The biology of intraerythrocytic Babesia parasites presents unique challenges for diagnosing human babesiosis. Antibody-based assays, while highly sensitive, fail to detect early-stage infections prior to seroconversion and cannot differentiate between active and previously resolved infections. Nucleic acid-based tests (NAT) may lack the sensitivity to detect low parasite burdens during the window period and asymptomatic low-grade infections. Recent technological advances have significantly improved the sensitivity, specificity, and high throughput of both NAT and antibody-based detection methods for Babesia. These advances include genomics approaches for identifying novel high-copy-number targets for NAT and immunodominant antigens for better antibody-based assays. Future advancements will likely involve next-generation sequencing and CRISPR technology to enhance Babesia detection.
Fig. 1 Technologies for detection of Babesia microti (Meredith S., et al. 2021).
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Understanding Babesia Microti
Historically identified by the intra-erythrocytic morphology of the parasites, Babesia microti has been shown to belong to a paraphyletic species complex. Initially, Babesia microti was considered a single species; however, phylogenetic analysis of 18S rDNA and beta-tubulin genes conducted in 2003 revealed that these parasites form a complex comprising at least three genetically distinct clades. Recent studies have expanded this complexity to include at least five clades, indicating a much broader diversity within the species than previously understood. This diversity complicates our understanding of the Babesia microti life cycle, as many biological studies predated the availability of DNA sequencing technology.
Babesia microti Biology and Detection Challenges
A comprehensive understanding of early-stage Babesia microti infections in humans is lacking. Observations from clinical, epidemiological studies, and data from asymptomatic blood donors in endemic areas suggest that most symptomatic cases develop within 1-4 weeks following exposure. However, the incubation period can be challenging to determine due to unnoticed tick bites and variable lengths of parasitemia and antibody presence.
The intraerythrocytic nature of Babesia microti poses challenges for effective detection. Blood film microscopy and xenodiagnosis are direct methods for Babesia diagnosis, but they lack the sensitivity required to detect low-grade infections or identify parasites during the window period before seroconversion.
Detection Techniques
The transition from microscopic examination of stained blood films in 1888 to modern molecular techniques marked significant progress in detecting Babesia infections. Advances made particularly in the latter half of the 20th century, have improved detection accuracy and sensitivity. Modern molecular techniques are 10- to 20-fold more sensitive than earlier methods, and innovations promise further enhancements in sensitivity, specificity, and throughput.
Direct Detection of Babesia microti Parasites
Experimental inoculation and xenodiagnosis have been traditional tools for diagnosing babesial infections. Early methods involved inoculating animal models and observing for infection. While effective, this approach is resource-intensive and not practical for routine diagnostics.
The identification of the Babesia parasite by microscopy has historical importance. On Giemsa-stained blood films, Babesia microti presents as ring-like trophozoites similar to P. falciparum. A distinctive cruciform structure known as the "Maltese Cross" is pathognomonic of Babesia infection. Although microscopy offers a direct method of detection, its sensitivity is limited, especially for low parasitemia levels common in asymptomatic or early-stage infections.
Nucleic Acid-Based Assays
Since the early 1990s, PCR-based molecular detection methods have outperformed microscopy in sensitivity. Early PCR protocols for P. falciparum detection, for example, demonstrated far greater sensitivity than thick blood smears. Recent advances such as real-time PCR (RT-PCR) and droplet digital PCR have further enhanced sensitivity, enabling the reliable detection of Babesia microti even at low parasitemia levels. For instance, high-copy-number BMN family genes have been exploited to achieve limits of detection as low as 10.0 parasites per mL.
The completion of the Babesia microti genome sequence in 2012 enabled genomics-based detection advancements, identifying novel high-copy-number targets like BMN genes. The development of genome-wide sequencing of parasite isolates has revealed extensive genetic diversity, contributing to more accurate molecular surveillance and diagnostics.
Antigen Detection Assays
Antigen-based detection offers a promising approach for active infections. A significant milestone was the identification of Babesia microti alpha-helical cell surface protein 1 (BmBAHCS1), which showed high sensitivity as a biomarker. Antigen capture assays based on BmBAHCS1 could potentially reduce the window period before seroconversion, yet challenges remain in enhancing sensitivity.
Greater emphasis on genome-wide screening for immunodominant Babesia microti antigens has been suggested for creating a robust antigen detection system. Lessons from malaria diagnostics, including potential pitfalls due to emerging antigen polymorphism, play a crucial role in guiding the development of Babesia antigen-detection assays.
Antibody-Based Assays
Indirect immunofluorescence assay (IFA) has been the most sensitive and reliable method for detecting antibodies against Babesia microti. However, the inability to differentiate between active and past infections and the delayed antibody response pose limitations. Enzyme-linked immunosorbent assay (ELISA) protocols using recombinant Babesia microti proteins have been developed, showing promise in improving sensitivity and specificity. Combining multiple immunodominant antigens may achieve the desired efficiency for surveillance and diagnostic purposes.
Novel and Future Technologies
Next Generation Sequencing (NGS)
NGS offers an unbiased approach for detecting a broad range of pathogens in clinical samples through metagenomics. This method can help understand Babesia microti genome-wide genetic diversity and evolutionary relationships, providing insight into potential species differentiation and transmission dynamics. However, cost and sensitivity considerations must be addressed for routine diagnostic use.
CRISPR Technology
CRISPR-based diagnostics offer rapid, low-cost, and highly sensitive detection. Systems like DETECTR and SHERLOCK have shown promise for detecting Plasmodium spp. and could be adapted for Babesia detection. The ability to simultaneously identify species and genotypes makes CRISPR technology a robust tool for both diagnostic and surveillance purposes.
Conclusion
The understanding of Babesia microti and its detection has come a long way since the parasite's discovery in 1888. Phylogenetic analyses have revealed the complexity and diversity of the Babesia microti species complex. Advances in detection technologies, including genomics, NGS, and CRISPR, have improved diagnostic capabilities, making early detection and treatment more feasible. However, challenges remain, future research and technological advancements will be crucial in addressing these challenges, ensuring better diagnosis, treatment, and prevention of babesiosis.
References
- Meredith S., et al. Technologies for detection of Babesia microti: Advances and challenges. Pathogens. 2021, 10 (12): 1563.
- Goethert H. K. What Babesia microti is now. Pathogens. 2021, 10 (9): 1168.
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