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.

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.

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.

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.

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.

Comparison of three PCR-based methods to detect Loa loa and Mansonella perstans in long-term frozen storage dried blood spots.

OBJECTIVES: Loa loa and Mansonella perstans are two very common filarial species in Africa. Although microscopy is the traditional diagnostic method for human filariasis, several polymerase chain reaction (PCR) methods have emerged as an alternative approach for identifying filarial parasites. The aim of this study is to compare three molecular methods and decide which is the most suitable for diagnosing human loiasis and mansonellosis in non-endemic regions using dried blood spot (DBS) as a medium for sample collection and storage. METHODS: A total of 100 DBS samples, with their corresponding thin and thick blood smears, were selected for this study. Microscopy was used as the reference method to diagnose and calculate the microfilaraemia. Filarial DNA was extracted using the saponin/Chelex method and the DNA isolated was assayed by Filaria-real time-PCR, filaria-nested PCR, and cytochrome oxidase I PCR. All PCR products were subsequently purified and sequenced. The statistical values for each molecular test were calculated and compared. RESULTS: Overall, 64 samples were identified as negative by all tests and a further 36 samples were positive by at least one of the methods used. The sensitivity and specificity were similar for the different molecular methods, all of which demonstrated good agreement with microscopy. CONCLUSIONS: Based on this study, and from a practical point of view (single and short amplification round), the optimal technique for diagnosing filarial infection in non-endemic regions is filaria-real time-PCR, which presents high sensitivity and specificity and is also able to detect a wide range of human filariae.

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples.

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.

Application of a Genus-Specific LAMP Assay for Schistosome Species to Detect Schistosoma haematobium x Schistosoma bovis Hybrids.

Schistosomiasis is a disease of great medical and veterinary importance in tropical and subtropical regions caused by different species of parasitic flatworms of the genus Schistosoma. The emergence of natural hybrids of schistosomes indicate the risk of possible infection to humans and their zoonotic potential, specifically for Schistosoma haematobium and S. bovis. Hybrid schistosomes have the potential to replace existing species, generate new resistances, pathologies and extending host ranges. Hybrids may also confuse the serological, molecular and parasitological diagnosis. Currently, LAMP technology based on detection of nucleic acids is used for detection of many agents, including schistosomes. Here, we evaluate our previously developed species-specific LAMP assays for S. haematobium, S. mansoni, S. bovis and also the genus-specific LAMP for the simultaneous detection of several Schistosoma species against both DNA from pure and, for the first time, S. haematobium x S. bovis hybrids. Proper operation was evaluated with DNA from hybrid schistosomes and with human urine samples artificially contaminated with parasites' DNA. LAMP was performed with and without prior DNA extraction. The genus-specific LAMP properly amplified pure Schistosoma species and different S. haematobium-S. bovis hybrids with different sensitivity. The Schistosoma spp.-LAMP method is potentially adaptable for field diagnosis and disease surveillance in schistosomiasis endemic areas where human infections by schistosome hybrids are increasingly common.

Loop-Mediated Isothermal Amplification in Schistosomiasis.

Human schistosomiasis is one of the most important parasitic diseases, causing around 250 million cases (mostly in Africa) and 280,000-500,000 deaths every year. Due to the limited resources and the far-removed nature of many endemic areas, the implementation of new, sensitive and specific diagnostic tools has had little success. This is particularly true for PCR-based molecular methods that require expensive equipment and trained personnel to be executed. Loop-mediated isothermal amplification (LAMP) along with other isothermal techniques appeared in the early 21st century as an alternative to those methods, overcoming some of the aforementioned limitations and achieving a more inexpensive diagnostic. However, to this date, neither LAMP nor any other isothermal technique have signified a meaningful change in the way schistosomiasis diagnosis is routinely performed. Here, we present the recent developments in LAMP-based schistosomiasis diagnosis. We expose the main advantages and disadvantages of LAMP technology over PCR and other classical diagnostic methods focusing in various research approaches on intermediate hosts, animal models and patients. We also examine its potential clinical application in post-therapy monitoring, as well as its usefulness as a point-of-care test.

Detection of SARS-CoV-2 RNA in Urine by RT-LAMP: A Very Rare Finding.

Detection of SARS-CoV-2 is routinely performed in naso/oropharyngeal swabs samples from patients via RT-qPCR. The RT-LAMP technology has also been used for viral RNA detection in respiratory specimens with both high sensitivity and specificity. Recently, we developed a novel RT-LAMP test for SARS-CoV-2 RNA detection in nasopharyngeal swab specimens (named, N15-RT-LAMP) that can be performed as a single-tube colorimetric method, in a real-time platform, and as dry-LAMP. To date, there has been very little success in detecting SARS-CoV-2 RNA in urine by RT-qPCR, and the information regarding urine viral excretion is still scarce and not comprehensive. Here, we tested our N15-RT-LAMP on the urine of 300 patients admitted to the Hospital of Salamanca, Spain with clinical suspicion of COVID-19, who had a nasopharyngeal swab RT-qPCR-positive (n = 100), negative (n = 100), and positive with disease recovery (n = 100) result. The positive group was also tested by RT-qPCR for comparison to N15-RT-LAMP. Only a 4% positivity rate was found in the positive group via colorimetric N15-RT-LAMP and 2% via RT-qPCR. Our results are consistent with those obtained in other studies that the presence of SARS-CoV-2 RNA in urine is a very rare finding. The absence of SARS-CoV-2 RNA in urine in the recovered patients might suggest that the urinary route is very rarely used for viral particle clearance.

Whip-LAMP: a novel LAMP assay for the detection of Trichuris muris-derived DNA in stool and urine samples in a murine experimental infection model.

BACKGROUND: Trichuris trichiura (human whipworm) infects an estimated 477 million individuals worldwide. In addition to T. trichiura, other Trichuris species can cause an uncommon zoonosis and a number of human cases have been reported. The diagnosis of trichuriasis has relied traditionally on microscopy. Recently, there is an effort to use molecular diagnostic methods, mainly qPCR. LAMP technology could be an alternative for qPCR especially in low-income endemic areas. Trichuris muris, the causative agent of trichuriasis in mice, is of great importance as a model for human trichuriasis. Here, we evaluate the diagnostic utility of a new LAMP assay in an active experimental mouse trichuriasis in parallel with parasitological method by using stool and, for the first time, urine samples. METHODS: Stool and urine samples were collected from mice infected with eggs of T. muris. The dynamics of infection was determined by counting the number of eggs per gram of faeces. A LAMP based on the 18S rRNA gene from T. muris was designed. Sensitivity and specificity of LAMP was tested and compared with PCR. Stool and urine samples were analysed by both LAMP and PCR techniques. RESULTS: Trichuris muris eggs were detected for the first time in faeces 35 days post-infection. LAMP resulted specific and no cross-reactions were found when using 18 DNA samples from different parasites. The detection limit of the LAMP assay was 2 pg of T. muris DNA. When testing stool samples by LAMP we obtained positive results on day 35 p.i. and urine samples showed amplification results on day 20 p.i., i.e. 15 days before the onset of T. muris eggs in faeces. CONCLUSIONS: To the best of our knowledge, we report, for the first time, a novel LAMP assay (Whip-LAMP) for sensitive detection of T. muris DNA in both stool and urine samples in a well-established mice experimental infection model. Considering the advantages of urine in molecular diagnosis in comparison to stool samples, should make us consider the possibility of starting the use urine specimens in molecular diagnosis and for field-based studies of human trichuriasis where possible. Further studies with clinical samples are still needed.

Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification.

Schistosomiasis is considered a neglected parasitic disease. Around 280,000 people die from it annually, and more than 779 million people are at risk of getting infected. The schistosome species which infect human beings are Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma guineensis, and Schistosoma mekongi. This disease is also of veterinary significance; the most important species being Schistosoma bovis since it causes the disease in around 160 million livestock in Africa and Asia. This work was aimed at designing and developing a genus-specific loop-mediated isothermal amplification (LAMP) method for detecting the most important schistosome species affecting humans and for the species-specific detection of S. bovis. Bioinformatics tools were used for primer design, and the LAMP method was standardised for detecting the ITS-1 region from S. intercalatum, S. haematobium, S. mansoni, S. japonicum, and S. bovis DNA (generic test) and the NADH 1 gene for specifically detecting S. bovis (at different DNA concentrations). Detection limits achieved were 1 pg DNA for S. mansoni, 0.1 pg for S. haematobium, 1 pg for S. intercalatum, and 10 pg for S. bovis. No amplification for S. japonicum DNA was obtained. The LAMP designed for the amplification of S. bovis NADH-1 worked specifically for this species, and no other DNA from other schistosome species included in the study was amplified. Two highly sensitive LAMP methods for detecting different Schistosoma species important for human and veterinary health were standardised. These methods could be very useful for the diagnosis and surveillance of schistosome infections.

A Trypanosoma cruzi Genome Tandem Repetitive Satellite DNA Sequence as a Molecular Marker for a LAMP Assay for Diagnosing Chagas' Disease.

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi which is endemic throughout Latin America and is spread by worldwide migration. Diagnosis is currently limited to serological and molecular techniques having variations regarding their sensitivity and specificity. This work was aimed at developing a new sensitive, applicable, and cost-effective molecular diagnosis technique for loop-mediated isothermal amplification-based detection of T. cruzi (Tc-LAMP). The results led to determining a highly homologous satellite repeat region (231 bp) among parasite strains as a molecular marker for diagnosing the disease. Tc-LAMP was performed correctly for detecting parasite DNA (5 fg for the CL Brener strain and 50 fg for the DM28, TcVI, and TcI strains). Assay results proved negative for DNA from 16 helminth species and 7 protozoa, including Leishmania spp. Tc-LAMP based on the highly repeated T. cruzi satellite region is thus proposed as an important alternative for diagnosing T. cruzi infection, overcoming other methods' limitations such as their analytic capability, speed, and requiring specialized equipment or highly trained personnel. Tc-LAMP could be easily adapted for point-of-care testing in areas having limited resources.