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Purpose: We report the clinical findings and molecular identification of ocular Thelazia callipaeda from Niigata Prefecture in the Hokuriku area of Japan during winter. Observations: A 77-year-old male visited an ophthalmology clinic in Niigata Prefecture in January 2022 after a 2-week-duration of a conjunctival injection in the left eye and foreign body sensation. Slit-lamp microscopy revealed 11 active nematodes in the left conjunctival sac. Morphological characteristics included longer female body length than male, buccal cavity lacking teeth and lips, and serrated striations along the body surface. The specimens were determined to be T. callipaeda. Genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed an h9 haplotype. Conclusions and Importance: T. callipaeda infection, especially the h9 haplotype, commonly occurs in western Japan owing to its higher incidence in warmer climates, suggesting the origin of the case. Here, we report a human case of Thelaziasis diagnosed in a cold region of Japan (the Hokuriku area) during winter. This human case of T. callipaeda infection from a cold, previously unassociated region, raises concerns about the potential geographical widening of its distribution, and further investigation may be warranted to prevent its spread.

Abstract Background Understanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC). Methods A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3. Results Nine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia

Abstract Background Loss of efficacy of diagnostic tests may lead to untreated or mistreated malaria cases, compromising case management and control. There is an increasing reliance on rapid diagnostic tests (RDTs) for malaria diagnosis, with the most widely used of these targeting the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). There are numerous reports of the deletion of this gene in P. falciparum parasites in some populations, rendering them undetectable by PfHRP2 RDTs. The aim of this study was to identify P. falciparum parasites lacking the P. falciparum histidine rich protein 2 and 3 genes (pfhrp2/3) isolated from asymptomatic and symptomatic school-age children in Kinshasa, Democratic Republic of Congo. Methods The performance of PfHRP2-based RDTs in comparison to microscopy and PCR was assessed using blood samples collected and spotted on Whatman 903™ filter papers between October and November 2019 from school-age children aged 6–14 years. PCR was then used to identify parasite isolates lacking pfhrp2/3 genes. Results Among asymptomatic malaria carriers (N = 266), 49%, 65%, and 70% were microscopy, PfHRP2_RDT, and pfldh-qPCR positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 80% and 70% while the sensitivity and specificity of RDTs compared to microscopy were 92% and 60%, respectively. Among symptomatic malaria carriers (N = 196), 62%, 67%, and 87% were microscopy, PfHRP2-based RDT, pfldh-qPCR and positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 75% and 88%, whereas the sensitivity and specificity of RDTs compared to microscopy were 93% and 77%, respectively. Of 173 samples with sufficient DNA for PCR amplification of pfhrp2/3, deletions of pfhrp2 and pfhrp3 were identified in 2% and 1%, respectively. Three (4%) of samples harboured deletions of the pfhrp2 gene in asymptomatic parasite carriers and one (1%) isolate lacked the pfhrp3 gene among symptomatic parasite carriers in the RDT positive subgroup. No parasites lacking the pfhrp2/3 genes were found in the RDT negative subgroup. Conclusion Plasmodium falciparum histidine-rich protein 2/3 gene deletions are uncommon in the surveyed population, and do not result in diagnostic failure. The use of rigorous PCR methods to identify pfhrp2/3 gene deletions is encouraged in order to minimize the overestimation of their prevalence.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Abstract Background Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. Methods A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. Results The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p

Abstract Background The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG. Methods In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016–2018. Results A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10–3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10–16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10–6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia. Conclusions Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important.

Background Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276). Results ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8–100.0] and 97.8% specificity 95% CI [95.5–99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8–100.0]) and a slightly higher specificity (99.1% 95% CI [97.3–99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar’s test; p > 0.05), with a concordance of 98.2%, represented by a Cohen’s kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.

Abstract Background Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. Methods Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. Results Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4–24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10−8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. Conclusion This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Elucidation of the mechanisms of drug resistance in malaria parasites is crucial for combatting the emergence and spread of resistant parasites, which can be achieved by tracing resistance-associated mutations and providing useful information for drug development. Previously, we produced a novel genetic tool, a Plasmodium berghei mutator (PbMut), whose base substitution rate is 36.5 times higher than that of wild-type parasites. Here, we report the isolation of a mutant with reduced susceptibility to piperaquine (PPQ) from PbMut under PPQ pressure by sequential nine-cycle screening and named it PbMut-PPQ-R-P9. The ED50 of PbMut-PPQ-R-P9 was 1.79 times higher than that of wild-type parasites, suggesting that its PPQ resistance is weak. In the 1st screen, recrudescence occurred in the mice infected with PbMut but not in those infected with wild-type parasites, suggesting earlier emergence of PPQ-resistant parasites from PbMut. Whole-genome sequence analysis of PbMut-PPQ-R-P9 clones revealed that eight nonsynonymous mutations were conserved in all clones, including N331I in PbCRT, the gene encoding chloroquine resistance transporter (CRT). The PbCRT(N331I) mutation already existed in the parasite population after the 2nd screen and was predominant in the population after the 8th screen. An artificially inserted PbCRT(N331I) mutation gave rise to reduced PPQ susceptibility in genome-edited parasites (PbCRT-N331I). The PPQ susceptibility and growth rates of PbCRT-N331I parasites were significantly lower than those of PbMut-PPQ-R-P9, implying that additional mutations in the PbMut-PPQ-R9 parasites could compensate for the fitness cost of the PbCRT(N331I) mutation and contribute to reduced PPQ susceptibility. In summary, PbMut could serve as a novel genetic tool for predicting gene mutations responsible for drug resistance. Further study on PbMut-PPQ-R-P9 could identify genetic changes that compensate for fitness costs owing to drug resistance acquisition.

Despite a decade of sustained malaria control, malaria remains a serious public health problem in the Democratic Republic of Congo (DRC). Children under five years of age and school-age children aged 5–15 years remain at high risk of symptomatic and asymptomatic malaria infections. The World Health Organization’s malaria control, elimination, and eradication recommendations are still only partially implemented in DRC. For better malaria control and eventual elimination, the integration of all individuals into the national malaria control programme will strengthen malaria control and elimination strategies in the country. Thus, inclusion of schools and school-age children in DRC malaria control interventions is needed.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.

Abstract Background Chloroquine treatment for Plasmodium falciparum has been discontinued in almost all endemic regions due to the spread of resistant isolates. Reversal of chloroquine susceptibility after chloroquine discontinuation has been reported in dozens of endemic regions. However, this phenomenon has been mostly observed in Africa and is not well documented in other malaria endemic regions. To investigate this, an ex vivo study on susceptibility to chloroquine and lumefantrine was conducted during 2016–2018 in Wewak, Papua New Guinea where chloroquine had been removed from the official malaria treatment regimen in 2010. Genotyping of pfcrt and pfmdr1 was also performed. Results In total, 368 patients were enrolled in this study. Average IC50 values for chloroquine were 106.6, 80.5, and 87.6 nM in 2016, 2017, and 2018, respectively. These values were not significantly changed from those obtained in 2002/2003 (108 nM). The majority of parasites harboured a pfcrt K76T the mutation responsible for chloroquine resistance. However, a significant upward trend was observed in the frequency of the K76 (wild) allele from 2.3% in 2016 to 11.7% in 2018 (P = 0.008; Cochran–Armitage trend test). Conclusions Eight years of chloroquine withdrawal has not induced a significant recovery of susceptibility in Papua New Guinea. However, an increasing tendency of parasites harbouring chloroquine-susceptible K76 suggests a possibility of resurgence of chloroquine susceptibility in the future.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014–2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage–specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.

Regulation of intracellular Ca2+ concentration ([Ca2+]i) is vital for eukaryotic organisms. Recently, we identified a Ca2+ channel (TcIP3R) associated with intracellular Ca2+ stores in Trypanosoma cruzi, the parasitic protist that causes Chagas disease. In this study, we measured [Ca2+]i during the parasite life cycle and determined whether TcIP3R is involved in the observed variations. Parasites expressing R‐GECO1, a red fluorescent, genetically encoded Ca2+ indicator for optical imaging that fluoresces when bound to Ca2+, were produced. Using these R‐GECO1‐expressing parasites to measure [Ca2+]i, we found that the [Ca2+]i in epimastigotes was significantly higher than that in trypomastigotes and lower than that in amastigotes, and we observed a positive correlation between TcIP3R mRNA expression and [Ca2+]i during the parasite life cycle both in vitro and in vivo. We also generated R‐GECO1‐expressing parasites with TcIP3R expression levels that were approximately 65% of wild‐type (wt) levels (SKO parasites), and [Ca2+]i in the wt and SKO parasites was compared. The [Ca2+]i in SKO parasites was reduced to approximately 50–65% of that in wt parasites. These results show that TcIP3R is the determinant of [Ca2+]i in T. cruzi. Since Ca2+ signaling is vital for these parasites, TcIP3R is a promising drug target for Chagas disease.

Abstract Background In the Lao PDR, malaria morbidity and mortality have remarkably decreased over the past decade. However, asymptomatic infections in rural villages contribute to the on-going local transmission. The primary objective of this study was to explore the characteristics of infections in a malaria-endemic district of the Lao PDR. The specific objectives were to investigate the prevalence and species of malaria parasites using molecular methods and to assess individual and household parasite levels and the characteristics associated with malaria infection. Methods The study population included 870 participants from 236 households in 10 villages of the Xepon district. Interviews, blood examinations and body temperature measurements were conducted between August and September 2013. A multilevel logistic regression model, with adjustment for clustering effects, was used to assess the association between predictor variables and an outcome variable (malaria infection status as principally determined by PCR). The predictive factors included individual-level factors (age, gender, past fever episode, and forest activity during night time) and household-level factors (household member size, household bed net usage/density and a household with one other malaria-infected member). Results Fifty-two participants (including 26 children) tested positive (positive rate: 6.0 %): Plasmodium falciparum mono-infection was the most common infection (n = 41, 78.8 %), followed by P. falciparum and Plasmodium vivax mixed infections (n = 9, 17.3 %). The majority of infected participants (n = 42, 80.8 %) had no fever episodes in the two previous weeks or a measurable fever (>37 °C) at the time of survey. Living in a household with one other malaria-infected member significantly increased the odds of infection (odds ratio 24.33, 95 % confidence interval 10.15–58.32). Among the 40 households that had at least one infected member, nine households were responsible for 40.4 % of the total infections. Conclusions Plasmodium vivax was detected more frequently than it was reported from the district hospital. Most infections were asymptomatic and sub-microscopic and were highly clustered within households. To further eliminate malaria in Xepon and other similar settings in the country, the National Malaria Control Programme should consider household-based strategies, including reactive case detection targeting the household members of index cases.

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/μL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, in this study, we investigated if a commercially available fluorescent cell counter, LUNA-FL, was useful for the detection of Plasmodium parasite and the estimation of parasitemia. Whole blood samples from uninfected persons, spiked with P. falciparum-infected erythrocytes, were analysed. Most of the leucocytes and platelets were removed from whole blood samples with SiO2-nanofiber filters set on spin columns. The filtered samples were stained with acridine orange, and automatic detection, as well as counting of erythrocytes and parasites, were performed using LUNA-FL. Whole blood, with various levels of parasites, was analysed by Giemsa microscopy or with LUNA-FL to estimate parasitemia, and a comparative analysis was performed. The coefficient determination value of the regression line was high (R2 = 0.98), indicating that accurate quantitative parasite detection could be performed using LUNA-FL. LUNA-FL has a low running cost; it is compact, fast, and easy to operate, and may therefore be useful for point-of-care testing in the endemic areas.

Background. Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods. A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (nD14), healthy individuals (nD40), and patients with other diseases (nD276). Results. ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8-100.0] and 97.8% specificity 95% CI [95.5-99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8-100.0]) and a slightly higher specificity (99.1% 95% CI [97.3-99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar's test; p > 0.05), with a concordance of 98.2%, represented by a Cohen's kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion. ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis. [ABSTRACT FROM AUTHOR]