BACKGROUND: In resource-limited regions, relying on individual clinical results to monitor community diseases is sometimes not possible. Establishing wastewater and non-sewered sanitation surveillance systems can offer opportunities to improve community health.
OBJECTIVE: We provide our experience of establishing a wastewater and non-sewered sanitation surveillance laboratory in Malawi, a resource-limited region, for Vibrio cholerae and Salmonella serotype Typhi.
METHODS: Three locations (inclusive of 8 discrete sample collection sites in total) in the Blantyre District were studied for nine weeks, from September 6 to November 1, 2022. Grab samples were collected weekly. We piloted locally available culture-based medical diagnostic methods for V. cholerae and S. Typhi in wastewater, followed by confirmation analysis of the isolates using reverse transcription polymerase chain reaction (RT-PCR).
RESULTS: Bacterial counts ranged from up to 106 colony-forming units/mL for V. cholerae and up to 107 colony-forming units/mL for S. Typhi. RT-PCR of the isolates showed that the available culture-based medical diagnostic methods were successful in detecting V. cholerae but were less accurate for S. Typhi in wastewater.
UNASSIGNED: This experience serves as a catalyst for the development and validation of alternative wastewater surveillance analytical methods that are not dependent solely on RT-PCR. In this field trial conducted in Africa, new data-driven approaches were developed to promote early-level wastewater research and expand analysis options in resource-limited settings. Although culture-based methods are labor-intensive and have some limitations, we suggest initially leveraging the overlap with the locally available medical testing capacity for V. cholerae, whereas S. Typhi with RT-PCR may still be required. Wastewater analysis may be acceptable for V. cholerae and S. Typhi, which have a high degree of clinical case underreporting, fecal shedding, short incubation periods, and clear outbreak trends, predominantly in low- and middle-income countries.

The American Society of Tropical Medicine and Hygiene\'s American Committee of Medical Entomology has released their updated edition (Version 3.2) of the Arthropod Containment Guidelines. The Guidelines were written to provide pertinent risk assessment and risk management information for the safe handling and rearing of arthropods used in research. The format of the Guidelines is like the outline used in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories. Four Arthropod Containment Levels (ACLs) are described, with increasing requirements for safety and security from ACL-1 to ACL-4. Each containment level provides information on standard practices, including standard and special practices, storage, labeling, monitoring and trapping escaped arthropods, training, medical surveillance, safety equipment, and facility design.

Research with infectious SARS-CoV-2 is complicated because it must be conducted under biosafety level 3 (BSL-3) conditions. Recently, we constructed a live attenuated SARS-CoV-2 virus by rational design through partial recoding of the SARS-CoV-2 genome and showed that the attenuated virus, designated sCPD9, was highly attenuated in preclinical animal models. The recoded sequence was designed by codon pair deoptimization and is located at the distal end of gene ORF1ab. Codon pair deoptimization involves recoding of the viral sequence with underrepresented codon pairs but without altering the amino acid sequence of the encoded proteins. Thus, parental and attenuated viruses produce exactly the same proteins. In Germany, the live attenuated SARS-CoV-2 mutant sCPD9 was recently classified as a BSL-2 pathogen based on its genetic stability and strong attenuation in preclinical animal models. Despite its high attenuation in vivo, sCPD9 grows to high titers in common cell lines, making it suitable as substitute for virulent SARS-CoV-2 in many experimental setups. Consequently, sCPD9 can ease and accelerate SARS-CoV-2 research under BSL-2 conditions, particularly in experiments requiring replicating virus, such as diagnostics and development of antiviral drugs.

The current study examined the stability of several antidoping prohibited substances analytes in urine after 15-min exposure to UV-C light in a Biosafety Level 2 cabinet. The urine matrices were exposed within the original antidoping bottles with the aim to destroy DNA/RNA and possible SARS CoV-2. The analytes small molecules Phase I and Phase II metabolites and peptides, in total 444, endogenous, internal standards, and prohibited substances, pH, and specific gravity in urine were studied. The accredited analytical methods were used by Anti-Doping Laboratory Qatar for the comparison of data of the same urine samples analyzed with and without UV-C exposure. In the study conditions, no problems of stability were detected in the substances spiked in the urine samples exposed in the UV-C irradiation.

Although currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) have significantly contributed to our understanding of tuberculosis (TB) biology, these models have limitations such as differences in genome size, growth rates and virulence. However, attenuated Mycobacterium tuberculosis strains may provide more representative, safer models to study M. tuberculosis biology. For example, the M. tuberculosis ΔleuDΔpanCD double auxotroph, has undergone rigorous in vitro and in vivo safety testing. Like other auxotrophic strains, this has subsequently been approved for use in biosafety level (BSL) 2 facilities. Auxotrophic strains have been assessed as models for drug-resistant M. tuberculosis and for studying latent TB. These offer the potential as safe and useful models, but it is important to understand how well these recapitulate salient features of non-attenuated M. tuberculosis. We therefore performed a comprehensive comparison of M. tuberculosis H37Rv and M. tuberculosisΔleuDΔpanCD. These strains demonstrated similar in vitro and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that M. tuberculosisΔleuDΔpanCD has a heightened stress response that leads to reduced bacterial replication during exposure to acid stress, which has been verified using a dual-fluorescent replication reporter assay. Importantly, infection of human peripheral blood mononuclear cells with the 2 strains elicited comparable cytokine production, demonstrating the suitability of M. tuberculosisΔleuDΔpanCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosisΔleuDΔpanCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.

Vaccinia virus, the prototype Orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology and virus-host interactions, as a protein expression system, as a vaccine vector, and as an oncolytic agent. The ubiquitous use of vaccinia viruses in laboratories around the world raises certain safety concerns because the virus can be a pathogen in individuals with immunological and dermatological abnormalities, and on occasion can cause serious problems in normal hosts. This chapter reviews standard operating procedures when working with vaccinia virus and reviews published cases of accidental laboratory infections with poxviruses.