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Infection by Toxoplasma gondii is prevalent worldwide. The parasite can infect a broad spectrum of vertebrate hosts, but infection of fetuses and immunocompromised patients is of particular concern. Easy-to-perform, robust, and highly sensitive and specific methods to detect Toxoplasma infection are important for the treatment and management of patients. Rapid diagnostic methods that do not sacrifice the accuracy of the assay and give reproducible results in a short time are highly desirable. In this context, rapid diagnostic tests (RDTs), especially with point-of-care (POC) features, are promising diagnostic methods in clinical microbiology laboratories, especially in areas with minimal laboratory facilities. More advanced methods using microfluidics and sensor technology will be the future trend. In this review, we discuss serological and molecular-based rapid diagnostic tests for detecting Toxoplasma infection in humans as well as animals.

Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries, causing up to 100,000 fatal cases annually. Detection of the pathogenic E. histolytica and its differentiation from the non-pathogenic Entamoeba spp. play a crucial role in the clinical management of patients. Laboratory diagnosis of intestinal amoebiasis in developing countries still relies on labour-intensive and insensitive methods involving staining of stool sample and microscopy. Newer and more sensitive methods include a variety of antigen detection ELISAs and rapid tests; however, their diagnostic sensitivity and specificity seem to vary between studies, and some tests do not distinguish among the Entamoeba species. Molecular detection techniques are highly sensitive and specific and isothermal amplification approaches may be developed into field-applicable tests; however, cost is still a barrier for their use as a routine laboratory test method in most endemic areas. Laboratory diagnosis of extraintestinal amoebiasis faces challenges of lack of definitive detection of current infection and commercially available point-of-care tests. For both types of amoebiasis, there is still a need for highly sensitive and specific tests that are rapid and cost-effective for use in developing countries where the disease is prevalent. In recent years, new molecules of diagnostic value are being discovered and new tests developed. The advances in ‘omics’ technologies are enabling discoveries of new biomarkers that may help distinguish between different infection stages.

Laboratory diagnosis of Strongyloides infections can be grouped into direct and indirect detection methods, and a combination of the two methods is often needed to reach an accurate and timely diagnosis. This review focuses on non-conventional direct detection via molecular and antigen detection assays. Conventional PCR is the most commonly used molecular diagnostic for Strongyloides. Real-time PCR is accurate and highly sensitive for quantitative and qualitative analysis. Meanwhile, PCR-RFLP can efficiently distinguish human and dog isolates of S. stercoralis, S. fuelleborni (from monkey), and S. ratti (from rodent). Loop-mediated isothermal amplification (LAMP) amplifies DNA isothermally with high specificity, efficiency, and rapidity, and has potential for point-of-care (POC) translation. As for antigen detection assay, coproantigen detection ELISAs for strongyloidiasis traditionally relied on raising rabbit polyclonal antibodies against the parasite antigens for use as capture or detection reagents. Subsequently, hybridoma technology using animals has enabled the discovery of monoclonal antibodies specific to Strongyloides antigens and was utilised to develop antigen detection assays. In recent times, phage display technology has facilitated the discovery of scFv antibody against Strongyloides protein that can accelerate the development of such assays. Improvements in both direct detection methods are being made. Strongyloides molecular diagnostics is moving from the detection of a single infection to the simultaneous detection of soil-transmitted helminths. Meanwhile, antigen detection assays can also be multiplexed and aptamers can be used as antigen binders. In the near future, these two direct detection methods may be more widely used as diagnostic tools for strongyloidiasis.

Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.

Detection of Strogyloides -specific IgG antibodies in urine and serum has been used in diagnostic and epidemiological studies on strongyloidiasis. However, the usefulness of these assays in assessing responses to anthelmintic treatment is unclear. Thus, we evaluated the diagnostic performance and temporal profiles of Strongyloides -specific IgG antibodies in a cohort of participants at baseline and post-treatment. The participants were prospectively screened for baseline parasitic infections by fecal examination [agar plate culture technique (APCT) and formalin-ethyl acetate concentration technique (FECT)] and digital droplet polymerase reaction (ddPCR) for Strongyloides stercoralis . At each sampling point, Strongyloides -specific IgG in urine and serum were measured by an in-house S . ratti -based enzyme-linked immunosorbent assay (ELISA). At baseline, 169 of 351 participants (48.1%) had S . stercoralis infection by the combined fecal examination and ddPCR. The diagnostic sensitivities of IgG in urine and serum were 91.1% and 88.2%, respectively. The participants were given treatment with a single oral dose of ivermectin (IVM, 200 μg/kg) and were followed up by fecal and immunological diagnosis at 3 to 18 months post-treatment. The cure rate of IVM treatment evaluated by APCT and ddPCR was 88.3% at three months post-treatment. The profiles of IgG in urine in the curative treatment group showed a significant trend of decline with time post-treatment (Kruskal-Wallis test = 113.4–212.6, p value < 0.0001) and the lowest levels were seen 12 months post-treatment. The treatment response (> 50% reduction in urinary IgG antibody units) was 100%, and conversion from positive to negative results was 65.4%. The treatment response and conversion to negative assessed by serum IgG-ELISA were similar to those by urine IgG-ELISA. The results from this long-term diagnostic study highlight the utility of urinary IgG and serum IgG for screening and monitoring treatment outcomes in strongyloidiasis.

A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.

Background Strongyloidiasis, a nematode infection which is mainly caused by Strongyloides stercoralis in humans, can lead to a fatal syndrome in immunocompromised individuals. Its diagnosis is challenging due to the absence of a diagnostic gold standard. The infection is highly prevalent in migrants from endemic countries in tropical and subtropical areas, and a rapid diagnostic test would be helpful for screening purposes. The aim of this study was to estimate the accuracy of a novel immunochromatographic test (ICT) for the diagnosis of S. stercoralis infection. Methods A single-centre diagnostic accuracy study was undertaken using well-characterized frozen sera available from the biobank of a referral hospital for parasitic diseases in Italy. The included sera were from migrants from sub-Saharan Africa, and matching results were available for agar plate culture and/or polymerase chain reaction for S. stercoralis ; moreover, the results of both a commercial enzyme-linked immunosorbent assay test and an in-house immunofluorescence test for strongyloidiasis were made available. Laboratory staff who read the ICT results were blinded as regards the results of the other tests. Two readers independently read the ICT, and a third one was involved when results were discrepant. The accuracy of the ICT was assessed both against the results of the panel of faecal tests and by latent class analysis (LCA). Results Agreement between the readers was excellent [Cohen’s κ = 92.7%, 95% confidence interval (CI) 88.3–97.1%]. When assessed against the results of the faecal tests, the sensitivity and specificity of the ICT were 82.4% (95% CI 75.7–89.0%) and 73.8% (95% CI 66.8–80.9%), respectively. According to the LCA, the sensitivity and specificity were 86.3% (95% CI 80.1–92.5%) and 73.9% (95% CI 67.0–80.8%), respectively. Conclusions The results of the ICT demonstrated ease of interpretation. The accuracy proved good, though the sensitivity might be further improved for screening purposes. Graphical Abstract

Background Strongyloides stercoralis is a pathogenic nematode affecting the human intestine. Chronic strongyloidiasis often remains asymptomatic, posing diagnostic challenges due to the low sensitivity of conventional methods. Using traditional methods, this study investigated the prevalence of strongyloidiasis in Khuzestan Province, southwestern Iran. We also studied the effectiveness of a Strongyloides immunoglobulin G4 (IgG4) rapid diagnostic test (RDT) for timely infection detection before and after treatment. Methods This cross-sectional study, conducted during 2022–2024, evaluated 520 participants with eosinophilia (> 5%) for S. stercoralis infection. Coprological methods used were direct smear stool microscopy and agar plate culture. Serological methods were enzyme-linked immunosorbent assay (ELISA) (NovaTec ® kit) and a prototype IgG4 RDT using a recombinant antigen (NIE) . Traditional coprology and composite references were used to assess the diagnostic power. Among copro-positive patients, 30 cases were followed up at least 3 months after treatment using the same methods. Results Of the 373 participants who submitted stool samples, coprological methods identified 95 positive cases, with culture proving to be more sensitive than direct smear (24.9%, 93/373 versus 7.5%, 28/373). Of the 520 participants, 35.2% (183/520) and 43.7% (227/520) tested positive for S. stercoralis using ELISA and IgG4 RDT, respectively. Spearman’s rank correlation between the IgG4 RDT and ELISA was significant ( ρ  = 0.772; P  < 0.001). Despite minor discrepancies, the IgG4 RDT showed substantial agreement with the ELISA ( κ  = 0.776). Increased eosinophil counts were strongly associated with Strongyloides infection with a mean of 20.48% in copro-positives versus 15.22 in copro-negatives and area under the curve (AUC) of 0.741 and 0.701 for coprology and the combination of coprology and serology methods (CRS), respectively. In the 30 follow-up patients, a significant reduction in eosinophil counts ( P  < 0.001) was observed. Five cases (17%) remained larva-positive, and serological tests significantly increased readings/scores. Three copro-negative patients showed strong positive results on ELISA and IgG4 RDT. Conclusions On the basis of the obtained results, the prevalence of S. stercoralis infection among the eosinophilic population was high. This study showed that the IgG4 RDT is a reliable and efficient diagnostic tool for S. stercoralis infection. The rapid test results demonstrated significant agreement with the ELISA and effectively detected infection in eosinophilic patients, making it a suitable diagnostic test for screening, particularly in resource-limited settings. Graphical Abstract

Malaysia successfully achieved zero indigenous human malaria cases since 2018. However, challenges persist from Plasmodium knowlesi (zoonotic malaria) and low-density infections, posing reintroduction risks in previously malaria-free areas. Addressing these hidden infections is critical for sustaining Malaysia’s elimination gains. This study investigated the persistence of low-density malaria transmission in high-risk localities declared malaria-free for at least three consecutive years. A community-based cross-sectional survey was conducted from June to October 2020 in 23 high-receptivity localities across Sabah, Perak, Kelantan, and Johor. Blood samples from asymptomatic residents were screened via conventional microscopy and nested PCR (nPCR) targeting the Plasmodium 18S rRNA gene, with positive nPCR products species-determined. Sociodemographic and geospatial data were analyzed for associations with infection status. Of 3,322 asymptomatic individuals, no infections were detected by microscopy, whereas nPCR revealed a low-density malaria prevalence of 1.86% (62/3,322). Infections comprised P. malariae (40.3%), P. vivax (29.0%), P. knowlesi (24.2%), P. falciparum (1.6%), P. cynomolgi (1.6%), and mixed P. vivax/P. knowlesi (3.2%). All PCR-positive cases originated from Sabah and an Orang Asli settlement in Perak. Adults (≥17 years) constituted the majority (~68%), with no significant difference in prevalence by gender or previous malaria history (p > 0.05). Asymptomatic low-density malaria infections persist in purportedly malaria-free communities, remaining undetectable by routine microscopy. These hidden parasite reservoirs pose a risk for malaria reintroduction, especially in receptive areas. Malaria surveillance programs must thus incorporate highly sensitive diagnostic tools to detect low-density infections and safeguard elimination gains. Intensified, targeted interventions in identified “malaria hotspots”, including community engagement and vector control, are crucial to eliminate residual foci and prevent disease resurgence.