Aroa Silgado

We sincerely thank Dr. Taufiq and Dr. Pascoal (1) for their interest in our study and for their thoughtful and constructive comments regarding our article, Performance of the Techcyte AI-based image analysis system for coproparasitological diagnosis in clinical stool specimens: a retrospective evaluation study (2). We appreciate the opportunity to further discuss and clarify the rationale behind our interpretation of the results.

Background: Diagnosis of chronic Trypanosoma cruzi infection relies on serological techniques using invasive blood samples (serum or plasma), which can be impractical in remote areas. Dried blood spot (DBS) sampling offer advantages over invasive samples in terms of ease of collection (finger prick), transport and storage. This study aimed to compare the performance of anti- T. cruzi antibody detection from DBS samples collected on two different devices: Whatman cards and Cobas Plasma Separation Cards (PSC). Methodology/principal findings: A prospective study using 382 paired serum and whole blood samples was conducted. Whole blood were used to obtain DBS collected on Whatman and PSC cards. All samples were tested for IgG anti- T. cruzi using the Elecsys Chagas assay, with serum serving as the gold standard. Both DBS collection systems showed high concordance with serum samples (>96%). However, antibody index values detected in DBS samples were lower than those detected in serum samples (p < 0.05). Qualitative results from DBS were highly concordant between the collection systems, with only two discrepancies (2/382), both positive on Whatman and negative on PSC. Antibody index values from Whatman-collected samples were higher than those from PSC-collected samples (median 96.45 vs. 63.33; p < 0.05). Conclusions/significance: DBS sampling is a feasible alternative to invasive blood collection, particularly in settings with limited access to health systems. Both DBS-collection devices showed high diagnostic accuracy; however, the lower antibody index values from PSC-collected samples may compromise the sensitivity of the technique in samples with a low antibody index.

Diagnosis of chronic Trypanosoma cruzi infection relies on the concordant results of two serological tests based on different antigens and/or principles. Currently, the only accepted cure criterion is the switch from positive to negative serology, but anti- T . cruzi IgG may persist for years, even after treatment. Recently, a novel chemiluminescence immunoassay based on native T. cruzi excreted-secreted antigens (TESA)—Chagas TESA VirClia—was developed. This study assessed the performance of this assay for the detection of anti- T . cruzi antibodies for the diagnosis and post-treatment follow-up of chronic T. cruzi infection. A retrospective diagnostic accuracy study was conducted using 227 serum samples, including samples from individuals tested for chronic T. cruzi infection and from individuals with other parasitic or viral infections. T. cruzi infection status was established using two reference serological assays based on recombinant and native antigens. All samples were analyzed by TESA-based assay and compared with the reference tests. Concordance between the TESA-based assay and the reference tests was 93.8%, with a sensitivity and specificity of 92% and 100%, respectively. Limited cross-reactivity was observed in samples positive for Leishmania spp. antibodies, while no cross-reactivity was observed in viral infections. A decrease in antibody index over time was observed more frequently in treated patients when tested by the TESA-based assay. These findings support the use of the Chagas TESA VirClia assay as a reliable confirmatory tool within the diagnostic algorithm for chronic T. cruzi infection. Moreover, this assay was the only test showing a consistent post-treatment decline in antibody index over time. IMPORTANCE: The diagnosis and monitoring of chronic Trypanosoma cruzi infection remain challenging because no single serological test achieves 100% sensitivity and specificity, and the antibodies may persist for many years even after treatment. This study highlights the value of a novel serological assay based on native antigens excreted by the parasite, which more effectively reflects changes in antibody levels over time. Its high diagnostic accuracy and greater ability to detect declining antibody values in treated patients support its use as a reliable diagnostic tool as well as a promising method for serological follow-up.

Coproparasitological stool analysis based on microscopic examination is the reference diagnostic technique routinely performed in clinical microbiology laboratories. Automated image analysis systems could offer a suitable solution, reducing technologist workload and improving screening efficiency. The Human Fecal Ova & Parasite (O&P) Detection Solution (Techcyte) is an artificial intelligence (AI) software employing imaging-based algorithms to provide screening presumptive diagnostic results. This study aimed to validate the Wet Mount Iodine Solution software for detecting and diagnosing protozoan and helminthic infections in stool specimens. A total of 178 clinical stool specimens were retrospectively analyzed between July and October 2024 for comparative evaluation of the AI-based system. Positive specimens with confirmed mono ( n = 116) and mixed ( n = 31) protozoa and helminth infections, as well as negative specimens ( n = 31), were included. Conventional microscopy results were compared with AI presumptive detection and identification outcomes. In addition, operator-reviewed AI results were evaluated against the reference diagnosis. The system achieved 96.62% positive percent agreement (PPA) and 93.33% negative percent agreement (NPA) for overall detection considering operator-reviewed interpretation. The kappa coefficient was 0.865, indicating strong agreement with conventional microscopy. The AI system detected additional parasites in 74/178 (41.57%) specimens, providing a diagnostic improvement compared to traditional microscopy. AI-assisted microscopy enhances intestinal parasite diagnosis, providing reliable results when combined with operator review. Despite limitations such as specimen low volume analysis and artifact detection, the system shows strong potential for improving diagnostic workflows in clinical microbiology laboratories. IMPORTANCE: Microscopic examination of stool specimens remains the reference technique for diagnosing intestinal parasitic infections although it is labor-intensive and operator-dependent methodology. The implementation of artificial intelligence-assisted microscopy offers a promising approach to streamline diagnostic workflows, improve consistency, and enhance traditional methods. Our evaluation study provides valuable clinical evaluation data of the Human Fecal Ova & Parasite (O&P) Detection Wet Mount Iodine Solution (Techcyte), demonstrating high sensitivity, specificity, and agreement with conventional microscopy. Importantly, AI-assisted review yielded diagnostic gains in a substantial proportion of specimens, underscoring its value as a screening and complementary tool for routine parasitological diagnostics. These findings highlight the potential of AI-powered image analysis to improve the accuracy and efficiency of coproparasitological testing, thereby supporting clinical decision-making and optimizing resource utilization in microbiology laboratories.

Background: Schistosomiasis is a neglected tropical disease that mostly affects inhabitants of sub-Saharan Africa. With rising global migration, imported cases of schistosomiasis are increasingly being reported in non-endemic countries, where diagnosis is hindered by low parasite burdens and multiple Schistosoma species. Microscopy remains the gold standard, despite its limitations, whereas molecular techniques offer greater sensitivity. The aim of this study was to assess the performance of real-time polymerase chain reaction (PCR) protocols for the detection, at an international health centre in Barcelona, of imported cases of urogenital and intestinal schistosomiasis. Methods: This cross-sectional study included 75 adults from sub-Saharan Africa attending the Drassanes-Vall d’Hebron International Health Unit, Barcelona, between May 2023 and February 2024. Paired urine and stool samples were collected. Microscopy was performed on all samples. Urine was analysed by real-time PCR using the Dra1 target sequence. Stool was tested by three protocols targeting, respectively, Dra1, Sm1-7, and 28S rRNA. Schistosoma infection was confirmed by microscopic identification of eggs and/or parasite DNA detection by real-time PCR. Results: Schistosomiasis was confirmed in 12/75 patients (16%). Urogenital schistosomiasis was diagnosed in 3/75 cases; the performance values of real-time PCR in urine samples were not assessed. In stool, the pan- Schistosoma real-time PCR showed 55.6% sensitivity and 98.5% specificity, with a moderate agreement ( κ = 0.631) with microscopy. The Sm1-7 assay fully matched microscopy for Schistosoma mansoni detection, and reached 100% sensitivity and specificity. A novel contribution of this study is the application of a real-time PCR assay targeting the Dra1 repetitive sequence in stool samples for the detection of Schistosoma intercalatum/Schistosoma guineensis . All of the microscopy-positive cases were real-time PCR positive, and one additional infection was detected by real-time PCR, which meant that 100% sensitivity and 98.6% specificity were achieved with this technique. Conclusions: Our findings underscore the need for accurate diagnostic tools for cases of imported schistosomiasis in non-endemic settings. Microscopy remains the reference standard, while the pan- Schistosoma real-time PCR showed limited sensitivity for stool samples. In contrast, the Sm1-7 and Dra1 assays demonstrated higher sensitivity and strong concordance with microscopy, with Dra1 also proving useful for the detection of S. intercalatum / S. guineensis in stool. Graphical

Dengue virus (DENV) is the most important arbovirus worldwide. In 2019, a significant increase in dengue cases was reported worldwide, resulting in a peak of imported cases in some European countries such as Spain. We aimed to describe travel-associated and locally acquired DENV strains detected in 2019 in the Catalonia region (northeastern Spain), a hotspot for dengue introduction in Europe. Through sequencing and phylogenetic analysis of the envelope gene, 75 imported viremic cases and two local strains were described. Autochthonous transmission events included an infection of a local mosquito with an imported dengue strain and a locally acquired human dengue infection from a locally infected mosquito. Overall, all four DENV serotypes and up to 10 different genotypes were detected. Phylogenetic analysis revealed transcontinental circulations associated with DENV-1 and DENV-2 and the presence of DENV-4 genotype I in Indonesia, where few cases had been previously described. A molecular study of the autochthonous events determined that local Ae. albopictus mosquitoes were infected by an African DENV-1 genotype V strain, while the locally acquired human case was caused by a DENV-3 genotype I of Asian origin. These findings underline the wide variability of imported strains and the high risk of DENV introduction into this territory, emphasizing the importance and usefulness of molecular characterization and phylogenetics for both local and global surveillance of the disease.

The gold standard diagnosis for malaria is the microscopic visualization of blood smears to identify Plasmodium parasites, although it is an expert-dependent technique and could trigger diagnostic errors. Artificial intelligence (AI) tools based on digital image analysis were postulated as a suitable supportive alternative for automated malaria diagnosis. A diagnostic evaluation of the iMAGING AI-based system was conducted in the reference laboratory of the International Health Unit Drassanes-Vall d’Hebron in Barcelona, Spain. iMAGING is an automated device for the diagnosis of malaria by using artificial intelligence image analysis tools and a robotized microscope. A total of 54 Giemsa-stained thick blood smear samples from travelers and migrants coming from endemic areas were employed and analyzed to determine the presence/absence of Plasmodium parasites. AI diagnostic results were compared with expert light microscopy gold standard method results. The AI system shows 81.25% sensitivity and 92.11% specificity when compared with the conventional light microscopy gold standard method. Overall, 48/54 (88.89%) samples were correctly identified [13/16 (81.25%) as positives and 35/38 (92.11%) as negatives]. The mean time of the AI system to determine a positive malaria diagnosis was 3 min and 48 s, with an average of 7.38 FoV analyzed per sample. Statistical analyses showed the Kappa Index = 0.721, demonstrating a satisfactory correlation between the gold standard diagnostic method and iMAGING results. The AI system demonstrated reliable results for malaria diagnosis in a reference laboratory in Barcelona. Validation in malaria-endemic regions will be the next step to evaluate its potential in resource-poor settings.

Background: Malaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory. Methods: A total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format. Results: In field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%. Conclusions: This is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas. Graphical

Zika virus (ZIKV) is a vector-borne flavivirus with a known teratogenic effect, yet the full spectrum has not been delineated. Studies on endemic areas tried to characterize the clinical outcomes of ZIKV intrauterine exposure. We aimed to describe early neurodevelopmental outcomes on prenatally ZIKV-exposed children in a non-endemic ZIKV area. This is a prospective observational cohort study conducted from May 2016 to December 2021 at Hospital Universitari Vall d’Hebron in Barcelona, Catalonia, Spain. We monitored for up to 24 months 152 children extracted from a pregnant women cohort with suspected ZIKV infection; eleven women (11/150; 7.3%) fulfilled the criteria for a confirmed ZIKV infection. Among the 152 children included, we describe two cases of congenital ZIKV syndrome (CZS) born from women with a confirmed ZIKV infection. Additionally, we describe five cases of other potentially ZIKV-related outcomes (OPZROs), all with normal birth cranial circumference and born to women with probable ZIKV infection. The low exposed prevalence of adverse outcomes in asymptomatic children at birth in a non-endemic area suggests that close follow-up should be addressed by primary care pediatricians instead of pediatric specialists. Further studies are needed to assess the effects of ZIKV intrauterine exposure beyond two years of life.

Background: Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. Methodology/Principal findings: Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). Conclusions/Significance: An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.