Parasitic diagnosis: A journey from basic microscopy to cutting-edge technology

Abstract

Parasitic infections pose a significant public health challenge, particularly in tropical and subtropical regions, where they affect nearly a quarter of the global population. These infections can lead to various health issues, including malnutrition, anemia, and increased susceptibility to other diseases, thereby hindering development efforts. The World Health Organization highlights that a significant proportion of neglected tropical diseases are parasitic, underscoring the need for improved diagnostic methods. Early microscopy and staining techniques laid the groundwork for identifying parasites, paving the way for modern diagnostic approaches. Serodiagnostics have progressed from early 20^th-century tests to more advanced techniques, such as enzyme-linked immunosorbent assays and immunoblot. However, challenges remain, such as cross-reactivity and the difficulty in distinguishing between past and current infections. Currently, molecular diagnostics, utilizing technologies such as polymerase chain reaction, multiplex assays, and next-generation sequencing are increasingly used to improve sensitivity and specificity in detecting parasites. In the years to come, there is a growing emphasis on integrating artificial intelligence and deep learning, particularly convolutional neural networks, which are revolutionizing parasitic diagnostics by enhancing detection accuracy and efficiency. Innovative imaging technologies are enabling faster identification of parasites and addressing traditional diagnostic limitations. However, challenges persist, including the need for diverse datasets and infrastructure support in low-resource settings. Continued research and development are essential to overcome these obstacles and ensure better global health outcomes in the face of evolving parasitic threats.

INTRODUCTION

Parasitic infections remain a critical yet often neglected aspect of global health, particularly in tropical and subtropical regions where they significantly impact public health and economic stability.[1] These infections result in various health issues, including malnutrition, anemia, and increased susceptibility to diseases that hinder development efforts in affected regions.[2] The global burden of parasitic infections is significant, affecting nearly one-quarter of the world’s population. These infections, encompassing neglected tropical diseases (NTDs) and zoonotic diseases transmitted from animals, contribute substantially to illness and death.[3] Notably, out of the 20 NTDs listed by the World Health Organization (WHO), 13 are caused by parasites.[4]

Furthermore, parasitic infections account for four out of eight neglected zoonotic diseases listed by the WHO in 2013. These include cysticercosis, cystic echinococcosis (CE), leishmaniasis, and trypanosomiasis. One study estimated that approximately 151 million cases of schistosomiasis, 633,404 cases of CE, 4.36 million cases of cysticercosis, and about 44.47 million cases of food-borne trematodes occur globally in 2021.[5] These infections lead to impaired cognitive and physical development in children, perpetuating the cycle of poverty.[6] Chronic infections reduce productivity and learning, further entrenching socioeconomic disparities.

More than a parasite, the interplay between parasitic infections and other diseases complicates treatment and control efforts. Such interplay is complex and multifaceted, involving interactions with host immunity, microbiota, and co-infections with other pathogens. For example, parasitic infections can manipulate the host’s immune response, affecting susceptibility to other pathogens, such as HIV and tuberculosis.[7] Intestinal parasitic infections can increase susceptibility to tuberculosis by altering immune responses and nutritional status, leading to worse treatment outcomes.[8] Co-infections with bacteria and parasites can lead to secondary infections, complicating the clinical picture, as observed in malaria and visceral leishmaniasis.[9]

Collectively, these parasitic infections drain enormous money from developing economies, perpetuating cycles of poverty and disease. The economic burden is particularly severe in regions with high prevalence, where healthcare resources are already limited. This impacts healthcare systems, productivity, and overall economic stability. Various studies highlight the direct and indirect costs associated with different parasitic infections, revealing a substantial financial toll on affected populations. A recent study reported that India spends 0.34 of the total consumption expenditure on infectious diseases that includes parasitic infections.[10] In 2014, India lost US$ 1940 million to counter malaria.[11] In the state of Bihar, visceral leishmaniasis drains 11% of annual household expenditures, and prompting families to take load to cover expenses.[12] In the US, the economic burden from ectoparasitic infections (Pediculosis pubis and Scabies infections) is considerable, with outpatient treatment costs contributing significantly to healthcare expenses.[13] Likewise, in Iran, the annual cost associated with this infection is estimated at US$2.3 billion, including treatment and productivity losses.[14] In addition, parasitic infections have indirect economic impacts. For example, in Latin America, porcine cysticercosis results in economic losses exceeding US$164 million, affecting livestock production and human health costs.[15] India’s dairy production incurs a loss of US$787.63 million due to ticks and tick-borne diseases.[16] Likewise, neurocysticercosis leads to substantial societal costs, including healthcare and lost productivity, with estimates of over US$400 million annually in the US alone.[17]

DRIVERS FOR PARASITIC INFECTIONS

While the focus on parasitic infections is often overshadowed by more prominent diseases like viral and bacterial, their significant health and economic impacts warrant urgent attention and resource allocation to mitigate their effects on vulnerable populations. However, despite long-standing efforts to improve public health, multiple catalysts for spreading parasites exist. These are broadly influenced by parasites’ internal factors (life cycle and drug resistance) and external factors such as socioeconomic and environmental factors.

Internal factors

Parasites with complex life cycles often involve multiple hosts, which complicates their control and eradication. While the complexity of these life cycles poses significant challenges for control, it also highlights the adaptability of parasites. These life cycles can include various stages in different hosts and environments, making it challenging to target all potential reservoirs of infection. Parasites frequently manipulate host behavior to enhance their transmission, affecting predator-prey dynamics.[17] Coinfecting parasites may have conflicting interests, which can stabilize predator-prey systems rather than lead to extinction. Many zoonotic parasites, such as Taenia spp. and Toxoplasma gondii, have complex life cycles that span multiple food chains, complicating food safety measures.[18] Increased human-wildlife interactions heighten the risk of transmission, as these parasites can persist in various environments. Helminths adaptively divide growth between hosts, optimizing their life cycles for successful transmission and reproduction.[19] Longer life cycles can enhance transmission rates, often involving smaller, more abundant first hosts and larger definitive hosts.

The emergence of drug resistance in parasites poses significant challenges to public health, particularly in the context of NTDs and malaria. Research indicates that aggressive treatment strategies can inadvertently accelerate the evolution of drug-resistant strains, complicating control efforts. Understanding the mechanisms behind this resistance is important for developing effective treatment protocols. The genetic variability among parasites contributes to their ability to develop resistance, particularly in malaria, where competition between drug-sensitive and resistant strains is evident.[20] Parasites employ various mechanisms, such as altered drug uptake and metabolism, to survive against chemotherapeutic agents.[21] Moreover, long-term drug use produces various adverse effects. Continuous reliance on specific drugs, such as macrocyclic lactones for filarial infections, has led to resistance in certain regions.[22] While the Mass Drug Administration has effectively controlled helminth infections, prolonged use raises concerns about potential resistance.[23]

External factors

Parasitic diseases, particularly soil-transmitted helminths (STHs), are significantly influenced by poverty and poor sanitation, especially in low- and middle-income countries (LMICs). These conditions create an environment conducive to the transmission of parasites, leading to severe health consequences, particularly among vulnerable populations such as children. Poverty is a critical determinant of STH prevalence, with nearly one billion people affected globally.[24] Socioeconomic vulnerability correlates with increased transmission risk, as in Brazil, where lower GDP was linked to higher disease likelihood. In the U.S., areas with persistent poverty and inadequate sanitation still report high rates of hookworm infections.[25] Poor sanitation practices, such as open defecation and inadequate waste management, facilitate STH transmission. A study in Argentina found that unimproved sanitation significantly increased the odds of skin-penetrating STH infections.[6] The presence of raw sewage in homes was reported in 42.4% of households in a US study, highlighting the sanitation crisis.[26] Intestinal parasitic infections are prevalent in India and are significantly influenced by poverty and poor sanitation.[27] These infections are endemic and contribute to major health issues, particularly among school-age children, leading to nutritional deficiencies, anemia, and impaired learning. Factors such as low personal hygiene standards and the hot, humid tropical climate further exacerbate their spread.

Environmental factors like climate change can influence the distribution and transmission of parasites.[28] The steady and ongoing change in climatic patterns across the globe is triggering a cascade of climate-adaptive phenomena, both genetic and behavioral, in parasites and influencing the host-pathogen–transmission triangle. Parasite and vector traits are now heavily influenced by increasing temperatures that almost dissolved geospatial boundaries and impacted the basic reproductive number of parasites. Various studies indicate that alterations in temperature, rainfall, and host movement due to climate change can create favorable conditions for parasites, leading to increased transmission rates. Climate change affects the habitats of vectors such as triatomine insects, which transmit Trypanosoma cruzi, the causative agent of Chagas disease. Studies have shown that global warming can favor and hinder these vectors’ distribution in North and Central America.[29] In Australia, rising temperatures and altered rainfall patterns have been linked to increased habitats for parasites and their vectors, contributing to diseases like the Ross River virus and malaria.[30] Host movement plays a crucial role in parasite transmission dynamics. For instance, in montane ungulates, the movement of wildlife and livestock under changing climatic conditions significantly influences gastrointestinal nematode transmission.[31] Seasonal variations in temperature and rainfall also affect schistosomiasis transmission, with specific temperature ranges promoting parasite development.[32] Climate change has led to an increased prevalence of parasitic infections in urban areas, where improved sanitation may not fully mitigate the risks posed by climate-induced changes in parasite distribution.[33]

THE DIAGNOSTICS JOURNEY IN PARASITOLOGY

Amid rising risks of parasitic diseases across the globe, accurate and timely diagnosis is essential for effective treatment, disease control, and individual well-being. By enabling tailored therapy, preventing drug resistance, and facilitating surveillance efforts, parasite diagnosis plays a crucial role in reducing the burden of these diseases. Furthermore, early diagnosis and treatment can alleviate symptoms, prevent complications, and improve patients’ quality of life. In addition, accurate diagnosis can contribute to economic growth by reducing healthcare costs and improving workforce productivity. The diagnosis of parasitic infections has evolved significantly over centuries, driven by technological advancements and a deeper understanding of these microscopic organisms.

The dawn of the microscopic era in parasitology

The 17^th century marked a pivotal moment in the history of parasitology, thanks to the invention of the microscope. This revolutionary device, pioneered by Antonie van Leeuwenhoek, enabled researchers to visualize the microscopic world, including the intricate forms of parasites. Before the advent of the microscope, parasitic infections were often poorly understood and misdiagnosed. Symptoms were attributed to supernatural forces or imbalances in bodily humor. With the aid of the microscope, scientists began to observe and identify various parasites, such as protozoa, helminths, and arthropods, which caused a wide range of diseases.

In the early days of microscopy, the primary tool used for parasite visualization was a simple microscope. These early microscopes were relatively crude compared to modern instruments, but they were sufficient for observing large parasites and their basic structures. The magnification power of these early microscopes was limited, typically ranging from 10× to 40×. This level of magnification allowed scientists to demonstrate the overall morphology of parasites. However, it was not enough to resolve fine details of their internal structures.

Despite this, light microscopy has been extensively used to examine stool samples and identify parasites through appropriate training.[34] Staining is essential for differentiating parasites from the background material in fecal smears. Fresh and preserved specimens are analyzed, often requiring various stains and reagents. Common stains include trichrome and iron hematoxylin for protozoan cysts and trophozoites. In contrast, blood films are typically stained with Giemsa or Wright’s stain. The Giemsa stain requires prior fixation with absolute methanol, and thick films may need laking in distilled water or treatment with saponin before staining.[35] Likewise, in a study, Parija and Prabhakar investigated the effectiveness of lactophenol cotton blue (LPCB) stain in preparing wet mounts of stool samples to identify intestinal parasites through routine microscopy.[36] The findings indicate that LPCB stain is highly effective in highlighting trophozoites, cysts, and helminthic ova, making them easily detectable and identifiable in stool samples. As a result, the authors recommended the routine use of LPCB wet mount in parasitology laboratories for stool sample analysis. The group further confirmed this in another study involving LPCB staining in detecting Cyclospora and Isospora oocysts in stool samples.[36] LPCB staining successfully highlighted these parasites colored blue, allowing for clear differentiation of their internal structures, which aids in accurate identification. However, LPCB was not effective in identifying Cryptosporidium. The study suggests that LPCB is a useful method in laboratories that do not routinely perform acid-fast staining, particularly in rural or peripheral health centers in developing countries.

A study highlighted that Cryptosporidium spp. and Microsporidia have been the main research subjects over the past 40 years, with investigations into factors such as refractivity, staining unevenness, size, and temperature to improve staining protocols.[37] It concludes that while there is a wealth of studies on the efficacy of staining methods, there is a limited understanding of the physical–chemical interactions between dyes and parasites, suggesting the potential for computational tools to enhance interpretation accuracy in fecal smear analysis. Moreover, studying stool samples has low sensitivity, often requiring serial samples and specialized skills for accurate visualization.

Despite these limitations, the simple microscope proved to be a powerful tool for advancing the field of parasitology. It enabled scientists to make groundbreaking discoveries that laid the foundation for modern parasitology, enabling scientists to develop diagnostic techniques, treatments, and preventive measures to combat parasitic diseases. Leeuwenhoek observed and described various protozoa responsible for diseases like giardiasis. The microscope allowed for the detailed study of parasitic worms, such as roundworms, tapeworms, and flukes. By examining different life stages of parasites, researchers gained insights into their complex life cycles, which was crucial for developing effective control strategies.

Lately, scanning electron microscopy has been used to observe and study parasitic protozoa for its superior resolution, which enables detailed structural observations of parasites.[38] Environmental scanning electron microscopy (ESEM) is a valuable tool for taxonomically documenting helminth parasites, including roundworms, tapeworms, flukes, and spiny-headed worms. ESEM allows for observing samples in their natural, hydrated state without extensive sample preparation, such as drying or coating, which can alter the sample’s structure.[39] Moreover, ESEM can simulate various environmental conditions within the specimen chamber, providing insights into how parasites might behave in different environments. This capability is crucial for studying delicate parasitic structures and interactions with host organisms. Several noncommercial systems such as ESEM AQUASEM II, have been reported, allowing for controlled dynamic conditions during observation, preserving the morphological features of the parasites that are crucial for species determination, and enabling further molecular studies postexamination.[39] ESEM has been employed to study the interactions between parasites and host immune cells, providing insights into immune responses and parasite survivability. For instance, it has been used to observe how immune cells from different sheep breeds interact with helminth parasites, revealing breed-specific immune responses.[40] While ESEM provides unique advantages, it is often used with other microscopy techniques like STEM and HIM to provide a comprehensive view of parasitic structures and interactions. These techniques offer high-resolution imaging and 3D modeling capabilities, which are invaluable for detailed structural analysis.[38,41]

Although electron microscopy is a powerful tool in parasitic diagnostics, its application faces several challenges that limit its widespread use. It requires meticulous sample preparation, which is labor-intensive and time-consuming. Transmission EM preparation can take 1–3 days, while volume EM can take 3–5 days, involving frequent manual reagent exchanges.[42] A notable shortage of trained EM technologists and pathologists proficient in ultrastructural interpretation hampers the ability to effectively maintain and operate EM services. The costs associated with maintaining EM facilities are high, which can be prohibitive for many institutions, especially those with low caseloads that might not justify the expense.[43]

Serodiagnostics

Serological diagnostics for parasitic infections involve detecting specific antibodies or antigens in the blood, providing a noninvasive method to diagnose infections. These diagnostics are crucial for identifying both current and past infections, especially for tissue-invading parasites where direct recovery of the parasite is challenging.

The journey of serological tests can be traced back to the early 20^th century when the Wassermann test for syphilis laid the foundation for future parasitological applications. From the mid-20^th century, serological tests for parasitic diseases such as malaria, schistosomiasis, and trypanosomiasis started gaining prominence. Initially, serodiagnosis was primarily used for indirect diagnosis, especially when direct observation of parasites was challenging. Over time, the development of various serological tests has enhanced the ability to detect parasitic diseases early, even before clinical symptoms appear. This evolution has been marked by the introducing of numerous immunodiagnostic techniques and the refining of existing methods to increase sensitivity and specificity.

For parasitic diagnostics, early serodiagnostic methods included complement fixation tests and immunodiffusion, which laid the groundwork for more advanced techniques.[44] Afterward, the enzyme-linked immunosorbent assay (ELISA) and its variants, such as sandwich ELISA and competitive ELISA, have become standard due to their high sensitivity and ability to screen for multiple parasitic infections.[45] This is the most used serological test for diagnosing tissue-invading parasites such as clonorchiasis, paragonimiasis, cysticercosis, and sparganosis. It utilizes various antigens to detect specific IgGs in patient sera or cerebrospinal fluid (CSF).[46]

While serological diagnostics are efficient in detecting parasitic infections, they are not without limitations. A significant challenge in serological diagnostics is the potential for cross-reactivity with other pathogens, which can lead to false positives.[45,47] Likewise, the antibody-based serodiagnostic test’s inability to differentiate between past and current infections complicates diagnosis where the presence of antibodies may indicate past exposure rather than current infection.[48]

This necessitates the use of antigen-based serological tests. For example, histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) detect the HRP2 antigen, which is only present in circulation during active infection with the Plasmodium falciparum parasite.[49] Furthermore, serological outcomes need careful interpretation in conjunction with clinical findings.[50] It is recommended to correlate findings with the patient’s history, clinical signs, and other laboratory findings, especially in cases of unexplained eosinophilia.[45] In addition, ensuring the quality and reliability of serological tests requires standardization and technical regulation, as these tests are critical for public health and epidemiological surveillance.[51]

Despite limitations, serological RDTs that are immunochromatographic lateral flow devices have emerged as crucial tools in the diagnosis of parasitic infections. These tests offer a practical alternative to traditional microscopic methods that require specialized equipment and expertise. These tests are particularly valuable in resource-limited settings where access to laboratory facilities is restricted. The development and application of these tests have been explored for various parasitic diseases, including malaria, Chagas disease, and visceral leishmaniasis, among others. For example, a study involving Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas) achieving a sensitivity of 94% and specificity of 91% for diagnosing T. cruzi infection in dogs.[52] The test effectively identifies infections across various T. cruzi discrete typing units. It shows no cross-reactions with other tested parasites, making it a reliable, low-cost option for rapid serological diagnosis in both domestic and wild canids. Likewise, Mahdavi et al. 2023 discussed the development of a novel serological RDT for visceral leishmaniasis (VL) using a recombinant kinesin antigen (rKLi8.3).[50] This antigen was evaluated through ELISA and lateral flow test on sera from patients with VL and other diseases. The rKLi8.3-based tests demonstrated improved sensitivity (up to 97.1%) and specificity (up to 99.2%) without cross-reactivity with other parasitic diseases, enhancing diagnostic efficiency in endemic regions. In endemic and low-resource settings, WHO recommends using RDT for the detection of malarial parasites. In fact, in 2020, the WHO prequalified RDT manufacturers and supplied 419 million malaria RDTs across the countries in need. To date, there have been 24 WHO-prequalified RDTs available for commercial use.[53]

Molecular diagnostics

Molecular diagnostics have revolutionized the field of parasitology, offering several advantages over traditional methods.[54] These techniques provide increased sensitivity and specificity, enabling early detection and accurate identification of parasites, even in low-level infections. Molecular diagnostics leverage technologies such as polymerase chain reaction (PCR), multiplex assays, and next-generation sequencing to improve sensitivity and specificity in detecting parasites. These methods are particularly beneficial in identifying mixed infections and drug-resistant strains, which are challenging to diagnose with conventional methods. These tests are rapid, providing results within hours, accelerating patient care, and allowing for timely treatment initiation. They are versatile, applicable to a wide range of parasites, and can be adapted to detect emerging parasites or drug-resistant strains. Targeted genes for primer have been developed for genes such as 18S rRNA, internal transcribed spacer regions, and mitochondrial DNAs.[54] In addition, they often require only small, noninvasive samples, minimizing patient discomfort. These techniques are used for diagnosis, drug resistance testing, epidemiological studies, parasite characterization, and vaccine development.

PCR and its variants, such as nested PCR and real-time PCR, are widely used for their high sensitivity and specificity.[55] They are particularly effective in detecting low-level parasitemia and mixed infections, which are often missed by microscopy.[56] In contrast, multiplex PCR assays allow simultaneous detection of multiple pathogens in a single test, enhancing diagnostic efficiency. For instance, multiplex panels have been developed for Plasmodium species, Entamoeba species, etc., enabling accurate species differentiation that is crucial for appropriate treatment.[57,58] Likewise, Loop-mediated isothermal amplification (LAMP) is a cost-effective and rapid alternative to PCR, suitable for resource-limited settings. It does not require sophisticated equipment, making it accessible for field diagnostics.[54]

The introduction of molecular diagnostics in parasitology can be traced back to the late 20^th century, with the advent of techniques like PCR. While specific DNA probes were used in research and diagnostics before PCR, their sensitivity was limited by the absence of amplification. PCR revolutionized the field by enabling the specific amplification of minute amounts of parasite DNA, significantly enhancing detection capabilities. In the early 1990s, PCR-based methods began to be applied to various parasitic infections, initially focusing on tissue parasites such as those causing malaria, leishmaniasis, trypanosomiasis, and toxoplasmosis. Over the past few decades, the application of molecular techniques has expanded to include a wider array of parasites, and these methods have become increasingly integrated into clinical microbiology laboratories.

Today, molecular diagnostics has been considered the gold standard for almost all parasites. Molecular diagnostics have transformed malaria detection, especially in identifying drug-resistant strains and mixed infections. Techniques such as PCR and LAMP are pivotal in areas where RDTs fail due to genetic deletions in the parasite. Likewise, molecular methods, such PCR, have improved the differentiation of Entamoeba histolytica from non-pathogenic species, which is critical for accurate diagnosis and treatment.[55,59] In another study, Parija and Khairnar investigated a novel, non-invasive method for diagnosing amoebic liver abscess (ALA) by detecting E. histolytica DNA in urine samples using PCR.[60] This approach is significant because traditional methods require invasive procedures such as collecting blood or liver abscess pus. The research found that E. histolytica DNA was present in 39.6% of urine samples from ALA patients, suggesting that the kidney barrier allows the passage of this DNA molecule. In addition, the PCR method can be used as a prognostic marker to monitor the disease’s progression following metronidazole treatment. This innovative diagnostic method could reduce the need for invasive procedures in diagnosing ALA. Advances in molecular diagnostics have also impacted veterinary parasitology, aiding in the early detection of parasites and the development of targeted control strategies, including vaccines.[61]

While challenges like standardization and cost-effectiveness remain, the future of molecular diagnostics in parasitology holds promise for further advancements, including point-of-care testing and integration with traditional methods, ultimately improving the diagnosis and management of parasitic diseases.

Artificial intelligence and deep tech

Artificial intelligence (AI) and deep tech are rapidly evolving fields with significant implications across various sectors. AI, mainly through deep learning, is transforming industries by enabling machines to learn and make decisions independently, thereby enhancing efficiency and innovation. Deep learning, a subset of machine learning, uses neural networks to process large datasets, allowing for high-level model abstractions and nonlinear transformations.[62] This has led to significant advancements in AI capabilities. AI, machine learning, and deep learning are catalysts for Industry 4.0 and 5.0, driving sustainability, efficiency, and innovation. These technologies enable continuous monitoring, predictive maintenance, and improved product quality, contributing to more resilient and adaptive industrial ecosystems.

These technologies are revolutionizing parasite diagnostics by enhancing the accuracy, efficiency, and accessibility of diagnostic methods.[62] The integration of AI, particularly deep learning models like convolutional neural networks, has been successfully applied to automate the detection and classification of parasites from microscopic images.[63] This automation reduces human error and speeds up the diagnostic process, leading to improved patient outcomes. AI has been applied to various parasitic diseases, including malaria, leishmaniasis, and trypanosomiasis. For instance, AI models have shown high accuracy in detecting malaria parasites from blood smears and leishmaniasis from Giemsa-stained images, significantly aiding in timely and accurate diagnosis.[64] AI-driven tools, such as the YOLOV5 model for leishmaniasis detection, have demonstrated superior performance in identifying parasites, even in complex samples. These tools are particularly beneficial in low-resource settings, where they can assist in reducing diagnostic time and workload.[64]

Traditional methods for diagnosing parasitic infections often rely on optical microscopy to examine bodily fluid samples. However, this technique can be limited by factors such as low parasite concentration or insufficient microscope sensitivity, potentially delaying early disease detection. Furthermore, identifying parasites within a sample containing billions of blood cells can be likened to finding a needle in a haystack. To address these challenges, researchers at the UCLA Samueli School of Engineering have developed a novel device capable of automatically detecting moving parasites within a sample.[65] This innovative technology offers several advantages over traditional microscopy. The device can detect parasite concentrations as low as ten parasites per milliliter of whole blood, significantly surpassing the capabilities of current methods. Moreover, the device can analyze over 3 mL of fluid in just 20 min, providing significantly faster results compared to conventional imaging techniques.

Likewise, Zhang et al. 2018 present a novel motility-based label-free computational imaging platform designed for the rapid detection of motile parasites in optically dense bodily fluids, utilizing the locomotion of parasites as a specific biomarker.[66] This platform is capable of screening approximately 3.2 mL of fluid samples in three dimensions, achieving a limit of detection of ten trypanosomes per mL of whole blood and three trypanosomes per mL of CSF, significantly outperforming current parasitological methods. The technology employs holographic speckle analysis combined with deep learning for sensitive detection and classification of parasites. It was used successfully for the identification of trypanosomes and Trichomonas vaginalis. Its cost-effective and portable design makes it suitable for use in resource-limited regions, potentially facilitating the timely diagnosis of NTDs caused by motile parasites.

Despite promising, AI-based parasite diagnostics face several significant challenges. First, a significant challenge in AI-based parasite diagnostics is the need for more diverse and representative datasets. Many AI models are trained on datasets from high-income countries, which may not accurately reflect the epidemiological and genetic diversity found in LMICs, particularly in Africa. Second, there are issues in ensuring the robustness and scalability of AI algorithms that are crucial for their widespread adoption. Models must be able to perform consistently across different geographic regions and healthcare settings. Finally, the implementation of AI technologies in diagnostics requires significant infrastructure, which can be a barrier in resource-limited settings. In addition, the cost of developing and maintaining AI systems can be prohibitive.[67] Furthermore, intellectual property (IP) considerations pose additional challenges. Unclear data ownership and access can hinder collaboration and innovation. This is more evident at the present time when the evolving legal frameworks for IP protection of AI models is creating uncertainty and discouraging investment.[68]

CONCLUSION

Parasitic diagnostics is a rapidly evolving field due to the emergence of new parasites, increasing drug resistance, technological advancements, changing epidemiology, global health initiatives, and ongoing research. New diagnostic tools are constantly being developed to address these challenges, including molecular diagnostics, point-of-care testing, and advanced imaging techniques involving AI and Deep technologies. These innovations, some of which are described here, aim to improve parasite detection accuracy, sensitivity, and speed, leading to earlier diagnosis, targeted treatment, and better patient outcomes. With time, many new feasible innovations are occupying the space and planning for a decentralized diagnostic ecosystem. For example, Smartphones, with their built-in sensors, offer a promising alternative to traditional diagnostic equipment, especially in LMICs. These devices can provide cost-effective, point-of-care diagnostic solutions, although their application in parasitic diagnostics is still in its early stages.[69]

Despite such innovations emerging with the potential of non-microscopic diagnostic methods, their integration into routine laboratory work is hindered by high infrastructure demands. This complicated their adoption in resource-limited settings. Moreover, these emerging diagnostic technologies need more consensus on protocols and results. The implementation of advanced diagnostic methods is often limited by factors such as Internet access and steady Wi-Fi coverage, particularly in developing countries. However, the challenges of today should not be a barrier for tomorrow. Rather, there is a constant need to find opportunities to integrate innovations in clinical settings. However, such integration of advanced technologies must be balanced with the practical realities of resource-limited settings, where traditional methods still play a crucial role. Moreover, the ongoing evolution of diagnostic techniques must be accompanied by efforts to standardize and assess their quality to ensure they effectively meet the needs of diverse populations. There is a need for harmonized external quality assessment schemes to ensure reliable and reproducible diagnostic outcomes.[70]

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.