Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

BACKGROUND: Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. METHODS: The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. RESULTS: The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7-100), and 99.7% (95% CI, 99.2-100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1-100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1-100) and specificity was 99.7% (95% CI, 99.2-100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3-99.5) and 100% respectively. CONCLUSIONS: The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns

Differential expression of cold- and diet-specific genes encoding two carp liver delta 9-acyl-CoA desaturase isoforms.

Carp respond to cold by the upregulated expression of Delta9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second Delta9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of delta9-monounsaturates, confirming their identity as delta9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30 degrees C to 10 degrees C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15 degrees C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of delta9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment

Low-Quality Housing Is Associated With Increased Risk of Malaria Infection: A National Population-Based Study From the Low Transmission Setting of Swaziland.

BackgroundLow-quality housing may confer risk of malaria infection, but evidence in low transmission settings is limited.MethodsTo examine the relationship between individual level housing quality and locally acquired infection in children and adults, a population-based cross-sectional analysis was performed using existing surveillance data from the low transmission setting of Swaziland. From 2012 to 2015, cases were identified through standard diagnostics in health facilities and by loop-mediated isothermal amplification in active surveillance, with uninfected subjects being household members and neighbors. Housing was visually assessed in a home visit and then classified as low, high, or medium quality, based on housing components being traditional, modern, or both, respectively.ResultsOverall, 11426 individuals were included in the study: 10960 uninfected and 466 infected (301 symptomatic and 165 asymptomatic). Six percent resided in low-quality houses, 26% in medium-quality houses, and 68% in high-quality houses. In adjusted models, low- and medium-quality construction was associated with increased risk of malaria compared with high-quality construction (adjusted odds ratio [AOR], 2.11 and 95% confidence interval [CI], 1.26-3.53 for low vs high; AOR, 1.56 and 95% CI, 1.15-2.11 for medium vs high). The relationship was independent of vector control, which also conferred a protective effect (AOR, 0.67; 95% CI, .50-.90) for sleeping under an insecticide-treated bed net or a sprayed structure compared with neither.ConclusionsOur study adds to the limited literature on housing quality and malaria risk from low transmission settings. Housing improvements may offer an attractive and sustainable additional strategy to support countries in malaria elimination

The burden of congenital Chagas disease and implementation of molecular diagnostic tools in Latin America

It is estimated that between 8000 and 15 000 Trypanosoma cruzi infected babies are born every year to infected mothers in Chagas disease endemic countries. Currently, poor access to and performance of the current diagnostic algorithm, based on microscopy at birth and serology at 8-12 months after delivery, is one of the barriers to congenital Chagas disease (CCD) control. Detection of parasite DNA using molecular diagnostic tools could be an alternative or complement to current diagnostic methods, but its implementation in endemic regions remains limited. Prompt diagnosis and treatment of CCD cases would have a positive clinical and epidemiological impact. In this paper, we analysed the burden of CCD in Latin America, and the potential use of molecular tests to improve access to early diagnosis and treatment of T. cruzi infected newborns.Fil: Picado, Albert. Foundation for Innovative New Diagnostics; SuizaFil: Cruz, Israel. Foundation for Innovative New Diagnostics; SuizaFil: Redard Jacot, Maël. Foundation for Innovative New Diagnostics; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Torrico, Faustino. Universidad Mayor de San Simón; Bolivia. Fundación CEADES; BoliviaFil: Sosa-Estani, Sergio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentina. Drugs for Neglected Diseases initiative; BrasilFil: Katz, Zachary. Foundation for Innovative New Diagnostics; SuizaFil: Ndung'u, Joseph Mathu. Foundation for Innovative New Diagnostics; Suiz

A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination.

BACKGROUND: To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. METHODS: A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. RESULTS: The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95-99) and specificity 83 % (n = 15/18, 95 % CI 59-96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92-99) and specificity of 96 % (n = 151/157, 95 % CI 92-99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80-100) and specificity of 95 % (n = 123/128, 95 % CI 90-98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69-97). CONCLUSION: This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination

Point of Care Diagnosis of Multiple Schistosome Parasites: Species-specific DNA Detection in Urine by Loop-mediated Isothermal Amplification (LAMP)

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% − 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis

Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions

Assessing the presence of Wuchereria bancrofti in vector and human populations from urban communities in Conakry, Guinea

The Global Programme to Eliminate Lymphatic Filariasis was launched in 2000 with the goal of interrupting transmission of lymphatic filariasis (LF) through multiple rounds of mass drug administration (MDA). In Guinea, there is evidence of ongoing LF transmission, but little is known about the most densely populated parts of the country, including the capital Conakry. In order to guide the LF control and elimination efforts, serological and entomological surveys were carried out to determine whether or not LF transmission occurs in Conakry.; The prevalence of circulating filarial antigen (CFA) of Wuchereria bancrofti was assessed by an immuno-chromatography test (ICT) in people recruited from all five districts of Conakry. Mosquitoes were collected over a 1-year period, in 195 households in 15 communities. A proportion of mosquitoes were analysed for W. bancrofti, using dissection, loop-mediated isothermal amplification (LAMP) assay and conventional polymerase chain reaction (PCR).; CFA test revealed no infection in the 611 individuals examined. A total of 14,334 mosquitoes were collected; 14,135 Culex (98.6 %), 161 Anopheles (1.1 %) and a few other species. Out of 1,312 Culex spp. (9.3 %) and 51 An. gambiae (31.7 %) dissected, none was infected with any stage of the W. bancrofti parasite. However, the LAMP assay revealed that 1.8 % of An. gambiae and 0.31 % of Culex spp. were positive, while PCR determined respective prevalences of 0 % and 0.19 %.; This study revealed the presence of W. bancrofti DNA in mosquitoes, despite the apparent absence of infection in the human population. Although MDA interventions are not recommended where the prevalence of ICT is below 1 %, the entomological results are suggestive of the circulation of the parasite in the population of Conakry. Therefore, rigorous surveillance is still warranted so that LF transmission in Conakry would be identified rapidly and adequate responses being implemented

Diagnostic performance of a novel loop-mediated isothermal amplification (LAMP) assay targeting the apicoplast genome for malaria diagnosis in a field setting in sub-Saharan Africa.

BACKGROUND: New diagnostic tools to detect reliably and rapidly asymptomatic and low-density malaria infections are needed as their treatment could interrupt transmission. Isothermal amplification techniques are being explored for field diagnosis of malaria. In this study, a novel molecular tool (loop-mediated isothermal amplification-LAMP) targeting the apicoplast genome of Plasmodium falciparum was evaluated for the detection of asymptomatic malaria-infected individuals in a rural setting in The Gambia. METHODS: A blood was collected from 341 subjects (median age 9 years, range 1-68 years) screened for malaria. On site, a rapid diagnostic test (RDT, SD Bioline Malaria Antigen P.f) was performed, thick blood films (TBF) slides for microscopy were prepared and dry blood spots (DBS) were collected on Whatman(®) 903 Specimen collection paper. The TBF and DBS were transported to the field laboratory where microscopy and LAMP testing were performed. The latter was done on DNA extracted from the DBS using a crude (methanol/heating) extraction method. A laboratory-based PCR amplification was done on all the samples using DNA extracted with the Qiagen kit and its results were taken as reference for all the other tests. RESULTS: Plasmodium falciparum malaria prevalence was 37 % (127/341) as detected by LAMP, 30 % (104/341) by microscopy and 37 % (126/341) by RDT. Compared to the reference PCR method, sensitivity was 92 % for LAMP, 78 % for microscopy, and 76 % for RDT; specificity was 97 % for LAMP, 99 % for microscopy, and 88 % for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference standard was 0.94 for LAMP, 0.88 for microscopy and 0.81 for RDT. Turn-around time for the entire LAMP assay was approximately 3 h and 30 min for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over 8 h by microscopy. CONCLUSION: The LAMP assay could produce reliable results the same day of the screening. It could detect a higher proportion of low density malaria infections than the other methods tested and may be used for large campaigns of systematic screening and treatment