Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has affected more than 15 million people and, as of 22 July 2019, caused deaths of more than 0.6 million individuals globally. With the excretion of SARS-CoV-2 in the stool of symptomatic and asymptomatic COVID-19 patients, its genome detection in the sewage water can be used as a powerful epidemiological tool to predict the number of positive cases in a population. This study was conducted to detect SARS-CoV-2 genome in sewage water during the lockdown. Sewage samples, from 28 pre-selected sites, were collected on alternate days from 13-25 July, 2020 from two selected areas [Johar Town (n = 05) and Township (n = 23)], where smart lockdown were implemented by the government authorities on 9 ^th July, 2020. Genomic RNA was extracted and the SARS-CoV-2 was detected and quantified using commercially available kit through Real-Time PCR. Out of 28, sixteen samples were positive on day one while 19, 17, 23, 17, 05 and 09 samples were positive on day 3, 5, 7, 9, 11, and 13, respectively. Results revealed a decreased positivity rate and SARS CoV-2 genome copies in sewage towards the end of lockdown however few sampling sites did not follow a clear pattern indicating the complexities in sewage water based surveillance i.e time of sampling etc. Hourly sampling from two sites for 24 hours also revealed the impact of sampling time on detection of SARS-CoV-2 genome in sewage. Results of current study insinuate a possible role of sewage-based COVID-19 surveillance in monitoring and execution of smart lockdowns.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 10⁶ TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (10^1.5 genome copies/mL) compared to group C (10³ genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.