Advances in point-of-care and molecular techniques to detect waterborne pathogens

Abstract

Rapid and precise detection of waterborne pathogens is critical for effective public health management and environmental safety. Traditional methods for water quality monitoring, considered the “Gold Standard,” are time-consuming, costly, and rely on centralized laboratories and expertise. These methods are impractical for on-site, real-time monitoring, and requiring further improvements. This review explores various waterborne pathogens and cutting-edge molecular detection techniques. It highlights the growing importance of point-of-care and point-of-application methods to expedite results and improve health risk management. Loop-mediated isothermal amplification emerges as a reliable, rapid, and accessible tool in the realms of on-site diagnostics and surveillance. Moreover, the review emphasizes the crucial role of water sample preparation and in-field nucleic acid isolation in augmenting pathogen detection to enable precise assessments of water quality. The advancement of these techniques will guarantee access to safe water and improve the management of waterborne diseases.

Introduction

Understanding the impact of water contamination: risks, causes, and potential solutions

The escalating water stress worldwide presents a critical issue, driven by climate change, population growth, and urbanization^1,2 Globally, over two billion people consume unsafe drinking water, posing significant threats to human health, social stability, and economic productivity. Unsafe drinking water contributes to 485,000 deaths annually, predominantly from waterborne diseases like cholera and diarrhea, underscoring the urgent need for enhanced water quality and sanitation efforts¹. The broader economic impacts include reduced workforce productivity due to illness and substantial costs to healthcare systems in treating water-related diseases³. Microbial contamination in water is primarily caused by fecal pollution, a result of contamination of recreational or drinking water with wastewater. This pollution introduces a variety of harmful pathogens into water sources, posing significant health risks to humans¹.

In 2019, water contamination was a major contributor to global health issues, leading to approximately 1.4 million preventable deaths and 74 million disability-adjusted life years (DALYs) due to inadequate water, sanitation, and hygiene (WASH) services⁴. These figures represent 2.5% of all deaths and 2.9% of all DALYs worldwide, increasing to 7.6% of all deaths and 7.5% of all DALYs among children under 5⁵. Diarrheal diseases are a major contributor, causing over 1 million deaths and 55 million DALYs, with 69% being preventable. Unsafe hand hygiene also leads to around 356,000 deaths and 17 million DALYs due to acute respiratory infections^4,5. These numbers highlight the critical need for global efforts to enhance water quality and sanitation.

Water contamination arises from various sources beyond microbial pollution, encompassing chemical pollutants from industrial runoff, agricultural activities, and improper disposal of pharmaceuticals and personal care products⁶. Chemical pollutants in water can stem from industrial processes, such as heavy metals, solvents, and toxic chemicals used in manufacturing, which can seep into water sources through stormwater runoff or improper disposal⁷. Agricultural activities contribute to water pollution through the use of fertilizers, pesticides, and herbicides, which can leach into waterways and contaminate drinking water sources. Improper disposal of pharmaceuticals and personal care products, such as medicines, antibiotics, and cosmetics, can also lead to water contamination, introducing harmful substances into the environment ⁶.

Prevention of waterborne diseases primarily focuses on reducing or eliminating infectious microorganisms from water supplies. Effective water quality monitoring or surveillance programs are essential to prevent diseases originating from contaminated water (Fig. 1).

Fig. 1: Point-of-care testing for water pollution detection.

Water pollution is caused by various sources, including industrial waste, agricultural runoff, climate change, and wastewater. The POC testing devices can rapidly identify contaminants and pathogens on-site. These portable and user-friendly devices enable timely water quality assessments, allowing for immediate action to mitigate the adverse effects of pollutants. The integration of DNA amplification techniques ensures high sensitivity and specificity, making POC testing a vital tool for monitoring and managing water pollution in diverse environmental settings.

Drinking water may harbor a variety of biological hazards, such as viruses, bacteria, protozoa, and fungi ¹. Enteric viruses, such as Noroviruses, Hepatitis A and E, Adenoviruses, Astroviruses, Enteroviruses (Evs), Sapoviruses, Parechoviruses, and Rotaviruses, are the most common viral pathogens found in drinking water sources and can cause mild to severe diseases, including gastroenteritis and even cancers^1,8. The transmission of these viruses occurs through the fecal-oral route, with water sources becoming contaminated either through direct human fecal discharge or poor wastewater management⁹.

There are more than 500 species of bacteria known to be pathogenic to humans¹⁰. Among the most frequently encountered bacterial pathogens transmitted through drinking water are Burkholderia pseudomallei, Campylobacter coli and Campylobacter jejuni, Escherichia coli O157, Francisella tularensis, Legionella pneumophila, Mycobacterium avium, Salmonella typhi, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Vibrio cholerae O1 and O139^11,12. Protozoa are another group of waterborne pathogens capable of causing diseases and illnesses in humans when they are either ingested or come into contact with the skin. Common taxa include Cryptosporidium, Giardia, Entamoeba histolytica, and Toxoplasma gondii. These taxa can cause diarrhea and other gastrointestinal symptoms, and in severe cases can be life-threatening^1,13. Human health risks associated with these bacterial pathogens increase when either potable or recreational water becomes contaminated^12,14.

In contrast to developed nations, many developing countries face challenges with inadequate wastewater infrastructure and the absence of monitoring programs. This leads to increases in unsecured and contaminated water sources and increased human health risks¹⁰.

To reduce the incidence of waterborne diseases caused by these pathogens, it is essential to prioritize investments in water treatment infrastructure, water quality monitoring, and wastewater management. Regular monitoring of water quality and effective wastewater management are critical for ensuring the safety of water sources. For example, quantitative microbial risk assessment models indicate that certain levels of microbial source tracking (MST) markers in recreational waters, such as Bacteroides HF183 and human adenovirus (HAdV), correlate with a significant health risk from exposure to untreated or secondary treated sewage¹⁵. Routine monitoring of water quality is essential for understanding the true burden of fecal contamination, as evidenced by the variability in E. coli levels at the point of collection and use in different countries and seasons, highlighting the need for more frequent and seasonally adjusted data collection¹⁶. The implementation of real-time monitoring systems is effective for the early detection of water contamination. However, achieving safe drinking water for everyone requires routine and systematic monitoring to quickly guide improvements in water safety. This approach guarantees the provision of clean and safe drinking water, as well as maintaining the quality of recreational water. Advancements in water quality monitoring technologies are crucial for effective water management and will ultimately lead to improved global health outcomes.

Challenges and limitations in waterborne pathogen detection

The development of water analysis methods, particularly in pathogen detection, has experienced significant advancements over time. Early microbiological techniques, such as culture-based methods, were developed to identify and quantify microbial pathogens in water samples^17,18. In the mid-20th century, the advent of molecular biology revolutionized water analysis with the introduction of polymerase chain reaction (PCR) techniques. PCR allows for the rapid and specific detection of pathogens by amplifying their genetic material and provides high accuracy and precision in detecting target pathogens in most matrixes. PCR and its variants, including quantitative PCR, nested PCR, multiplex PCR, and digital PCR, have broad applications in genotyping, gene expression analysis, pathogen detection, and sequencing due to their ability to amplify target DNAs with high specificity and sensitivity^19,20.

Quantitative real-time PCR is considered one of the gold standard assays for the detection of pathogens in water²¹. However, PCR typically requires a dedicated laboratory for sample preparation and analysis. For instance, the USEPA has approved TaqMan qPCR for detecting Bacteroides as an indicator of human fecal pollution in environmental water²². While this method offers high sensitivity and specificity, its use in the field is limited due to its dependence on centralized laboratory facilities. Additionally, the water sample purification and DNA isolation techniques specified in this guideline are not suited for field deployment^23,24.

qPCR techniques are unable to distinguish between viable and non-viable bacterial cells, making them incomparable to standard microbiological methods that detect only viable cells²⁵. Some studies have attempted to develop qPCR methods using DNA intercalating dyes to specifically detect viable bacteria^26,27,28,29. However, the practicality of these methods for field use remains unresolved due to the additional steps they require.

qPCR assays are highly sensitive to the presence of inhibitors³⁰, which are frequently found in environmental water samples^31,32. While PCR inhibitor removal kits offer a potential solution³³, their use necessitates complex sample preparation and access to sophisticated equipment, rendering the process time-consuming and complex, ultimately increasing the overall cost.

Furthermore, the high sensitivity of qPCR assays, when Cq values exceed 35, can increase the risk of false positive results³⁴. Although PCR is faster than traditional culture methods, its efficiency is reduced by the multiple thermal cycling steps required, resulting in a completion time of 2–3 h. These limitations highlight the urgent demand for advanced, rapid technologies that can provide quicker turnaround times for results, paving the way for future developments in water quality monitoring.

Rapid in-field diagnostics

In recent years, the adoption of point-of-care diagnostics (in clinical settings) and in-field diagnostics (in environmental monitoring) has become increasingly prevalent. The field of point-of-care diagnostics has evolved remarkably, from early glucose testing to sophisticated smartphone-based technologies, significantly impacting public health, environmental monitoring, and food safety analysis. The continuous development and evaluation of these technologies are essential to ensure their effectiveness and reliability in various clinical and environmental settings^35,36.

To address these constraints, alternative DNA amplification methods that can be quickly executed in non-laboratory settings have been ventured. The aim is to create uncomplicated, budget-friendly techniques that can be conveniently employed on-site with minimal equipment, all while maintaining a high degree of precision. Recent nucleic acid amplification techniques, with simplified procedures and faster results, have gained attention for point-of-care and in-field diagnostics, enhancing public health in various settings. To ensure the effectiveness of tests for use in both developed and developing countries, the World Health Organization (WHO) has recommended that molecular diagnostic tests for diseases adhere to the ASSURED criteria, which stands for affordable, sensitive, specific, user-friendly, robust, and rapid, equipment-free, and deliverable³⁷. By meeting these criteria, point-of-care tests can be cost-effective, precise, user-friendly, durable, quick, require minimal equipment, and easily accessible to those in need.

Efforts to develop new nucleic acid amplification assays have resulted in the development of several isothermal amplification techniques that can be utilized as point-of-application tests, meeting the ASSURED criteria for molecular diagnostics. Examples of assays include Helicase-dependent amplification³⁸, recombinase polymerase amplification (RPA)³⁹, rolling circle amplification (RCA), cross-priming amplification⁴⁰, nucleic acid sequence-based amplification (NASBA)⁴¹, and loop-mediated isothermal amplification (LAMP)⁴². Isothermal assays operate at a single temperature, which makes them a suitable choice for in-field implementation. However, most of them still require a centralized laboratory to perform.

Rapid diagnostics at the site of application are crucial for the effective management of waterborne diseases, and their importance is only now being recognized (Best et al. ⁴³; Caruso et al. 2023; and Khodaparast et al. ²³). Technological advancements in biosensors, molecular diagnostics, and POC technologies have significantly improved the speed and accuracy of pathogen detection. For instance, an in-field LAMP assay has been developed to detect Bacteroides as an indicator of human fecal pollution in water. This assay has been commercialized and is now used by water quality authorities in Australia, demonstrating the practical application and effectiveness of these diagnostics in monitoring environmental health²³.

Despite these successes, in-field diagnostics face specific challenges and limitations. These include varying environmental conditions that can affect the performance of assays, the need for thorough operator training to ensure accurate results, and logistical issues in deploying these technologies in remote or resource-poor areas^44,45. Ensuring robust sample processing and stability, along with the development of user-friendly, equipment-free technologies, will be crucial for the successful implementation of these diagnostic tools.

In addition to addressing these challenges, the integration of in-field diagnostics with existing health and environmental data systems plays a crucial role in enhancing monitoring and decision-making⁴⁶. These diagnostics facilitate data sharing, support real-time analytics, and connect with broader health information systems, ensuring that data collected in the field can be quickly analyzed and used to inform public health strategies and environmental interventions.

MST

Advancements in in-field diagnostics are invaluable for enhancing water quality monitoring. These innovations are crucial for identifying contamination sources in various environments, especially in water bodies. The precision and adaptability of in-field diagnostics are particularly vital for detecting waterborne pathogens, where rapid and accurate identification can guide timely interventions to protect public health and environmental integrity. Human and animal feces can contain a variety of microorganisms that can pose a threat to human health if present in water. However, detecting each individual pathogen organism in water can be expensive and technically challenging. An alternative approach is to measure the presence of fecal indicator bacteria (FIB) such as E. coli, as an indicator of fecal pollution in water⁴⁷. High levels of FIB can indicate the potential presence of other pathogens that are associated with fecal contamination. Measuring FIB concentration in water is a widely used approach for assessing water quality and is used in many countries as a standard method for monitoring water safety⁴⁸.

While E. coli is frequently used as an indicator for FIB, it has certain limitations in pinpointing the specific host animal and contamination source. This is because E. coli is naturally present in the gastrointestinal tracts of all warm-blooded animals. Furthermore, its ability to survive and proliferate outside an animal’s gut can complicate contamination source identification^48,49. Research has indicated that there may not always be a clear correlation between the E. coli levels in water and the risk of human illness, especially when the contamination source is unknown⁵⁰. Therefore, relying only on E. coli as a FIB may not offer a comprehensive assessment of water quality and the associated health risks⁵⁰.

Consequently, ongoing scientific investigations are exploring alternative methodologies, such as MST techniques, to accurately discern the origin of fecal contamination and deploy targeted and effective mitigation strategies. Bacteroides exhibit notable proficiency as the marker for source identification due to their exclusive association with warm-blooded animals, their prevalence in fecal matter, and their limited capability for significant re-growth in the environment owing to their anaerobic lifestyle^24,48,49,51. The existence of diverse Bacteroides strains that display host-specificity enables precise source identification. Bacteroides has emerged as a promising MST marker, achieved through the amplification of sequence-specific 16S rRNA utilizing DNA amplification techniques⁴⁹. Unlike E. coli, which is not exclusive to a particular host animal and can survive outside of the host’s body, the use of Bacteroides as a marker provides greater specificity and accuracy in identifying the source of fecal contamination⁵². Therefore, identifying Bacteroides strains in polluted waters holds the potential to quickly diagnose the contamination source, leading to more effective management. However, to fully comprehend this potential and establish Bacteroides as a widely accepted and reliable tool for MST and water quality evaluation, several challenges must be addressed.

The traditional method of detecting bacteria in water involved cultivating the sample on solid gelatin media and manually counting the resulting colonies after 48 h of incubation^53,54. Several studies have attempted to establish culture-based methodologies for investigating microbial contamination in water (Table 1). Although the culture-based method has been the standard for bacterial detection in natural water for almost a century⁵⁴ and is well-established for detecting most FIB, it has several limitations.

Table 1 Most common culture-based methods for indicator bacteria detection

Firstly, it is time-consuming and may take up to 48 h or more to get the results. Secondly, it provides no information on the source of the detected bacteria, which is critical for MST²⁴. Additionally, there is the possibility that the microbe may transition to a viable but non-culturable state in water, which can lead to false-negative results and impact the method’s accuracy⁵³.

In 2012, the USEPA incorporated a TaqMan® quantitative polymerase chain reaction (qPCR) assay as a standard method for detecting Enterococci for water quality assessment⁵⁵. This molecular detection approach offers several advantages over culture-based techniques, including enhanced specificity, greater output without the need for biochemical confirmation, and significantly quicker results^24,56,57. qPCR is a powerful technique that allows for the quantification of a wide range of DNA targets with high sensitivity, capable of detecting as few copies of the target sequence. Compared to traditional detection methods, qPCR is faster, highly automated, and more accurate in detecting and quantifying nucleic acids⁵⁸.

While qPCRs provide benefits over traditional culture-based methods, they also have limitations. The detection of indicator bacteria can be expensive and still time-consuming due to the required cold-chain logistics, expertise, and equipment. Additionally, meticulous sample preparation and data interpretation are crucial to prevent contamination and guarantee accuracy^24,54,56,57.

Advancements and applications of LAMP assay in pathogen detection and MST

LAMP, developed by Notomi et al.⁴², offers an alternative to PCR with several advantages. A key benefit of LAMP is its shorter runtime, as it eliminates the need for the thermocycling steps required in PCR, making it a faster and more efficient DNA amplification method. Additionally, LAMP’s simplicity enables non-technical personnel to perform the assay, allowing its use in a broader range of environments, including resource-limited areas^24,59. LAMP eliminates the need for complex DNA isolation from the sample, thereby streamlining the pathogen detection process. This is because the LAMP DNA polymerase, Bacillus stearothermophilus (Bst), has a higher tolerance to amplification inhibitors that usually inhibit other DNA polymerases used in PCR⁵³. The common biological DNA amplification inhibitors include whole-blood, hemin, blood culture media, N-acetyl cysteine, NaCl, urine, stools, and anticoagulants. Humic acids, tannins, metal ions, and other organic and inorganic compounds are common inhibitors in environmental water^60,43. These inhibitors can often interfere with PCR reactions and hinder DNA amplification, but Bst’s ability to tolerate inhibitors can make LAMP a more effective option for amplifying DNA from challenging and dirty samples^43,61. Additionally, the use of 4–6 primers in LAMP enables highly specific amplification of the target DNA, comparable to the gold standard assays⁶². Therefore, LAMP is more likely to provide accurate results in water samples with quality variation⁶¹.

The four main primers included in the LAMP reaction are the forward and backward inner primers (FIP and BIP) the forward outer primer (F3) and backward outer primer (B3). The FIP and BIP primers bind to the target region and form loop structures, which help initiate the amplification reaction more efficiently. Additionally, loop forward (LF) and loop backward (LB) primers can be added to target two more regions on the target sequence, further enhancing the reaction speed (Fig. 2)^42,63. The specific polymerases used in LAMP reactions have a strand displacement activity that eliminates the need for thermocycling steps⁴². This allows the LAMP reaction to proceed at a constant temperature, typically around 60–65 °C. This results in faster amplification times and lower power requirements compared to PCR methods, making LAMP more attractive for remote deployments and point-of-application diagnostics.

Fig. 2: Schematic representation of loop-mediated amplification reaction and its principle.

The LAMP reaction utilizes four main primers designed to recognize six distinct regions of the target DNA. The FIP contains an F2 region at the 3’-end and an F1c region at the 5’-end, while the F3 includes an F3 region complementary to the F3c region of the target sequence. The BIP is composed of a B2 region at the 3’-end and a B1c region at the 5’-end, and the B3 contains a B3 region complementary to the B3c region of the template sequence. The amplification process begins when the F2 region of FIP anneals to the F2c region of the target DNA, allowing Bst DNA polymerase to initiate complementary strand synthesis. Subsequently, the F3 primer anneals to the F3c region of the target and extends, displacing the FIP-linked strand and forming a loop at the 5’-end. This loop serves as a template for BIP, which anneals its B2 region to the B2c region of the template, initiating DNA synthesis and opening the 5’-end loop. Next, the B3 primer anneals to the B3c region of the target DNA and extends, displacing the BIP-linked strand and forming a dumbbell-shaped DNA structure. This dumbbell-shaped DNA is converted into a stem-loop structure, initiating the second stage of the LAMP reaction known as LAMP cycling. The resulting amplicons consist of a mixture of stem-loop and cauliflower-like structures with multiple loops. To further accelerate the LAMP reaction, loop primers (LF and LB) are designed to anneal to the loop regions on the stem-loop structures. The annealing of these loop primers provides additional binding sites for DNA polymerase, enhancing the exponential amplification of the target DNA. This allows multiple rounds of synthesis to occur simultaneously, significantly increasing the reaction speed and yield.

A wide variety of detection methods are available for LAMP products, enabling selection based on the specific application of the assay. These methods include turbidity, agarose gel electrophoresis, calorimetric, and UV light detection, as well as the use of intercalating dyes^64,65. The incorporation of intercalating fluorescent dyes in commercially available master mixes has revolutionized the LAMP assay by enabling real-time amplification detection, offering a swift and precise method for measuring DNA amplification. LAMP, known for its high specificity and sensitivity, has gained recognition as a robust alternative to conventional DNA amplification techniques for in-field pathogen detection^42,63. The combination of microfluidic chips with LAMP reactions empowers rapid and accurate infectious disease diagnosis, particularly suited for point-of-application diagnostics⁶⁶. Lateral flow devices are also being developed and used for the detection of LAMP products, offering a simple and easy-to-use method for detection in resource-limited settings⁶⁵. The inherent simplicity of the LAMP assay makes it feasible to perform even without specialized expertise, enabling its application outside centralized laboratories. This advantage proves crucial in swiftly evaluating and effectively tackling health or environmental issues. These features make LAMP an attractive option for on-site pathogen detection, particularly in resource-limited settings where sophisticated laboratory infrastructure is not readily available. LAMP assays have been shown to be an effective method in MST by detecting various indicator bacteria. One such example is the work by Martzy et al. ⁵³, who developed a LAMP assay for Enterococcus spp., a standard fecal indicator bacterium, in water. The assay was shown to have equal sensitivity and specificity to qPCR, making it suitable for in-field testing. The development of LAMP assay to detect Bacteroides, an indicator bacterium for MST in waterways was first achieved by Jiang et al. ²⁴ and further optimized for in-field detection of human fecal contamination of water by Khodaparast et al. ²³. These findings highlight the potential of LAMP assay in MST applications for water quality monitoring.

LAMP assays have demonstrated promising results in detecting several waterborne pathogens such as E. coli O157:H7⁶⁷, Campylobacter spp.⁶⁸, Salmonella spp.⁶⁹, Pseudomonas aeruginosa⁷⁰, Shigella flexneri⁷¹, Adenoviruses⁷², Hepatitis A⁷³, Hepatitis B⁷⁴, Noroviruses⁷⁵, Rotaviruses⁷⁶, or other microscopic parasites that are transmitted through water such as Giardia lamblia⁷⁷, E. histolytica⁷⁸, and Cryptosporidium parvum⁷⁹.

Reverse transcription LAMP (RT-LAMP) integrates reverse transcription with the LAMP amplification technique to identify RNA targets, enabling the detection of RNA-based viruses. Numerous RT-LAMP assays have been specifically developed to identify various RNA viruses. For instance, RT-LAMP assays have been developed to detect viruses such as SARS-CoV-2^36,80,81,82 and Zika virus⁸³ improving point-of-care diagnostics and facilitating the establishment of more effective control strategies for these devastating pathogens.

Significant advancements in LAMP assay include the development of multiplex versions that detect multiple targets simultaneously, improving diagnostic efficiency and reducing pathogen identification time^84,85,86,87. Techniques such as loop-primer endonuclease cleavage (LEC)–LAMP and dual-sample microfluidic chips have been successfully employed to detect multiple targets in a single reaction, demonstrating high sensitivity and specificity^87,88. Research on LAMP has evolved significantly since the pivotal studies in 2000 demonstrated its diagnostic potential. Since then, numerous studies have applied LAMP assays for pathogen detection, significantly advancing POC diagnostics. Due to their superior capabilities and continuous advancements, LAMP assays have become an attractive technique for these applications. The increasing number of publications on LAMP assays underscores their growing influence and importance in the field of POC diagnostics (Figs. 3–5).

Fig. 3: Innovations in LAMP assays for detecting waterborne pathogens.

Significant advancements in LAMP assays have enhanced the detection of pathogens, improving sensitivity, specificity, speed, and practicality for field applications. Key achievements include increased sensitivity and specificity, allowing for the detection of low pathogen concentrations and reducing false positives. LAMP assays offer rapid detection within 30–60 min, making them suitable for timely water quality assessments. Field-friendly protocols and portable devices enable on-site pathogen detection, while modifications like propidium monoazide differentiate between live and dead pathogens. Multiplexing capabilities allow simultaneous detection of multiple pathogens, enhancing efficiency. Integration with biosensors facilitates easy and rapid detection, and robustness improvements ensure consistent performance across varying conditions. Automated systems reduce human error and increase throughput. The cost-effectiveness and commercialization of standardized LAMP kits make these assays accessible and effective for widespread use, ensuring water safety and public health.

Fig. 4: Isothermal assays published on PubMed.

This pie chart represents the distribution of published articles on the development of isothermal amplification assays on PubMed. Each segment of the pie chart is presented in a different color, representing a type of isothermal amplification assay. The numbers indicate the number of research articles published for each assay so far.

Fig. 5: Comparison of LAMP, qPCR, and culturing methods.

This figure compares three diagnostic methods—culturing, qPCR, and LAMP—across several criteria relevant to their use in detecting waterborne pathogens. The criteria include water sample preparation, DNA/RNA extraction, laboratory instrument requirements, time required, cost, complexity, and field deployability, highlighting the more resource-intensive nature of qPCR and culturing methods compared to the LAMP assay. Water sample preparation: all three methods require preparation, such as filtration of water samples to enrich the target pathogens. DNA/RNA extraction: required for qPCR but not for culturing; most LAMP assays can be completed without using lab-based DNA extraction. Laboratory instrument needed: required for culturing (growth media and controlled environment) and qPCR (thermocycler), but not for LAMP (can be operated with a simple heat block). Time required: culturing takes 48–72 h, qPCR takes 2–3 h, and LAMP takes 30–60 min. Cost: culturing and LAMP have lower costs (indicated by fewer ‘+‘ signs) compared to qPCR. Complexity: culturing and LAMP are less complex than qPCR. Field deployability: only LAMP is suitable for field deployment. H hours, M minutes.

Common freshwater pathogens

Several human pathogens are present in freshwater environments. Some of the most prevalent ones are listed in Table 2.

Table 2 The main human pathogens reported in fresh water^1,13

Giardia

Giardiasis, a common diarrheal disorder caused by the enteric protozoan parasite Giardia, affects both humans and animals. It is responsible for numerous gastrointestinal outbreaks annually in both developed and developing countries^89,90. The impact of giardiasis can be severe, especially for vulnerable populations such as young children, pregnant women, and individuals with weakened immune systems⁹¹. In addition, giardia infections are highly prevalent in domestic animals such as cats, dogs, and livestock⁹², as the cysts of this parasite can spread through surface water that has been contaminated by either human or animal fecal materials⁸⁹. Contact between agricultural animals, such as cattle, and the contaminated water can lead to diarrhea, resulting in production losses⁹³. The challenge in detecting these infections occurs because infected animals can be asymptomatic, serving as a source of transmission⁹⁴.

Various diagnostic methods have been developed to detect giardiasis (Table 3). While direct stool examination is commonly used in laboratories, antigen detection methods are suggested as a complementary assay for giardia detection in fecal samples. However, direct stool examination and serologic tests may lack sensitivity and specificity, potentially leading to false-negative or false-positive results⁹⁵.

Table 3 Methods for the detection of pathogens found in freshwater and commercially available tests

Verweij et al.⁹⁶ developed a qPCR assay to detect the ribosomal RNA (rRNA) gene sequence of G. lamblia, which reported a specificity of 100% and sensitivity of 98%. The assay was found to be more sensitive (98%) than the microscopy analysis (89%) of the samples. This qPCR assay proved useful in detecting patients with giardiasis symptoms but who were negative with microscopy and antigen detection tests⁹⁶. Later, a multiplex real-time PCR assay was developed for the detection of three parasites responsible for most cases of diarrhea, including E. histolytica, G. lamblia, and C. parvum⁹⁷.

To enhance the efficiency of Giardia detection, a LAMP assay was developed for detecting G. duodenalis in fecal and environmental water samples (Table 4). This assay has demonstrated superior performance in the presence of DNA amplification inhibitors compared to PCR and qPCR⁸⁹. When combined with the ARAD microfiber filtration protocol, the LAMP assay enabled the filtration of large volumes of drinking water, reaching up to 1000 L, for the detection of G. duodenalis⁹⁸. As the LAMP assay can be implemented in settings where laboratory resources are limited, such as in developing countries with a high incidence of giardia infections, it has the potential to significantly improve the detection and control of waterborne diseases in these regions.

Table 4 Specific characteristics of LAMP assays developed for waterborne pathogens and human fecal pollution indicators

Molina-Gonzalez et al.⁹⁹ improved the accuracy of a previously developed RPA assay for detecting G. duodenalis¹⁰⁰ by optimizing sample preparation and adding betaine into the reaction. However, the authors recommended future research to explore the potential of field deployable DNA extraction for in-field application of the assay and further analysis to improve the assay’s specificity.

While DNA-based detection methods offer high sensitivity and specificity for detecting Giardia, the necessity for sample preparation and DNA extraction presents limitations when it comes to conducting the assay in the field. Nevertheless, there is still a lack of detection methods suitable for point-of-application or point-of-care scenarios where rapid and accurate detection is imperative.

Cryptosporidium

Cryptosporidium species are a common cause of diarrhea in children in most developing countries due to poor hygiene and lack of access to safe food and drinking water¹⁰¹, with approximately 30 different species reported to infect both humans and animals through contamination of drinking or environmental water with host feces^{102,103,104,105}. Cryptosporidiosis is responsible for up to half of all deaths in children under the age of 5 due to diarrhea and is the second leading cause of diarrhea-related deaths in children¹⁰⁶. Several detection methods have been optimized for the detection of Cryptosporidium in water and food samples, as reviewed in detail by Ahmed and Karanis¹⁰⁷, and DNA-based detection techniques utilized to investigate or genotype Cryptosporidium (Table 3).

Molecular detection assays have emerged as powerful tools for detecting cryptosporidium in water due to their high sensitivity and specificity. DNA-based amplification techniques such as PCR enable the differentiation of host species genotypes, aiding in tracking the source of the infection^105,108.

In the last decade, PCR-based assays have been improved for detecting Cryptosporidium in water and stool samples. One such improvement is the development of qPCR, which has demonstrated high sensitivity and specificity (100%)¹⁰⁹. The qPCR assay reported a limit of detection (LoD) of 1 oocyst for C. hominis and C. parvum and showed a higher sensitivity on water samples tested compared to nested PCR.

Multiplex PCR assays have been developed to simultaneously detect Cryptosporidium along with other pathogens, and some of these assays have become commercially available¹¹⁰. For example, the BD Max parasitic panel (EPP) test can detect Giardia duodenalis, Cryptosporidium hominis, C. parvum, and E. histolytica using multiplex PCR. The test has a high specificity of 99.6% and a sensitivity of 95.5% for C. parvum or C. hominis. The use of multiplex PCR assays for detecting multiple pathogens at once can be advantageous in terms of time and cost efficiency, making them a valuable tool for the surveillance and diagnosis of infectious diseases¹¹¹.

Droplet digital PCR (ddPCR) was designed for investigating Cryptosporidium oocysts in fecal samples by Yang et al.¹¹². ddPCR was shown to be more robust in the presence of inhibitors compared to qPCR and did not require the generation of a standard curve for the interpretation of results. However, ddPCR is estimated to cost approximately twice as much as qPCR. Another nucleic acid-based detection method, Fluorescence in situ hybridization (FISH), has been optimized for detecting Cryptosporidium oocysts by using fluorescent probes that target the ribosomal rRNA of C. parvum. This method demonstrated the ability to differentiate viable oocysts in water and fecal samples^113,114. However, the accuracy of viable determination using FISH is dependent on the decay rate of the rRNA from recently deceased oocysts, leading to the potential for false-positive results for viable oocysts. The susceptibility to environmental factors such as temperature, chlorination, light exposure, and sample preparation should be considered when interpreting FISH results¹¹⁵.

A LAMP assay which was supplemented with stem primers, similar to the previous study by Bakheit et al. ¹¹⁶ achieved a higher sensitivity (10 oocysts/ml) compared to nested PCR in detecting human pathogens C. parvum, C. hominis, and C. meleagridis with a time to positive of about 80 min from reaction preparation¹⁰¹ (Table 4). The assay showed no off-target activity when tested on non-target DNAs and due to the use of lateral flow dipstick (LFD) for detecting the amplified target, this assay may be an alternative for point of care diagnostics especially in the decentralized laboratories, however, the LFD may need improvement to prevent opening the reaction tubes. Similar to Giardia, commercially available DNA-based detection methods for Cryptosporidium remain complex and lack streamlined sample processing and simple field-deployable DNA isolation procedures for point-of-care and point-of-application purposes.

E. histolytica

E. histolytica is a parasitic protozoan that can infect humans and other primates, causing amebiasis. Amongst 40 species of this parasite, eight of them are pathogens to humans¹¹⁷. This protozoan is found in contaminated water and food and can cause a range of symptoms from mild diarrhea to severe abdominal pain and dysentery. In some cases, the parasite can spread beyond the intestines and invade other organs such as the liver, lungs, and brain, causing potentially life-threatening complications^118,119. The treatment of E. histolytica infection typically involves the use of antibiotics. It is important to note that asymptomatic carriers of E. histolytica may not require treatment, but they should be monitored for any signs or symptoms of infection. Additionally, prevention measures such as practicing good hygiene, washing hands regularly, and avoiding contaminated food and water sources can help reduce the risk of infection^120,121.

E. histolytica can be detected by various methods (Table 3). The microscopy method involves examining stool samples for the presence of E. histolytica cysts or trophozoites. While this approach is common and cost-effective, it cannot distinguish between different species of the parasite¹¹⁸ and suffers from low sensitivity¹²². On the other hand, culturing methods are highly sensitive but time-consuming, costly, and complex¹²².

The serology method, which detects antibodies against E. histolytica, is well-established but offers lower sensitivity compared to molecular assays¹¹⁹. Imaging techniques such as ultrasound and computed tomography (CT) scans are useful for detecting liver abscesses potentially caused by E. histolytica infection^118,119,123. Antigen detection tests are fast and simple to perform but may have lower sensitivity compared to molecular tests¹¹⁹. ELISAs methods using amebic antigens detection are highly specific however, they are expensive when used as the commercial kit¹²².

PCR assays were also developed to detect E. histolytica DNA in stool or tissue samples. These tests are highly sensitive and specific for this parasite and can detect very low levels of the parasite¹²⁴. For example, Ali and Roy¹¹⁷ designed a multiplex real-time PCR assay using hydrolysis probes to detect four human pathogen species of E. histolytica with similar morphology and that are otherwise indistinguishable. The LoD of the assay was 0.1 trophozoite of the parasite. Although multiplex PCR assays offer high sensitivity and specificity, their cost and complexity make them unsuitable for use in resource-limited settings¹²⁵.

Several studies have evaluated the performance of LAMP assay for E. histolytica detection, finding it to be highly sensitive and specific, which can be performed directly on stool samples without the need for DNA extraction, further simplifying the procedure^126,127,128 (Table 4). Foo et al.¹²⁹ conducted a study to compare the performance of the LAMP assay with three PCR methods (conventional PCR, nested PCR, and qPCR) for detecting E. histolytica DNA in stool samples. Their findings revealed that the LAMP assay demonstrated superior sensitivity and a lower LoD compared to the other PCR methods. Furthermore, the LAMP assay exhibited a shorter turnaround time, suggesting it could serve as a reliable alternative platform for point-of-care E. histolytica DNA detection.

T. gondii

T. gondii is an intracellular protozoan parasite that can infect warm-blooded animals, including humans. It is transmitted through ingestion of oocysts shed by infected cats, ingestion of tissue cysts in undercooked meat from infected animals, or congenital transmission from mother to fetus^130,131.

In humans, T. gondii infection, known as toxoplasmosis, can cause a range of clinical manifestations, from asymptomatic infection to severe disease. Acute infection in immunocompetent individuals is usually mild and self-limited, but it can cause flu-like symptoms, swollen lymph nodes, and muscle aches. However, in immunocompromised individuals, T. gondii infection can cause severe disease, including encephalitis, myocarditis, and pneumonitis, and in pregnant women, T. gondii infection can result in congenital infection and fetal abnormalities^130,132. Toxoplasmosis is typically treated with antibiotics such as pyrimethamine and sulfadiazine, along with folinic acid^133,134. However, with the increasing prevalence of antibiotic-resistant strains, it is crucial to improve diagnostic methods and prevent infections, particularly in high-risk populations such as pregnant women and immunocompromised individuals. Previous studies have highlighted the increasing significance of waterborne transmission of T. gondii to humans through the dispersal of oocysts in surface water. This mode of transmission has significant epidemiological implications and deserves close attention¹³⁵. Therefore, it is critical to develop effective diagnostic methods and treatment options that can address antibiotic resistance to effectively manage and prevent toxoplasmosis^133,134.

Serological tests like ELISA, indirect fluorescent antibody test (IFAT), and immunoblotting were developed to detect the presence of antibodies against T. gondii in serum as the rapid tests, but they cannot distinguish between active and inactive infections¹³¹. PCR is a sensitive and specific method for the detection of T. gondii DNA in various samples such as blood, tissue, cerebrospinal fluid, and amniotic fluid¹³⁶. PCR is also used for the detection of T. gondii in food samples, which can be a potential source of infection¹³⁷. Microscopic examination of stained smears or tissue sections can be used to detect the presence of T. gondii in various samples such as blood, cerebrospinal fluid, and tissue biopsy specimens¹³⁸. T. gondii can be cultured in vitro in specialized cell culture systems. This method is not routinely used for diagnostic purposes due to its low sensitivity and the requirement for specialized expertise and equipment^131,139. Rapid diagnostic tests such as lateral flow assays are available for the detection of T. gondii-specific antibodies in serum or plasma. These tests are simple to perform and do not require specialized equipment, making them useful for point-of-care and point-of-application testing¹⁴⁰.

Commercially available tests for detecting T. gondii in freshwater include PCR¹⁴¹ and immunological assays such as ELISA¹³¹. Detecting T. gondii in freshwater can be challenging due to the low abundance of the parasite in water. Additionally, the detection of the parasite may be affected by various factors such as the presence of other microorganisms and the sampling method employed¹⁴².

LAMP assay has been developed to detect T. gondii in various biological samples, including blood, cerebrospinal fluid, and tissue samples, and has proven to be a sensitive and specific diagnostic tool¹⁴³ (Table 4). Several studies demonstrate the utility of the LAMP assay for detecting T. gondii in water samples with high sensitivity and specificity, and provide valuable information for the development of reliable and efficient methods for water quality monitoring^144,145, but it’s still unavailable as a commercial test.

Hepatitis A virus (HAV)

HAV is a single-stranded RNA virus and highly contagious and causes acute liver inflammation. The virus is typically spread through contaminated food and water or close contact with an infected individual^146,147. Symptoms of HAV infection include fatigue, fever, nausea, vomiting, abdominal pain, dark urine, and yellowing of the skin and eyes. In most cases, the infection resolves on its own within a few weeks, and there is no specific treatment for hepatitis A¹⁴⁶. However, in severe cases or for individuals with underlying liver disease, hospitalization, and supportive care may be necessary¹⁴⁶.

Detection of HAV infection typically involves serologic testing to detect antibodies to the virus in the blood. In addition to serologic testing, molecular methods such as PCR are used to detect HAV RNA in blood or stool samples. PCR can be useful for diagnosing acute infection or for monitoring treatment response in individuals with chronic HAV infection¹⁴⁷. Kozak et al.¹⁴⁸ conducted a study to develop an RT-PCR assay for detecting HAV RNA using two different platforms: the Qiagen Rotor-Gene and the BD MAX. The sensitivity of the assay was reported to be 94.8% and 87%, while the specificity was 100% and 94.7%, respectively. The LoD of the assay was 4.3 × 10² copies/ml when tested on a spiked serum with the known concentration of plasmid harboring target region. In the study of Persson et al.¹⁴⁹, the 5′ untranslated regions (UTR) of HAV have been identified as the most conserved genomic region. In silico inclusivity, analysis revealed that the assay developed in this study was more accurate than the one recommended by the International Organization for Standardization (ISO) method (ISO 15216-1:2017) and demonstrated increased effectiveness in detecting globally emerging genotype III strains. When it comes to detecting low levels of HAV, a comparison between Reverse transcription PCR (RT-qPCR) and RT-ddPCR revealed that RT-qPCR is preferable for qualitative detection, although RT-ddPCR exhibited higher precision.

HAV can be detected in freshwater sources, such as rivers, lakes, and ponds, where it can be introduced through fecal contamination from infected individuals or animals¹⁵⁰. Detection of HAV in fresh water typically involves collecting water samples and testing for the presence of HAV RNA using PCR, which can provide rapid and sensitive detection of HAV¹⁵¹.

RT-LAMP assay has demonstrated high sensitivity and specificity in detecting IA, IB, and IIIB sub-genotypes of HAV in clinical samples (fecal), with detection limits ranging from 0.4 to 0.8 focus forming units (FFU) per reaction⁷³. In addition, other isothermal assays, such as the NASBA assay have been developed to detect HAV¹⁵² with a detection limit of 1 PFU of viral RNA. The assay was reported to have high specificity and comparable sensitivity to the real-time RT-PCR assay. However, in some cases, the time to result for NASBA may be longer than other amplification-based assays, and it requires several steps and optimization of reaction conditions for optimal performance¹⁵³.

Norovirus

Norovirus, a single-stranded RNA virus, induces gastroenteritis (L¹⁵⁴.). It has six genogroups, with GI and GII being the most associated with human diseases¹⁵⁴. The virus is highly contagious and is a well-known cause of acute gastroenteritis outbreaks in settings where individuals are in close proximity, including hospitals, nursing homes, schools, and cruise ships¹⁵⁵. Norovirus transmission occurs via the fecal-oral route, whereby the virus is disseminated through contact with fecal matter or vomit from infected individuals. Additionally, it can be spread via the consumption of contaminated food or water, or through contact with contaminated surfaces, followed by subsequent transfer to one’s nose or mouth^155,156. Norovirus infection is characterized by a wide range of symptoms, including nausea, vomiting, diarrhea, abdominal cramps, low-grade fever, headache, and body aches. These symptoms typically manifest within 12–48 h post-exposure to the virus and may persist for a duration of 1–3 days. Norovirus infection is highly transmissible, causing nearly 20% of acute gastroenteritis cases¹⁵⁷ and individuals who have been infected can serve as reservoirs of the virus for an extended period. Currently, there is no specific antiviral therapy available for norovirus infection, and the illness is generally self-limited, meaning that it will resolve spontaneously without medical intervention¹⁵⁶. However, epidemiological investigations assessing the prevalence of norovirus infections in the United States have demonstrated that this virus imposes a substantial economic burden on society. Specifically, it has been estimated that the total cost associated with norovirus infection in the United States is approximately $10.6 billion¹⁵⁸. Given the high degree of contagiousness associated with human noroviruses, timely identification of the virus and its source is of paramount importance during an outbreak. However, the development of rapid and reliable assays for detecting infectious viruses poses significant technical challenges¹⁵⁴.

RT-qPCR was developed to detect norovirus, owing to its high sensitivity and specificity. The ISO has recommended the use of RT-qPCR as the preferred method for detecting norovirus in both food and water samples, underscoring its effectiveness and accuracy^159,160. Additionally, multiplex real-time RT-PCR has been developed to detect human pathogen genogroups of noroviruses^161,162.

Other molecular-based methods that have been developed to detect norovirus include RT-LAMP¹⁶³ and NASBA¹⁶⁴. The colorimetric LAMP assay utilizing hydroxy naphthol blue dye demonstrated a LoD of 10³ copies per reaction, comparable to conventional RT-PCR. Additionally, the assay exhibited 100% specificity when tested against other common gastroenteritis viruses¹⁶³ (Table 4). The NASBA assay exhibited high sensitivity, accurately detecting 100% of positive samples that were confirmed using electron microscopy¹⁶⁴. The assay demonstrated remarkable specificity (100%) from fecal samples. Additionally, the LoD for the assay was 5 pg/ml of RNA in fecal samples. A more sensitive RT-LAMP assay was later developed by Jeon et al.¹⁶⁵ with a LoD of 10¹ copies/µl of genogroups GI and GII human norovirus in oyster samples. The assay was reported to be highly specific with time to result of under 60 min.

Moore and Jaykus¹⁶⁶ developed an RPA assay to detect human norovirus strain GII.4. Their study found that the assay’s LoD was 3.40 ± 0.20 log¹⁰ copies and that it was more resistant to the presence of inhibitors when compared to other RT-qPCR assays. Additionally, the assay demonstrated high specificity and could be completed in under 30 min.

Rotavirus

Rotavirus is a double-stranded RNA virus, highly contagious that can cause severe diarrhea, vomiting, and fever, particularly in infants and young children. It belongs to the Reoviridae family of viruses and is one of the most common causes of gastroenteritis worldwide^167,168.

Rotavirus is primarily transmitted through the fecal-oral route and can happen through direct contact with an infected person or through contact with contaminated surfaces. The virus can also be spread through the air by coughing or sneezing and through contaminated food or water¹⁶⁷. Due to its high contagiousness, rotavirus outbreaks often occur in crowded settings, such as daycare centers, schools, and nursing homes. Vaccines are also available to protect against rotavirus infection¹⁶⁹. The cost of treating rotavirus infection may vary depending on several factors such as the severity of the illness, the location of the treatment facility, and the type of treatment received.

One of the recommended tests to detect rotavirus infection involves identifying rotavirus antigens in the stool sample using an enzyme immunoassay (EIA) or a rapid antigen detection test¹⁷⁰. Isothermal DNA amplification techniques were developed to detect rotavirus such as RT-PCR¹⁷¹ and RT-LAMP¹⁷². The RT-PCR assay was designed to detect group F adenovirus, rotavirus A, and rotavirus C in stool samples. The PCR results were evaluated by electron microscopy and latex agglutination antigen tests. The RT-PCR assay outperformed in detecting adenovirus F and rotavirus A compared to the other assays, however, rotavirus C was not detected in any samples. The LoD of the PCR assays were ten copies of target DNA/reaction for all assays.

The RT-LAMP assay was reported to be highly specific (100%) and showed higher sensitivity than the PCR assay with a detection limit of 1.26 × 10⁴ copies of standard plasmid compared to PCR with LoD of 1.26 × 10⁵ copies¹⁷² (Table 4).

An RT-RPA assay and an RT-RPA assay combined with a lateral flow strip to detect Bovine rotavirus A. The LoD of the assays was 1.4 × 10² copies per reaction and 1.4 × 10¹ copies per reaction, respectively when tested on the standard RNA of the virus. Both assays demonstrated 100% specificity when compared to RT-PCR and have shown diagnostic sensitivity of 98.39% and 100%, respectively¹⁷³.

RCA was also developed to detect rotaviruses with a sensitivity of 10³ copies using 58 different padlock probes¹⁷⁴. The assay showed comparable results to PCR assays when tested on fecal samples, but it required approximately 3.5 h to complete the entire process.

Adenovirus

Adenovirus is a double-stranded DNA virus that can cause a wide range of diseases in humans, including respiratory infections, conjunctivitis, gastroenteritis, and even severe illnesses such as pneumonia, meningitis, and hepatitis¹⁷⁵. Adenovirus is highly contagious and is spread through direct contact with infected individuals or their bodily fluids, such as respiratory secretions, saliva, or feces. The virus can also survive on surfaces for extended periods, making it possible for individuals to contract the virus by touching contaminated surfaces^175,176.

The pathogenicity of adenoviruses is due to their ability to invade host cells and replicate rapidly, leading to tissue damage and inflammation. Adenovirus infections are often characterized by fever, cough, sore throat, runny nose, diarrhea, and conjunctivitis. In severe cases, adenovirus infections can cause respiratory distress, liver failure, and even death¹⁷⁷. Certain populations, such as young children, the elderly, and individuals with weakened immune systems, are at higher risk for developing severe adenovirus infections^176,177. Treatment for adenovirus infections is typically supportive and aimed at relieving symptoms. In some cases, antiviral medications may be used to treat severe infections^175,176. However, the cost may vary based on the severity of the infection and healthcare resources¹⁷⁸.

The choice of detection method depends on factors such as the clinical sample type, method sensitivity, specificity, and available resources. PCR is often the preferred method for detecting adenovirus due to its high sensitivity and specificity. However, it cannot distinguish between infectious and non-infectious viral particles. To address this, a combination of cell culture and PCR is recommended for identifying slow-growing or non-cytopathic adenoviruses^179,180.

A TaqMan rtPCR assay was developed to detect six adenovirus species (A-F) with high sensitivity and specificity¹⁸¹. The LoD of the assay was 5 copies of AdV40, 8 copies of AdV41, and 350 copies of AdV3. In this study, a fluorescence resonance energy transfer (FRET) probe was used to differentiate between AdV40 and AdV41 serotypes by analyzing the melting curve¹⁸¹. A highly sensitive and specific multiplex PCR-enzyme hybridization assay was developed to detect all six adenovirus species with a LoD of 10²–10³ copies/ml of control DNA and 100% specificity against 12 other respiratory pathogens¹⁸². However, the assay had a relatively long turnaround time of around 5 h.

A LAMP assay was optimized by Shuryaeva et al.¹⁸³ to monitor the amplification of a specific region of the hexon gene in HAdV (Table 4). When clinical samples were tested, the assay showed a sensitivity and specificity comparable to that of PCR (LoD of 10³ copies/ml). Colorimetric and fluorescent probes LAMP both were successfully optimized to detect the LAMP assay product in the study however, the colorimetric LAMP assay showed a lower sensitivity than the PCR. In another study, Rames and Macdonald¹⁸⁴ have developed an RPA assay to detect HAdV as an indicator of viral water quality. Lateral flow detection was combined with the RPA assay with a total time to positive of 20 min. The assay showed a sensitivity of 10–50 copies of the viral plasmid and compared to qPCR the assay was 100% sensitive and specific.

The detection of HAdV was also investigated using a duplex real-time recombinase-aided amplification (RAA) assays by Wang et al. ¹⁸⁵. The HAdV 3 and HAdV 7 were targeted by RAA combined with an internal amplification control (IAC) with a LoD of 5.0 and 14.8 copies per reaction, respectively. The use of IAC improved the assay efficacy in reducing the rate of false negative results. The reactions were carried out at 39 °C and completed in 20 min.

Enterovirus

Evs are a group of single-stranded RNA viruses that primarily infect the gastrointestinal tract and can cause a range of illnesses from mild to severe^186,187. Evs are spread through contact with fecal matter, respiratory secretions, or contaminated surfaces. In severe cases, enterovirus infection can lead to meningitis, encephalitis, myocarditis, or respiratory failure^186,188. Young children, infants, and people with weakened immune systems are at a higher risk of developing severe enterovirus infection¹⁸⁸. There is no specific treatment for enterovirus infection, and treatment is usually supportive.

RT-PCR is currently the most used method for Evs detection. Jacques et al.¹⁸⁹ have developed an RT-PCR for detecting enterovirus and improved the detection limit of previous cell culture methods enhancing the clinical diagnosis of EV-related meningitis. Using minor groove binder probes and primers modified with complementary locked primer technology, Hong et al.²¹ have further improved the sensitivity of the previously developed RT-PCR assay¹⁹⁰. Compared to the previous TaqMan probe real-time RT-PCR, the assay showed higher sensitivity on clinical samples (62/73 and 29/73).

LAMP assay was optimized to detect Evs in water and stool samples targeting the highly conserved 5′ untranslated region of the human Evs with a sensitivity of ten copies of plasmid¹⁹¹. Evs were detected in all water samples tested 9/9 (100%) and 18/19 (94.7%) in stool samples collected from patients, demonstrating greater sensitivity than conventional PCR. Additionally, several studies developed RT-LAMP assay to detect different species of Evs with high sensitivity and specificity^192,193,194.

Yin et al.¹⁹⁵ developed an RPA assay to detect enterovirus 71 (EV71) with 100% specificity and 3.767 log¹⁰ genomic DNA/reaction sensitivity and performed comparable sensitivity to the commercial RT-qPCR on clinical samples. For reliable molecular detection assays, it’s crucial to employ suitable viral isolation techniques for each sample type, especially in stool samples, to eliminate potential nucleic acid inhibitors. Additionally, careful primer design is essential to ensure the detection of all enterovirus types¹⁹⁶.

Blue-green algae

Blue-green algae, or cyanobacteria, are photosynthetic prokaryotes found in various aquatic habitats, including freshwater lakes, rivers, and marine environments. While they are important for maintaining the ecological balance of aquatic systems, they can also generate harmful algal blooms (HABs) under specific environmental conditions. Certain species, such as Planktothrix rubescens, can produce potent toxins called cyanotoxins that pose a significant risk to human and animal health^197,198. Exposure to these toxins can occur through ingestion of contaminated water, inhalation of aerosolized toxins, or skin contact with contaminated water. HABs can also deplete oxygen in the water, leading to negative effects on aquatic life. Therefore, it is crucial to monitor and manage blue-green algae populations to prevent harmful blooms and protect both human health and the environment¹⁹⁷.

Health issues from blue-green algae are primarily caused by cyanotoxins. In humans, symptoms vary from skin, eye, and respiratory irritation to gastrointestinal issues like nausea, vomiting, or diarrhea, as well as headaches, dizziness, muscle weakness, and potential liver or kidney damage. In rare cases, high cyanotoxin exposure can be fatal. Those at greater risk include children, pets, and individuals with weakened immune systems^{197,199,200,201}.

In animals, exposure to blue-green algae toxins can cause a range of symptoms depending on the species and level of exposure. Some common symptoms include, difficulty breathing or respiratory distress, loss of appetite, vomiting or diarrhea, seizures or convulsions, weakness or lethargy, liver damage or failure^{197,199,200,202}. The cost of exposure to HABs can be significant, including health care expenses, lost tourism revenue, fisheries and aquaculture losses, water treatment costs, and expenditures for monitoring and research^199,203.

Monitoring and research are necessary to understand and manage HABs, which can require significant resources. Molecular techniques are utilized to identify specific types of algae, including harmful species, and to track their distribution and abundance over time. PCR methods have been developed for the detection and identification of blue-green algae to assess water quality^204,205. Johnson and Mutharasan²⁰⁶ reported the development and validation of a cantilever-based biosensor assay for detecting the presence of a toxin-producing cyanobacteria, Microcystis aeruginosa. Based on the specific hybridization of 16S rRNA probes (27-base DNA sequence) to the target cyanobacterial DNA, the study reported that the biosensor assay could detect M. aeruginosa at concentrations as low as 50 cell/ml, with a detection time of less than 3 h with low false negatives result.

The FISH technique was optimized to detect cyanotoxin-producing species from non-toxin-producing ones in freshwater targeting 16S rRNA and mcyA-mRNA using different DNA probes²⁰⁷. LAMP assay has been developed for detecting microcystin-producing cyanobacterial species. The assay targets the mcyE gene and has a LoD of 8.5 pg/μl DNA. The assay showed no cross-reactivity when tested on four non-toxic cyanobacterial strains²⁰⁸. In another study, the microcystin synthetase B gene (mcyB) was targeted using LAMP assay and demonstrated higher sensitivity than conventional PCR, with a LoD of 1 fg per reaction²⁰⁹.

Li et al.²¹⁰ demonstrated the efficacy of RPA assay to detect microcystins-producing cyanobacteria in water samples. The researchers utilized RPA combined with lateral flow strips, achieving a sensitivity of 10³ copies/ml mcyG without any cross-reactivity with non-toxin-producing cyanobacteria.

The study of Li et al.²¹¹ developed an insulated isothermal PCR to detect Raphidiopsis raciborskii—a toxin-producing cyanobacterium- with high sensitivity and specificity compared to qPCR. Using fast DNA isolation, the assay can be completed in 50 min. To enable the quantification of the DNA, the assay was optimized using a spectrometer (Andor spectrometer with Solis spectroscopy software) to measure the fluorescence intensity.

To ensure safe and accessible water for all, water managers require rapid and reliable methods for detecting waterborne pathogens to mitigate health risks. Notably, there is a growing prominence of point-of-care and point-of-application methods driven by the urgency for quicker results to enhance health risk management. Therefore, waterborne pathogen detection methods have evolved to offer speed, sensitivity, and specificity, aligning with their suitability for various applications.

Point-of-care methods using LAMP are known for their high accuracy, rapid analysis time, and cost-effectiveness compared to the gold-standard methods for pathogen detection. The LAMP assay demonstrated superior analytical sensitivity and specificity compared to conventional PCR, nPCR, and qPCR, with a LoD of one E. histolytica trophozoite, making it a highly effective platform for pathogen detection¹²⁹. Several studies have described the development of LAMP assays for rapid point-of-need testing, with completion times under 30 min^23,212. Advancements in LAMP technology have improved both the time and cost efficiency of the method^213,214. For example, integrating gold nanoparticles into LAMP assays has been demonstrated to enhance visual detection while keeping costs low, making it ideal for decentralized and resource-constrained laboratories²¹⁵. LAMP is highlighted as a rapid, sensitive, and specific method that is particularly beneficial in developing and low-income countries due to its affordability and ease of use without the need for sophisticated lab equipment or skilled personnel²¹⁴. For diseases like the Zika virus, where there is an urgent need for PoC diagnostics in resource-poor settings, LAMP has been recognized as a low-cost molecular system that can be freeze-dried for distribution, offering high specificity and sensitivity²¹³.

Advancements in LAMP assay for detecting eDNA and waterborne pathogens in environmental waters

LAMP simplifies pathogen detection by eliminating the need for complex DNA isolation. It can be applied directly to minimally processed samples, making it suitable for on-site use and streamlining the detection process (Table 4). For instance, an in-field LAMP assay was developed to detect Bacteroides, which serves as an indicator of human fecal contamination in environmental water²³. In this study, the bacterial cells from water samples were lysed in the field using KOH, streamlining the process for on-site completion.

Combining the LAMP assay with lateral flow devices or smartphones facilitates simple, cost-effective pathogen detection. This integration underscores the assay’s suitability for point-of-care applications, ultimately contributing to improved public health²¹⁶. For example, Thio et al.²¹⁷ developed a smartphone-based lab-on-a-chip system designed for on-site E. coli detection, for assessing fecal contamination in environmental waters. This system incorporated a plasmonic-enhanced optoelectrowetting device, a transparent heater, and a smartphone, all integrated to enable on-chip water quality monitoring utilizing the LAMP technique.

Microfluidic devices can revolutionize water quality monitoring by providing high sensitivity and rapid detection in a portable and cost-effective manner. Its advantages, including miniaturization, in-situ analysis, multiplexing, precise control, and cost-effective fabrication, are propelling microfluidics to the forefront of water monitoring²¹⁸. This technology facilitates a quick and effective response to water contamination, even in urgent scenarios, making it a promising tool for ensuring water safety. For instance, Jin et al.²¹⁹ optimized a microfluidic chip that incorporates LAMP (referred to as on-chip LAMP) for simultaneous detection of ten pathogenic bacteria typically found in coastal waters. The limits of detection for this method ranged from 7.92 × 10⁻³ picograms to 9.54 × 10⁻¹ picograms of pure bacterial genomic DNA per reaction. This dual-sample on-chip LAMP assay demonstrated its effectiveness for analyzing multiple waterborne bacterial pathogens, making it suitable for on-site detection and routine monitoring of aquatic environments.

Drawbacks of using LAMP for point-of-care testing in pathogen detection

While LAMP is known for its simplicity in terms of operation, interpreting the results can sometimes be challenging, especially for inexperienced users. The presence of non-specific amplification or primer–primer annealing may lead to false-positive results, requiring careful analysis^220,221. Similar to other nucleic acid amplification techniques, LAMP is susceptible to contamination issues or cross-contamination^65,222,223, Contamination can lead to false-positive results, which can be particularly problematic in point-of-care settings where quick and accurate diagnosis is crucial. LAMP is generally less amenable to multiplexing compared to other PCR-based techniques²²⁴. This limitation can restrict the ability to detect multiple pathogens simultaneously, which may be necessary for certain diagnostic applications. Another drawback is the perceived complexity of the methodology, which necessitates a sophisticated primer design system. This complexity can limit the selection of target sites and affect the specificity and sensitivity^223,225. While LAMP is generally considered simpler to perform than traditional PCR, it still requires a certain level of training and skill to ensure reliable results. This can be a limiting factor in settings where trained personnel are not readily available. For example, users must be cautious to avoid sample contamination and follow aseptic techniques carefully²²².

Water sample preparation and in-field nucleic acid isolation for LAMP assay

Water sample preparation is crucial for accurate and timely water quality assessment. Efficient preparation and nucleic acid isolation are key to successful molecular assays. To optimize this process, it is imperative to explore novel and streamlined methodologies for in-field sample preparation and nucleic acid isolation. Emphasis should be placed on enhancing the sensitivity, specificity, and speed of these processes, ensuring their suitability for on-site applications. The development of user-friendly, portable, and cost-effective tools and protocols is essential to empower a broader range of professionals and field operators in conducting real-time environmental monitoring and pathogen detection.

Microbial pathogen and eDNA detection in water samples often require target enrichment due to the typically low quantities of these targets. This process involves filtering large volumes of water to concentrate the target organisms on a membrane filter, which can be achieved using a vacuum pump and a filtration apparatus with replaceable membranes (Fig. 6)^60,226,227. In remote and resource-limited settings, a portable pumping system is an accessible option for water filtration²²⁸. In scenarios involving smaller water volumes, employing disposable syringe filters is a practical alternative (Fig. 7)^23,24.

Fig. 6: Water sample filtration using a backpack pump system.

A backpack pump system is used to filter water samples from a body of water. The inset illustrates the components of the filtration setup, including the filter holder and filter membrane, which are used to trap and collect cells from the water sample. The filter membrane can then be removed from the filter holder for further analysis. This filtration protocol allows for filtering a higher volume of water compared to the syringe filtration protocol.

Fig. 7: Water sample processing using a syringe filtration protocol.

A Drawing a water sample into a 60 ml syringe. B Passing the water sample through a syringe filter to trap cells on the membrane. C Back-flushing the filter with ~0.2 ml elution buffer using the syringe to collect any retained cells and eDNA. D Collect the eluted sample in a collection tube for further analysis.

It’s worth highlighting that the maximum water filtration volume is contingent on factors such as water turbidity and the filter membrane’s pore size. For instance, a filter membrane with a 0.2 μm pore size may become clogged even when filtering a small volume of water²²⁷. An eDNA analysis study demonstrated that filtering larger water volumes enhances the accuracy of eDNA recovery but can lead to filter clogging⁶⁰. To overcome this issue, the study recommended combining multiple filters during the isolation process, significantly improving eDNA yield. Notably, the CTAB-PCI method proved effective in increasing eDNA yield and removing inhibitors. However, these steps introduced complexity and time consumption, making on-site isolation less practical.

The eDNA recovers from filtration is also depends on filtration pressure and water volume filtered. Thomas et al.²²⁸ found a complex relationship between filtration pressure, water volume, and eDNA retention on the filter surface. Higher pressure led to a 39% decrease in eDNA concentration per liter of filtered water. While the impact of pressure was somewhat weak in their small sample, it hinted that higher pressures might reduce eDNA retention, possibly due to cell lysis. However, the decrease in eDNA retention was offset by an increased water volume that could be filtered at higher pressures, explaining the lack of a straightforward relationship between filtration pressure and total eDNA per filter. Additionally, the filtration efficiency peaked at a flow rate of 1.0 l/m, and the 5 μm filters captured significantly more target eDNA than the 1 μm filters.

Various pathogenic microorganisms necessitate distinct filtration methods. Francy et al.²²⁹ conducted a study to identify efficient filtration methods for the simultaneous sampling of various classes of pathogens (bacteria, viruses, and protozoa) in recreational lake water studies. Among the different filtration techniques tested, automatic ultrafiltration (UF) showed the highest median recoveries for E. coli, Enterococci, and protozoa. Glass wool filtration performed consistently well in recovering enteric viruses from lake water samples, particularly for adenovirus. The automatic UF method proved efficient for sampling large volumes of lake water, even in the presence of high turbidity or algae, and could be used for regional public health risk studies at beaches.

In-field implementation of isothermal nucleic acid assays faces challenges due to equipment and expertise requirements, limiting their use for real-time monitoring in resource-limited settings. To overcome this, studies have improved in-field DNA isolation methods. Lee et al.²³⁰ research enhanced DNA extraction using magnetic beads to detect FIB (i.e., E. coli and E. faecalis), improving purity, reducing hands-on work, and reagent use. This method offers high throughput, low contamination risk, and ease of use. Potential PCR inhibition was addressed using bovine serum albumin (BSA) and DNA dilution, enabling efficient FIB detection through LAMP assays. BSA is used in DNA amplification sample preparation to stabilize and protect DNA from degradation, reduce interference by inhibitors, and prevent non-specific binding²³¹.

He et al.²³² introduced an integrated colorimetric RT-LAMP assay for detecting SARS-CoV-2 by combining viral RNA purification, amplification, and detection within a single tube using a colorimetric RT-LAMP. Silicon hydroxyl magnetic beads (Si–OH MBs) were used for nucleic acid extraction, with 200-nm MBs showing the highest RNA extraction efficiency. The assay was optimized with a 5-min incubation time for efficient RNA capture and improved sensitivity was enhanced by increasing the initial sample volume. Furthermore, the RT-LAMP reaction exhibited robustness in the presence of Si–OH MBs, hinting at potential improvements.

A KOH-based method was employed by Hulse et al.²³³ to lyse Chlamydia pecorum cells from swab samples, releasing genomic DNA into the solution for direct use in the LAMP reaction. Similarly, KOH was used to lyse Bacteroides from syringe filters, with the lysate directly employed in the LAMP reaction for MST in environmental waters²³.

Alkaline polyethylene glycol (alkaline-PEG) serves as an effective and rapid field-deployable lysis buffer for efficient sample processing that is compatible with LAMP assays. Successful utilization of alkaline-PEG was demonstrated for detecting Dichelobacter nodosus in sheep swab samples using LAMP assay⁴³. This method was observed to effectively adjust the pH to a more suitable range for LAMP DNA amplification while also mitigating the presence of inhibitors (due to sample dilution). However, Alkaline-PEG may not be as effective when targeting low-concentration DNA, such as eDNA detection in water samples.

Heat treatment at 95 °C has proven effective in releasing viral DNA without the necessity of prior extraction, facilitating successful DNA amplification techniques^234,235. Although this method is suitable for direct point-of-care diagnostics, it’s primarily effective for detecting viruses and some bacteria. Therefore, careful consideration is essential when opting for heat treatment as a lysis step, depending on the specific diagnostic needs.

In eDNA monitoring, effectively communicating the sensitivity of sampling protocols is vital for resource managers. Schultz and Lance²³⁶ explored a simulation model to estimate sensitivity and assessed the impact of protocol changes on detection limits. Low target species density can result in significant false negatives in eDNA surveys. The study considered factors affecting sensitivity, such as target marker distribution, and suggested various strategies to reduce false negatives, including increasing replicates, sample volume, and sample numbers. It underscores the need for a balanced sampling protocol that considers sensitivity, feasibility, and cost to enhance survey accuracy, particularly when targeting low ambient target marker concentrations.

In general, ensuring precise and timely water quality assessment and pathogen detection relies on effective water sample preparation. Simplifying sample preparation is essential for streamlined in-field diagnostics, reducing time and complexity, and increasing the feasibility of real-time monitoring, especially in resource-limited settings.

Future applications of LAMP in analyzing waterborne pathogens

Molecular diagnostic tools may be further advanced by integrating them with other technologies, such as biosensors and artificial intelligence (AI), which are expected to enhance detection accuracy and enable real-time analysis, particularly beneficial for monitoring water quality¹⁷. Molecular diagnostic methods, including LAMP, are increasingly utilized for rapid and robust microbial water quality assessment, with ongoing technological advancements expanding their applications²³⁷. LAMP has demonstrated high efficiency in detecting Vibrio parahaemolyticus in various infection models, suggesting its potential as a primary method for rapid pathogen detection in clinical and environmental settings²³⁸. The future use of LAMP technology is likely to focus on field employability, integration with sample preparation, and automatic result analysis using smartphones, facilitated by technological innovations and integration with other advanced methods such as biosensors and AI. This trend indicates a shift towards more rapid, accurate, and on-site pathogen detection, which could significantly impact public health and water safety monitoring. Biosensors can provide rapid, on-site analysis, reducing the need for centralized laboratory facilities. For example, biosensors have combined LAMP with electrochemical detection for the rapid and sensitive detection of digestive bacterial pathogens such as E. coli and Salmonella²³⁹.

AI can improve the accuracy and efficiency of LAMP-based pathogen detection by analyzing complex data patterns. AI algorithms can interpret LAMP results, identify specific pathogens, and even predict potential outbreaks²⁴⁰. For instance, AI can be used to analyze the results of LAMP assays for waterborne pathogens and provide real-time alerts to water management authorities in case of contamination. Expanding beyond diagnostics at the individual level, PoC devices can leverage integrated blockchain technology²⁴¹. This integration allows PoC devices to function as decentralized and interoperable systems, ensuring data security. Consequently, PoC devices with blockchain integration can quickly map outbreaks and support informed public health responses²²¹.

The evolution of LAMP-on-a-chip technology, combining the LAMP technique with microfluidic chips, presents a significant stride forward in pathogen detection. Classical microfluidic chips, such as those made from polymethyl methacrylate⁸¹ and poly-dimethylsiloxane⁸², provide a protected environment for testing, reducing the risk of cross-contamination. Paper-based and paper-polymer hybrid microfluidic devices offer cost-effective alternatives with high biocompatibility and easy fabrication. Fully integrated chips combine nucleic acid extraction, purification, and LAMP amplification in a single device, enabling on-site detection, especially in resource-limited settings²⁴².

Conclusion

Waterborne pathogens present a substantial concern for both public health and environmental well-being. Ensuring the timely and precise detection of these pathogens is of paramount importance for effective risk management. In response to the pressing need for expedited results and enhanced water quality monitoring, the prominence of point-of-care and point-of-application methods is on the rise. Current approaches, including culture-based methods, offer invaluable insights into microorganism types and quantities, yet are hampered by the laborious nature of their processes and their dependency on specialized equipment and expertise. Molecular techniques, exemplified by PCR, excel in sensitivity and speed but are often confined to centralized laboratory facilities, incurring associated expenses. In contrast, the integration of modern isothermal techniques such as LAMP with portable or microfluidic devices, along with the development of in-field sample preparation and DNA isolation methods, offers a promising trajectory for water monitoring and pathogen detection. This progress is particularly vital in resource-limited settings, where conventional methods may not be practical. Efficient water sample preparation emerges as essential for accurate water quality assessments. The process of target enrichment through filtration techniques is indispensable, with various filtration methods tailored to distinct pathogen categories. Advancements in waterborne pathogen detection, extending from streamlined sample preparation to on-site nucleic acid isolation, are pivotal in enhancing sensitivity, specificity, cost-effectiveness, simplicity, and rapidity. These innovations ultimately underpin the assurance of safe and accessible water for all.