied region was developed by using ArcGIS 10.5. Spatial analyst tools were applied by using Kriging/Co-kriging techniques, followed by IDW (Inverse Distance Weight) techniques. Overall prevalence of T. gondii infection was found in 881 (24.56%) patients. A significant (<0.05) variation was found in prevalence of infection in different divisions and districts of the province. Prevalence of infection was significantly (<0.05) high 129 (30.07%) in Kohat Division, followed by 177 (29.06%), 80 (27.87%), 287 (26.72%), 81 (21.21%), 47 (21.07%), and 80 (13.71%) cases in Hazara Division, D.I Khan Division, Malakand Division, Mardan Division, Bannu Division, and Peshawar Division. Among various districts, a significant variation (<0.05) was found in prevalence of infection. Prevalence of infection was significantly (<0.05) high 49 (44.95%) in district Karak, while low (16 (10.81%) in district Nowshera. No significant (>0.05) seasonal and annual variation was found in prevalence of Toxoplasma infection. LAT, ICT and ELISA assays were evaluated for prevalence of infection, which significantly (<0.05) detected T. gondii antibodies. LAT, ICT and ELISA assays significantly (<0.05) detected infection, while no significant (>0.05) difference was found between positivity of LAT and ICT assays. A significant difference (<0.05) was found in positivity of Toxoplasma-specific (IgM), (IgG) and (IgM, IgG) immunoglobulin by ICT and ELISA assay. The current study provides comprehensive information about geographical distribution, seasonal and annual variation of Toxoplasmosis infection in various regions of Khyber Pakhtunkhwa province of Pakistan. Infection of T. gondii in women shows an alarming situation of disease transmission from infected animals in the studied region, which is not only a serious and potential threat for adverse pregnancy outcomes, but also cause socioeconomic burden and challenges for various public and animal health organizations in Pakistan and across the country.