Optimisation of analytical methods for tuberculosis drug detection in wastewater: A multinational study.

Wastewater-based epidemiology (WBE) is a robust tool for disease surveillance and monitoring of pharmaceutical consumption. However, monitoring tuberculosis (TB) drug consumption faces challenges due to limited data availability. This study aimed to optimise methods for detecting TB drugs in treated and untreated wastewater from four African countries: South Africa, Nigeria, Kenya, and Cameroon. The limit of detection (LOD) for these drugs ranged from a minimum of 2.20 (±1.02) for rifampicin to a maximum of 2.95 (±0.79) for pyrazinamide. A parallel trend was observed concerning the limit of quantification (LOQ), with rifampicin reporting the lowest average LOQ of 7.33 (±3.44) and pyrazinamide showing the highest average LOQ of 9.81 (±2.64). The variance in LOD and LOQ values could be attributed to factors such as drug polarity. Erythromycin and rifampicin exhibited moderately polar LogP values (2.6 and 2.95), indicating higher lipid affinity and lower water affinity. Conversely, ethambutol, pyrazinamide, and isoniazid displayed polar LogP values (-0.059, -0.6, and -0.7), suggesting lower lipid affinity and greater water affinity. The study revealed that storing wastewater samples for up to 5 days did not result in significant drug concentration loss, with concentration reduction remaining below 1 log throughout the storage period. Application of the optimised method for drug detection and quantification in both treated and untreated wastewater unveiled varied results. Detection frequencies varied among drugs, with ethambutol consistently most detected, while pyrazinamide and isoniazid were least detected in wastewater from only two countries. Most untreated wastewater samples had undetectable drug concentrations, ranging from 1.21 ng/mL for erythromycin to 54.61 ng/mL for isoniazid. This variability may suggest differences in drug consumption within connected communities. In treated wastewater samples, detectable drug concentrations ranged from 1.27 ng/mL for isoniazid to 10.20 ng/mL for ethambutol. Wastewater treatment plants exhibited variable removal efficiencies for different drugs, emphasising the need for further optimisation. Detecting these drugs in treated wastewater suggests potential surface water contamination and subsequent risks of human exposure, underscoring continued research's importance.

Profiling pathogenic protozoan and their functional pathways in wastewater using 18S rRNA and shotgun metagenomics.

Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance.

Surveillance of multidrug-resistant tuberculosis in sub-Saharan Africa through wastewater-based epidemiology.

The spread of multidrug-resistant tuberculosis (MDR-TB) is a serious public health issue, particularly in developing nations. The current methods of monitoring drug-resistant TB (DR-TB) using clinical diagnoses and hospital records are insufficient due to limited healthcare access and underreporting. This study proposes using Wastewater-Based Epidemiology (WBE) to monitor DR-TB in six African countries (Ghana, Nigeria, Kenya, Uganda, Cameroon, and South Africa) and examines the impact of treated wastewater on the spread of TB drug-resistant genes in the environment. Using droplet-digital polymerase chain reaction (ddPCR), the study evaluated untreated and treated wastewater samples in selected African countries for TB surveillance. There was a statistically significant difference in concentrations of genes conferring resistance to TB drugs in wastewater samples from the selected countries (p-value<0.05); South African samples exhibited the highest concentrations of 4.3(±2,77), 4.8(±2.96), 4.4(±3,10) and 4.7(±3,39) log copies/ml for genes conferring resistance to first-line TB drugs (katG, rpoB, embB and pncA respectively) in untreated wastewater. This may be attributed to the higher prevalence of TB/MDR-TB in SA compared to other African countries. Interestingly, genes conferring resistance to second-line TB drugs such as delamanid (ddn gene) and bedaquiline (atpE gene) were detected in relatively high concentrations (4.8(±3,67 and 3.2(±2,31 log copies/ml for ddn and atpE respectively) in countries, such as Cameroon, where these drugs are not part of the MDR-TB treatment regimens, perhaps due to migration or the unapproved use of these drugs in the country. The gene encoding resistance to streptomycin (rrs gene) was abundant in all countries, perhaps due to the common use of this antibiotic for infections other than TB. These results highlight the need for additional surveillance and monitoring, such as WBE, to gather data at a community level. Combining WBE with the One Health strategy and current TB surveillance systems can help prevent the spread of DR-TB in populations.

Exploring the role of wastewater-based epidemiology in understanding tuberculosis burdens in Africa.

Tuberculosis (TB) remains a persistent challenge to public health and presents a substantial menace, especially in developing nations of sub-Saharan Africa. It exerts a considerable strain on healthcare systems in these regions. Effective control requires reliable surveillance, which can be improved by incorporating environmental data alongside clinical data. Molecular advances have led to the development of alternative surveillance methods, such as wastewater-based epidemiology. This studyinvestigated the presence, concentration, and diversity of Mycobacterium tuberculosis complex, the cause of TB, in from six African countries: Ghana, Nigeria, Kenya, Uganda, Cameroon, and South Africa. Samples were collected from wastewater treatment plants. All samples were found to contain Mycobacterium species that have been linked to TB in both humans and animals, including Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium caprae, at varying concentrations. The highest median concentration was found in Ghana, reaching up to 4.7 Log copies/ml for MTBC, 4.6 Log copies/ml for M. bovis, and 3.4 Log copies/ml for M. africanum. The presence of M. africanum outside of West Africa was found in South Africa, Kenya, and Uganda and could indicate the spread of the pathogen. The study underscores the usefulness of wastewater-based epidemiology for tracking TB and shows that even treated wastewater may contain these pathogens, posing potential public health risks.

Fluorescence and colorimetric LAMP-based real-time detection of human pathogenic Cryptosporidium spp. from environmental samples.

Public health concerns related to pathogenic protozoa are widespread, causing significant morbidity and mortality worldwide. Due to the lack of rapid and cost-effective diagnostic methods, timely treatment and control interventions are hindered. In this study, loop mediated isothermal amplification (LAMP) methods were optimised and evaluated for the rapid detection of human pathogenic Cryptosporidium species in environmental samples. Real-time fluorescence and colorimetric detection were tested simultaneously. As a reference method, the results were compared to the well-established droplet digital polymerase chain reaction (ddPCR) method. Both LAMP-based methods successfully detected the Cryptosporidium parvum (GP60 gene) and the entire Cryptosporidium genus (SAM gene) from environmental samples with 100% specificity and no cross-reactivity. Furthermore, both colorimetric and fluorescent methods demonstrated a high sensitivity, with the same limit of detection (LOD) of 1.1 copies of C. parvum per 25 µl reaction (0.02 ng/µl). For real-time fluorescence monitoring, the LAMP reaction can be completed within 28 min, and for visual detection, within 30 min. In addition, both fluorescent and colorimetric LAMP methods showed substantial agreement with the reference method (ddPCR) used. The developed protocols were able to detect C. parvum and Cryptosporidium spp. in 50-85% (n = 60) of environmental samples (treated and untreated wastewater, sludge, and surface water) compared to 58-98% (n = 60) detected by ddPCR. The results further demonstrate that LAMP is an efficient technique for detecting Cryptosporidium spp. in environmental samples due to its simplicity, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease control and public health protection.

Molecular surveillance of tuberculosis-causing mycobacteria in wastewater.

The surveillance of tuberculosis infections has largely depended on clinical diagnostics and hospitalization data. The advancement in molecular methods creates an opportunity for the adoption of alternative surveillance systems, such as wastewater-based epidemiology. This study presents the use of conventional and advanced polymerase chain reaction techniques (droplet digital PCR) to determine the occurrence and concentration of total mycobacteria and members of the Mycobacterium tuberculosis complex (MTBC) in treated and untreated wastewater. Wastewater samples were taken from three wastewater treatment plants (WWTPs) in the city of Durban, South Africa, known for a high burden of TB/MDR-TB due to HIV infections. All untreated wastewater samples contained total mycobacteria and MTBC at varying percentages per WWTP studied. Other members of the MTBC related to tuberculosis infection in animals, M. bovis and M. caprae were also detected. The highest median concentration detected in untreated wastewater was up to 4.9 (±0.2) Log10 copies/ml for total mycobacteria, 4.0 (±0.85) Log10 copies/ml for MTBC, 3.9 (±0.54) Log10 copies/ml for M. tuberculosis, 2.7 (±0.42) Log10 copies/ml for M. africanum, 4.0 (±0.29) Log10 copies/ml for M. bovis and 4.5 (±0.52) Log10 copies/ml for M. caprae. Lower concentrations were detected in the treated wastewater, with a statistically significant difference (P-value ≤ 0.05) in concentrations observed. The log reduction achieved for these bacteria in the respective WWTPs was not statistically different, indicating that the treatment configuration did not have an impact on their removal. The detection of M. africanum in wastewater from South Africa shows that it is possible that some of the TB infections in the community could be caused by this mycobacterium. This study, therefore, highlights the potential of wastewater-based epidemiology for monitoring tuberculosis infections.

An assessment of the health risks associated with shared sanitation: a case study of the community ablution blocks in Durban, South Africa.

Shared sanitation facilities have been hailed as an innovative approach to solve the challenge with sanitation access. However, these facilities may act as hotspots for disease transmission due to unhygienic conditions. In this study we used quantitative (based on Escherichia coli contamination) techniques to assess the health risks associated with the use of community ablution blocks (CABs). The most contaminated surfaces were the cistern handle (5.7 Log10 cfu/cm2) and internal pull latch (5.8 Log10 cfu/cm2). Based on the E. coli contamination, at least two people out of 100 CAB users might be potentially infected when they touch "hot" surfaces. These risks were modelled assuming transfer of potentially pathogenic E. coli from these surfaces to the mouth. The incorporation of risk-reduction measures, such as wiping of these surfaces or washing of hands, could potentially result in significant reduction of infection risks. The most significant risk-reduction intervention was determined to be wiping of the contact surfaces, especially twice prior to contact. A combination of risk-reduction interventions could further reduce the risks. This study shows that contamination of contact surfaces within shared CABs could lead to increased risks of infections, requiring measures aimed at reducing the associated risks. The risk assessment framework used in this study could therefore be applied in similar settings to estimate associated health risks with the use of such facilities.

The source and fate of Mycobacterium tuberculosis complex in wastewater and possible routes of transmission.

BACKGROUND: The Mycobacterium tuberculosis complex (MTBC) consists of causative agents of both human and animal tuberculosis and is responsible for over 10 million annual infections globally. Infections occur mainly through airborne transmission, however, there are possible indirect transmissions through a faecal-oral route which is poorly reported. This faecal-oral transmission could be through the occurrence of the microbe in environments such as wastewater. This manuscript, therefore, reviews the source and fate of MTBC in the wastewater environment, including the current methods in use and the possible risks of infections. RESULTS: The reviewed literature indicates that about 20% of patients with pulmonary TB may have extra-pulmonary manifestations such as GITB, resulting in shedding in feaces and urine. This could potentially be the reason for the detection of MTBC in wastewater. MTBC concentrations of up to 5.5 × 105 (±3.9 × 105) copies/L of untreated wastewater have been reported. Studies have indicated that wastewater may provide these bacteria with the required nutrients for their growth and could potentially result in environmental transmission. However, 98.6 (± 2.7) %, removal during wastewater treatment, through physical-chemical decantation (primary treatment) and biofiltration (secondary treatment) has been reported. Despite these reports, several studies observed the presence of MTBC in treated wastewater via both culture-dependent and molecular techniques. CONCLUSION: The detection of viable MTBC cells in either treated or untreated wastewater, highlights the potential risks of infection for wastewater workers and communities close to these wastewater treatment plants. The generation of aerosols during wastewater treatment could be the main route of transmission. Additionally, direct exposure to the wastewater containing MTBC could potentially contribute to indirect transmissions which may lead to pulmonary or extra-pulmonary infections. This calls for the implementation of risk reduction measures aimed at protecting the exposed populations.

Wastewater-Based Surveillance of Antibiotic Resistance Genes Associated with Tuberculosis Treatment Regimen in KwaZulu Natal, South Africa.

Essential components of public health include strengthening the surveillance of infectious diseases and developing early detection and prevention policies. This is particularly important for drug-resistant tuberculosis (DR-TB), which can be explored by using wastewater-based surveillance. This study aimed to use molecular techniques to determine the occurrence and concentration of antibiotic-resistance genes (ARGs) associated with tuberculosis (TB) resistance in untreated and treated wastewater. Raw/untreated and treated (post-chlorination) wastewater samples were taken from three wastewater treatment plants (WWTPs) in South Africa. The ARGs were selected to target drugs used for first- and second-line TB treatment. Both conventional polymerase chain reaction (PCR) and the more advanced droplet digital PCR (ddPCR) were evaluated as surveillance strategies to determine the distribution and concentration of the selected ARGs. The most abundant ARG in the untreated wastewater was the rrs gene, associated with resistance to the aminoglycosides, specifically streptomycin, with median concentration ranges of 4.69-5.19 log copies/mL. In contrast, pncA gene, associated with resistance to the TB drug pyrazinamide, was the least detected (1.59 to 2.27 log copies/mL). Resistance genes associated with bedaquiline was detected, which is a significant finding because this is a new drug introduced in South Africa for the treatment of multi-drug resistant TB. This study, therefore, establishes the potential of molecular surveillance of wastewater for monitoring antibiotic resistance to TB treatment in communities.

Detection of multidrug resistant environmental isolates of acinetobacter and Stenotrophomonas maltophilia: a possible threat for community acquired infections?

Acinetobacter spp. and Stenotrophomonas maltophilia are bacteria commonly associated with infections at the clinical settings. Reports of infections caused by environmental isolates are rare. Therefore, this study focused on determination of the antibiotic resistance patterns, antibiotic resistance genes, efflux pumps and virulence signatures of Acinetobacter spp. and S. maltophilia recovered from river water, plant rhizosphere and river sediment samples. The isolates were identified and confirmed using biochemical tests and PCR. The antimicrobial resistance profiles of the isolates were determined using Kirby Bauer disk diffusion assay and presence of antibiotic resistance and virulence genes were detected using PCR. S. maltophilia was more frequent in plant rhizosphere and sediment samples than the water samples. Acinetobacter spp. were mostly resistant to trimethoprim-sulfamethoxazole (96% of isolates), followed by polymyxin b (86%), cefixime (54%), colistin (42%), ampicillin (35%) and meropenem (19%). The S. maltophilia isolates displayed total resistance (100%) to trimethoprim- sulfamethoxazole, meropenem, imipenem, ampicillin and cefixime, while 80% of the isolates were resistant to ceftazidime. Acinetobacter spp. contained different antibiotic resistance genes such as sul1 (24% of isolates), sul2 (29%), blaOXA 23/51 (21%) and blaTEM (29%), while S. maltophilia harbored sul1 (8%) and blaTEM (20%). Additionally, efflux pump genes were present in all S. maltophilia isolates. The presence of multidrug resistant Acinetobacter spp. and Stenotrophomonas maltophilia in surface water raises concerns for community-acquired infections as this water is directly been used by the community for various purposes. Therefore, there is the need to institute measures aimed at reducing the risks of these infections and the resulting burden this may have on the health care system within the study area.

Epidemiological Evidence and Health Risks Associated With Agricultural Reuse of Partially Treated and Untreated Wastewater: A Review.

The use of partially treated and untreated wastewater for irrigation is beneficial in agriculture but may be associated with human health risks. Reports from different locations globally have linked microbial outbreaks with agricultural reuse of wastewater. This article reviews the epidemiological evidence and health risks associated with this practice, aiming toward evidence-based conclusions. Exposure pathways that were addressed in this review included those relevant to agricultural workers and their families, consumers of crops, and residents close to areas irrigated with wastewater (partially treated or untreated). A meta-analysis gave an overall odds ratio of 1.65 (95% CI: 1.31, 2.06) for diarrheal disease and 5.49 (95% CI: 2.49, 12.10) for helminth infections for exposed agricultural workers and family members. The risks were higher among children and immunocompromised individuals than in immunocompetent adults. Predominantly skin and intestinal infections were prevalent among individuals infected mainly via occupational exposure and ingestion. Food-borne outbreaks as a result of crops (fruits and vegetables) irrigated with partially or untreated wastewater have been widely reported. Contamination of crops with enteric viruses, fecal coliforms, and bacterial pathogens, parasites including soil-transmitted helminthes (STHs), as well as occurrence of antibiotic residues and antibiotic resistance genes (ARGs) have also been evidenced. The antibiotic residues and ARGs may get internalized in crops along with pathogens and may select for antibiotic resistance, exert ecotoxicity, and lead to bioaccumulation in aquatic organisms with high risk quotient (RQ). Appropriate mitigation lies in adhering to existing guidelines such as the World Health Organization wastewater reuse guidelines and to Sanitation Safety Plans (SSPs). Additionally, improvement in hygiene practices will also provide measures against adverse health impacts.