Current drug strategies for the treatment and control of schistosomiasis.

INTRODUCTION: Schistosomiasis, one of the current Neglected Tropical Diseases (NTDs) affects over 230 million people globally, with nearly 700 million at risk in more than 74 countries. Praziquantel (PZQ) has served as the primary treatment for the past four decades; however, its effectiveness is limited as it solely eliminates adult worms. In regions where infections are frequent, PZQ exhibits only temporary efficacy and has restricted potential to disrupt the prolonged transmission of the disease. AREAS COVERED: A comprehensive exploration using the PubMed database was conducted to review current pharmacotherapy approaches for schistosomiasis. This review also encompasses recent research findings related to potential novel therapeutics and the repurposing of existing drugs. EXPERT OPINION: Current schistosoma treatment strategies, primarily relying on PZQ, face challenges like temporary effectiveness and limited impact on disease transmission. Drug repurposing, due to economic constraints, is decisive for NTDs. Despite PZQ's efficacy, its failure to prevent reinfection highlights the need for complementary strategies, especially in regions with persistent environmental foci. Integrating therapies against diverse schistosome stages boosts efficacy and impedes resistance. Uncovering novel agents is essential to address resistance concerns in tackling this neglected tropical disease. Integrated strategies present a comprehensive approach to navigate the complex challenges.

Evaluation of Loop-Mediated Isothermal Amplification (LAMP) in Urine Samples for the Diagnosis of Imported Schistosomiasis.

Migratory flows and international travel are triggering an increase in imported cases of schistosomiasis in non-endemic countries. The present study aims to evaluate the effectiveness of the LAMP technique on patients' urine samples for the diagnosis of imported schistosomiasis in a non-endemic area in comparison to a commercial immunochromatographic test and microscopic examination of feces and urine. A prospective observational study was conducted in sub-Saharan migrants attending the Tropical Medicine Unit, Almería, Spain. For schistosomiasis diagnosis, serum samples were tested using an immunochromatographic test (Schistosoma ICT IgG-IgM). Stool and urine samples were examined by microcopy. Urine samples were evaluated by combining three LAMP assays for the specific detection of Schistosoma mansoni, S. haematobium, and for the genus Schistosoma. To evaluate the diagnostic accuracy, a latent class analysis (LCA) was performed. In total, 115 patients were included (92.2% male; median age: 28.3 years). Of these, 21 patients (18.3%) were diagnosed with schistosomiasis confirmed by microscopy, with S. haematobium being the most frequent species identified (18/115; 15.7%). The Schistosoma ICT IgG-IgM test result was 100% positive and Schistosoma-LAMP was 61.9% positive, reaching as high as 72.2% for S. haematobium. The sensitivity and specificity estimated by LCA, respectively, were: 92% and 76% for Schistosoma ICT IgG-IgM, 68% and 44% for Schistosoma-LAMP, and 46% and 97% for microscopy. In conclusion, the Schistosoma-LAMP technique presented a higher sensitivity than microscopy for the diagnosis of imported urinary schistosomiasis, which could improve the diagnosis of active infection, both in referral centers and in centers with limited experience or scarce resources and infrastructure.

First field study using Strong-LAMP for diagnosis of strongyloidiasis in Cubal, Angola.

BACKGROUND: Strongyloides stercoralis infection is a common neglected tropical disease distributed worldwide, mainly in tropical and subtropical climates. The impact of S. stercoralis infections on human health ranges from mild asymptomatic infections to chronic strongyloidiasis unnoticeable until the host is immunosuppressed. In severe strongyloidiasis, a syndrome of hyperinfection and larval dissemination to various organs can occur with high mortality rates. The diagnosis of strongyloidiasis is challenging because of the absence of a single standard reference test with high sensitivity and specificity, which also makes it difficult to estimate the accuracy of other diagnostic tests. This study aimed to evaluate, for the first time, the use of an easy-to-perform loop-mediated isothermal amplification (LAMP) colorimetric assay (named Strong-LAMP) for the molecular screening of strongyloidiasis in stool samples from patients in a low-resource endemic area in Cubal, Angola. To compare different LAMP application scenarios, the performance of the Strong-LAMP under field conditions in Angola was reassessed in a well-equipped reference laboratory in Spain and compared with a quantitative polymerase chain reaction (qPCR) method. METHODS: A total of 192 stool samples were collected from adult population in Cubal, Angola, and examined by parasitological methods (direct saline microscopy and Baermann's technique). DNA was extracted from each stool sample using a commercial kit and tested by the colorimetric Strong-LAMP assay for the detection of Strongyloides spp. under field conditions. Furthermore, all samples were shipped to a well-equipped laboratory in Spain, reanalysed by the same procedure and compared with a qPCR method. The overall results after testing were compared. RESULTS: Strongyloides stercoralis larvae were identified by direct saline microscopy and Baermann in a total of 10/192 (5.2%) and 18/192 (9.4%) stool samples, respectively. Other helminth and protozoan species were also identified. The Strong-LAMP-positive results were visually detected in 69/192 (35.9%) stool samples. The comparison of Strong-LAMP results in field conditions and at a reference laboratory matched in a total of 146/192 (76.0%) samples. A total of 24/192 (12.5%) stool samples tested positive by qPCR. CONCLUSIONS: This is the first study in which colorimetric Strong-LAMP has been clinically evaluated in a resource-poor strongyloidiasis endemic area. Strong-LAMP has been shown to be more effective in screening for strongyloidiasis than parasitological methods under field conditions and qPCR in the laboratory. Our Strong-LAMP has proven to be a field-friendly and highly accurate molecular test for the diagnosis of strongyloidiasis.

Real-Time Polymerase Chain Reaction Method for the Detection of Onchocerca volvulus in Post-Elimination Surveillance of Onchocerciasis in Ecuador.

Onchocerciasis has been declared eliminated in Ecuador and surveillance measures are of great interest. In this study, we examined the infectivity rates of Simulium exiguum by Onchocerca volvulus in previously hyperendemic areas in Esmeraldas province of Ecuador. These areas had previously undergone mass administration of ivermectin, which led to the interruption of transmission in 2009 and the certification of elimination in 2014. The study included three communities in Río Cayapas and one in Río Canandé, and a total of 2,950 adult S. exiguum were collected in 2018. We used quantitative polymerase chain reaction with O. volvulus O-150 plasmid control DNA to analyze 59 pools. Our findings revealed that the infectivity rates were zero, indicating that the transmission of O. volvulus remained suspended in the area.

Molecular diagnosis of Chagas disease: a systematic review and meta-analysis.

BACKGROUND: The complexity of the Chagas disease and its phases is impossible to have a unique test for both phases and a lot of different epidemiological scenarios. Currently, serology is the reference standard technique; occasionally, results are inconclusive, and a different diagnostic technique is needed. Some guidelines recommend molecular testing. A systematic review and meta-analysis of available molecular tools/techniques for the diagnosis of Chagas disease was performed to measure their heterogeneity and efficacy in detecting Trypanosoma cruzi infection in blood samples. METHODS: A systematic review was conducted up to July 27, 2022, including studies published in international databases. Inclusion and exclusion criteria were defined to select eligible studies. Data were extracted and presented according to PRISMA 2020 guidelines. Study quality was assessed using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). A random-effects model was used to calculate pooled sensitivity, specificity, and diagnostic odds ratio (DOR). Forest plots and a summary of the receiving operating characteristics (SROC) curves displayed the outcomes. Heterogeneity was determined by I2 and Tau2 statistics and P values. Funnel plots and Deek's test were used to assess publication bias. A quantitative meta-analysis of the different outcomes in the two different clinical phases was performed. RESULTS: We identified 858 records and selected 32 papers. Studies pertained to endemic countries and nonendemic areas with adult and paediatric populations. The sample sizes ranged from 17 to 708 patients. There were no concerns regarding the risk of bias and applicability of all included studies. A positive and nonsignificant correlation coefficient (S = 0.020; P = 0.992) was obtained in the set of studies that evaluated diagnostic tests in the acute phase population (ACD). A positive and significant correlation coefficient (S = 0.597; P < 0.000) was obtained in the case of studies performed in the chronic phase population (CCD). This resulted in high heterogeneity between studies, with the master mix origin and guanidine addition representing significant sources. INTERPRETATION/CONCLUSIONS AND RELEVANCE: The results described in this meta-analysis (qualitative and quantitative analyses) do not allow the selection of the optimal protocol of molecular method for the study of Trypanosoma cruzi infection in any of its phases, among other reasons due to the complexity of this infection. Continuous analysis and optimization of the different molecular techniques is crucial to implement this efficient diagnosis in endemic areas.

First field and laboratory evaluation of LAMP assay for malaria diagnosis in Cubal, Angola.

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.

Identification of Genomic Regions Implicated in Susceptibility to Schistosoma mansoni Infection in a Murine Backcross Genetic Model.

Only a small number of infected people are highly susceptible to schistosomiasis, showing high levels of infection or severe liver fibrosis. The susceptibility to schistosome infection is influenced by genetic background. To assess the genetic basis of susceptibility and identify the chromosomal regions involved, a backcross strategy was employed to generate high variation in schistosomiasis susceptibility. This strategy involved crossing the resistant C57BL/6J mouse strain with the susceptible CBA/2J strain. The resulting F1 females (C57BL/6J × CBA/2J) were then backcrossed with CBA/2J males to generate the backcross (BX) cohort. The BX mice exhibited a range of phenotypes, with disease severity varying from mild to severe disease, lacking a fully resistant group. We observed four levels of infection intensity using cluster and principal component analyses and K-means based on parasitological, pathological, and immunological trait measurements. The mice were genotyped with 961 informative SNPs, leading to the identification of 19 new quantitative trait loci (QTL) associated with parasite burden, liver lesions, white blood cell populations, and antibody responses. Two QTLs located on chromosomes 15 and 18 were linked to the number of granulomas, liver lesions, and IgM levels. The corresponding syntenic human regions are located in chromosomes 8 and 18. None of the significant QTLs had been reported previously.

Examining the gut microbiota from several human-biting tick species in Northwestern Spain.

Tick-borne diseases have increased significantly in Europe and Spain in recent years. One strategy explored for tick surveillance and control is the study of the microbiota. The focus is on understanding the relationships between pathogens and endosymbionts within the microbiota and how these relationships can alter these arthropods' vectorial capacity. Thus, it is pivotal to depict the bacterial communities composing the microbiota of ticks present in specific territories. This work aimed to describe the microbiota present in 29 adult individuals of 5 tick species collected from 4 provinces of Castilla y Leon in northwestern Spain from 2015 to 2022. DNA extraction and sequencing of the V4 hypervariable region of 16S-rRNA was performed on the tick samples, with subsequent analysis of diversity, taxonomic composition, and correlations between genera of microorganisms. There were no differences in the alpha diversity of microbiota by tick species, nor were compositional changes evident at the phylum level for microorganisms. However, interindividual differences at the microbial genus level allowed spatial differentiation of the 5 tick species included in the study. Correlation analyses showed complex interactions between different genera of microbiota members. These findings provide an initial insight into the composition of the gut microbiota of various tick species in northwestern Spain, which can contribute to establishing surveillance and control measures to reduce diseases such as rickettsiosis, Lyme disease, and Crimean-Congo hemorrhagic fever.

The most prominent modulated Annexins during parasitic infections.

Annexins (ANXs) exert different functions in cell biological and pathological processes and are thus known as double or multi-faceted proteins. These sophisticated proteins might express on both parasite structure and secretion and in parasite-infected host cells. In addition to the characterization of these pivotal proteins, describing their mechanism of action can be also fruitful in recognizing their roles in the pathogenesis of parasitic infections. Accordingly, this study presents the most prominent ANXs thus far identified and their relevant functions in parasites and infected host cells during pathogenesis, especially in the most important intracellular protozoan parasitic infections including leishmaniasis, toxoplasmosis, malaria and trypanosomiasis. The data provided in this study demonstrate that the helminth parasites most probably express and secret ANXs to develop pathogenesis while the modulation of the host-ANXs could be employed as a crucial strategy by intracellular protozoan parasites. Moreover, such data highlight that the use of analogs of both parasite and host ANX peptides (which mimic or regulate ANXs physiological functions through various strategies) might suggest novel therapeutic insights into the treatment of parasitic infections. Furthermore, due to the prominent immunoregulatory activities of ANXs during most parasitic infections and the expression levels of these proteins in some parasitic infected tissues, such multifunctional proteins might be also potentially relevant as vaccine and diagnostic biomarkers. We also suggest some prospects and insights that could be useful and applicable to form the basis of future experimental studies.

Executive summary consensus statement of imported diseases group (GEPI) of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Spanish Society of Tropical Medicine and International Health (SETMSI), on the diagnostic and treatment of imported schistosomiasis.

Schistosomiasis is a highly prevalent disease, especially in immigrant populations, and is associated with significant morbidity and diagnostic delays outside endemic areas. For these reasons, the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Spanish Society of Tropical Medicine and International Health (SEMTSI) have developed a joint consensus document to serve as a guide for the screening, diagnosis and treatment of this disease outside endemic areas. A panel of experts from both societies identified the main questions to be answered and developed recommendations based on the scientific evidence available at the time. The document was reviewed by the members from both societies for final approval.

Impact of pneumocystosis on the Spanish health care system, 1997-2020: Profile of HIV and non-HIV immunocompromised patients.

BACKGROUND: Pneumocystis jirovecii is an opportunistic fungus recognized for causing P. jirovecii pneumonia. The global prevalence is thought to be higher than 400,000 annual cases, although detailed information about epidemiological patterns is scarce. METHODOLOGY: A retrospective longitudinal descriptive study was performed among patients with diagnosis of pneumocystosis according to Classification of Diseases 9th edition, Clinical Modification (code 136.3 for the cases from 1997 to 2015; and 10th edition code B59.0 for cases from 2016 to 2020 in Spanish public hospitals from 1 January 1997-31 December 2020. RESULTS: A total of 25289 cases were diagnosed. The period incidence rate was 2.36 (95 % CI, 2.33-2.39) cases per 100,000 person-years. Infection was more frequent among men (72.2 %) than among women (27.8 %). Comorbidity was the main characteristic of this cohort. Up to 72.3 % of pneumocystis-infected patients (18293) had HIV coinfection. During the study period, there was a progressive decrease in the number of HIV coinfected cases as the group of patients without HIV infection increased, with the largest group in 2017. The lethality rate in the cohort was 16.7 %. The global cost was €229,234,805 and the average ( ± SD) cost per patient was €9065 ( ± 9315). CONCLUSIONS: The epidemiology of pneumocystosis in Spain has changed in the last two decades. We noted in our study the possibility of a reemergence among non-HIV immunocompromised patients as patients with hematological and nonhematological neoplasia and other risk groups. The lethality of pneumocystosis continues to be high, and the underlying diseases are the main variable associated with lethality.

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean-Congo Haemorrhagic Fever Virus in Patients from Spain.

Crimean-Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs.

Is babesiosis a rare zoonosis in Spain? Its impact on the Spanish Health System over 23 years.

BACKGROUND: Babesiosis is a zoonosis caused by an intraerythrocytic protozoan of the genus Babesia and transmitted mainly by ticks of the Ixodes spp. complex. There is no comprehensive global incidence in the literature, although the United States, Europe and Asia are considered to be endemic areas. In Europe, the percentage of ticks infected with Babesia spp. ranges from 0.78% to 51.78%. The incidence of babesiosis in hospitalized patients in Spain is 2.35 cases per 10,000,000 inhabitants/year. The mortality rate is estimated to be approximately 9% in hospitalized patients but can reach 20% if the disease is transmitted by transfusion. OBJECTIVE: To analyze the epidemiological impact of inpatients diagnosed with babesiosis on the National Health System (NHS) of Spain between 1997 and 2019. METHODOLOGY: A retrospective longitudinal descriptive study that included inpatients diagnosed with babesiosis [ICD-9-CM code 088.82, ICD-10 code B60.0, cases ap2016-2019] in public Spanish NHS hospitals between 1 January 1997 and 31 December 2019 was developed. Data were obtained from the minimum basic dataset (CMBD in Spanish), which was provided by the Ministerio de Sanidad, Servicios Sociales e Igualdad after the receipt of a duly substantiated request and the signing of a confidentiality agreement. MAIN FINDINGS: Twenty-nine inpatients diagnosed with babesiosis were identified in Spain between 1997 and 2019 (IR: 0.28 cases/10,000,000 person-years). A total of 82.8% of the cases were men from urban areas who were approximately 46 years old. The rate of primary diagnoses was 55.2% and the number of readmissions was 79.3%. The mean hospital stay was 20.3±19.2 days, with an estimated cost of €186,925.66. Two patients, both with secondary diagnoses of babesiosis, died in our study. CONCLUSIONS: Human babesiosis is still a rare zoonosis in Spain, with an incidence rate that has been increasing over the years. Most cases occurred in middle-aged men from urban areas between summer and autumn. The Castilla-La-Mancha and Extremadura regions recorded the highest number of cases. Given the low rate of primary diagnoses (55.2%) and the high number of readmissions (79.3%), a low clinical suspicion is likely. There was a 6.9% mortality in our study. Both patients who died were patients with secondary diagnoses of the disease.

Crimean-Congo Hemorrhagic Fever, Spain, 2013-2021.

Crimean-Congo hemorrhagic fever (CCHF) is a viral infectious disease for which distribution of the main vector, Hyalomma spp. ticks, is expanding. We analyzed all 10 cases of CCHF diagnosed in Spain during 2013-2021; case-patient median age was 56.5 years, and 7 were men. We identified CCHF virus genotypes III and V. Six case-patients acquired the infection in urban areas. Sixty percent of patients were infected in summer and 40% in spring. Two patients met criteria for hemophagocytic syndrome. Seven patients survived. The epidemiologic pattern of CCHF in Spain is based on occasional cases with an elevated mortality rate. Genotype III and, to a less extent also genotype V, CCHF circulates in humans in a common geographic area in Spain. Those data suggest that the expansion pathways are complex and may change over time. Physicians should remain alert to the possibility of new CCHF cases.

Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care.

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5'-quencher modified LAMP primer annealed to 3'-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis.

The Defensive Interactions of Prominent Infectious Protozoan Parasites: The Host's Complement System.

The complement system exerts crucial functions both in innate immune responses and adaptive humoral immunity. This pivotal system plays a major role dealing with pathogen invasions including protozoan parasites. Different pathogens including parasites have developed sophisticated strategies to defend themselves against complement killing. Some of these strategies include the employment, mimicking or inhibition of host's complement regulatory proteins, leading to complement evasion. Therefore, parasites are proven to use the manipulation of the complement system to assist them during infection and persistence. Herein, we attempt to study the interaction´s mechanisms of some prominent infectious protozoan parasites including Plasmodium, Toxoplasma, Trypanosoma, and Leishmania dealing with the complement system. Moreover, several crucial proteins that are expressed, recruited or hijacked by parasites and are involved in the modulation of the host´s complement system are selected and their role for efficient complement killing or lysis evasion is discussed. In addition, parasite's complement regulatory proteins appear as plausible therapeutic and vaccine targets in protozoan parasitic infections. Accordingly, we also suggest some perspectives and insights useful in guiding future investigations.

The Future of Point-of-Care Nucleic Acid Amplification Diagnostics after COVID-19: Time to Walk the Walk.

Since the onset of the COVID-19 pandemic, over 610 million cases have been diagnosed and it has caused over 6.5 million deaths worldwide. The crisis has forced the scientific community to develop tools for disease control and management at a pace never seen before. The control of the pandemic heavily relies in the use of fast and accurate diagnostics, that allow testing at a large scale. The gold standard diagnosis of viral infections is the RT-qPCR. Although it provides consistent and reliable results, it is hampered by its limited throughput and technical requirements. Here, we discuss the main approaches to rapid and point-of-care diagnostics based on RT-qPCR and isothermal amplification diagnostics. We describe the main COVID-19 molecular diagnostic tests approved for self-testing at home or for point-of-care testing and compare the available options. We define the influence of specimen selection and processing, the clinical validation, result readout improvement strategies, the combination with CRISPR-based detection and the diagnostic challenge posed by SARS-CoV-2 variants for different isothermal amplification techniques, with a particular focus on LAMP and recombinase polymerase amplification (RPA). Finally, we try to shed light on the effect the improvement in molecular diagnostics during the COVID-19 pandemic could have in the future of other infectious diseases.

Evaluation of the Immunomodulatory Effect of the Recombinant 14-3-3 and Major Antigen Proteins of Strongyloides stercoralis against an Infection by S. venezuelensis.

Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected parasitic disease that represents a serious public health problem. In immunocompromised patients, this parasitosis can result in hyperinfection or disseminated disease with high levels of mortality. In previous studies, the mRNAs encoding for the 14-3-3 and major antigen proteins were found to be expressed at high levels in S. stercoralis L3 larvae, suggesting potential key roles in parasite-host interactions. We have produced them as recombinant proteins (rSs14-3-3 and rSsMA) in a bacterial protein expression system. The serum levels of anti-rSs14-3-3 and anti-rSsMA IgGs are increased upon infection with S. venezuelensis, validating the use of the mouse model since the native 14-3-3 and MA proteins induce an immune response. Each recombinant protein was formulated in the adjuvant adaptation (ADAD) vaccination system and injected twice, subcutaneously, in CD1 mice that were experimentally infected with 3000 S. venezuelensis L3 to evaluate their protective and immunomodulatory activity. Our results, including the number of parthenogenetic females, number of eggs in stool samples and the analysis of the splenic and intestinal indexes, show that the vaccines did not protect against infection. The immunization with rSs14-3-3 induced changes in the cytokine profile in mice, producing higher expression of IL-10, TGF-ß, IL-13 and TNF-α in the spleen, suggesting a Th2/Treg-type response with an increase in TNF-α levels, confirming its role as an immunomodulator.

SMART-LAMP: A Smartphone-Operated Handheld Device for Real-Time Colorimetric Point-of-Care Diagnosis of Infectious Diseases via Loop-Mediated Isothermal Amplification.

Nucleic acid amplification diagnostics offer outstanding features of sensitivity and specificity. However, they still lack speed and robustness, require extensive infrastructure, and are neither affordable nor user-friendly. Thus, they have not been extensively applied in point-of-care diagnostics, particularly in low-resource settings. In this work, we have combined the loop-mediated isothermal amplification (LAMP) technology with a handheld portable device (SMART-LAMP) developed to perform real-time isothermal nucleic acid amplification reactions, based on simple colorimetric measurements, all of which are Bluetooth-controlled by a dedicated smartphone app. We have validated its diagnostic utility regarding different infectious diseases, including Schistosomiasis, Strongyloidiasis, and COVID-19, and analyzed clinical samples from suspected COVID-19 patients. Finally, we have proved that the combination of long-term stabilized LAMP master mixes, stored and transported at room temperature with our developed SMART-LAMP device, provides an improvement towards true point-of-care diagnosis of infectious diseases in settings with limited infrastructure. Our proposal could be easily adapted to the diagnosis of other infectious diseases.

How Has the Aspergillosis Case Fatality Rate Changed over the Last Two Decades in Spain?

(1) Background: Aspergillus produces high morbidity and mortality, especially in at-risk populations. In Spain, the evolution of mortality in recent years due to this fungus is not well established. The aim of this study was to estimate the case fatality rate of aspergillosis in inpatients from 1997 to 2017 in Spain. (2) Methodology: A retrospective descriptive study was conducted with records of inpatients admitted to the National Health System with a diagnosis of aspergillosis. (3) Principal findings: Of 32,960 aspergillosis inpatients, 24.5% of deaths were registered, and 71% of the patients who died were men. The percentage of deaths increased progressively with age. The case fatality rate progressively decreased over the period, from 25.4 and 27.8% in 1997-1998 to values of 20.6 and 20.8% in 2016 and 2017. Influenza and pneumonia occurrence/association significantly increased case fatality rates in all cases. (4) Conclusions: Our study shows that lethality significantly decreased in the last two decades despite the increase in cases. This highlights the fact that patients with solid and/or hematological cancer do not have a much higher mortality rate than the group of patients with pneumonia or influenza alone, these two factors being the ones that cause the highest CFRs. We also need studies that analyze the causes of mortality to decrease it and studies that evaluate the impact of COVID-19.