Diagnostic performance of loop-mediated isothermal amplification and ultra-sensitive rapid diagnostic tests for malaria screening among pregnant women in Kenya

Background: Screen-and-treat strategies with sensitive diagnostic tests may reduce malaria-associated adverse pregnancy outcomes. We conducted a diagnostic accuracy study to evaluate new point-of-care tests to screen pregnant women for malaria at their first antenatal visit in western Kenya. Methods: Consecutively women were tested for Plasmodium infection by expert-microscopy, conventional rapid diagnostic test (cRDT), ultra-sensitive RDT (usRDT), and loop-mediated isothermal amplification (LAMP). Photo-induced electron-transfer polymerase-chain-reaction (PET-PCR) served as the reference standard. Diagnostic performance was calculated and modelled at low parasite densities. Results: Between May-September 2018, 172 out of 482 screened participants (35.7%) were PET-PCR positive. Relative to PET-PCR, expert-microscopy was least sensitive (40.1%, 95% CI 32.7-47.9), followed by cRDT (49.4%, 41.7-57.1), usRDT (54.7%, 46.9-62.2), and LAMP (68.6%, 61.1-75.5). Test sensitivities were comparable in febrile women (N=90). Among afebrile women (N=392), the geometric- mean parasite density was 29 parasites/μL and LAMP (sensitivity=61.9%) and usRDT (43.2%) detected 1.74 (1.31-2.30) and 1.21 (0.88-2.21) more infections than cRDT (35.6%). Per our model, tests performed similarly at densities >200 parasites/μL. At 50 parasites/μL, the sensitivities were 45%, 56%, 62% and 74% with expert-microscopy, cRDT, usRDT, and LAMP, respectively. Conclusions: This first-generation usRDT provided moderate improvement in detecting low-density infections in afebrile pregnant women compared to cRDTs

Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed

Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR.

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs

Expected results for the MG-LAMP assay.

<p>Expected results for the MG-LAMP assay.</p

Comparison of MG-LAMP to PET-PCR.

<p>Comparison of MG-LAMP to PET-PCR.</p

Determination of the optimum MG concentration for use in the LAMP assay.

<p>Determination of the optimum MG concentration for use in the LAMP assay.</p

Oligonucleotide primer sequences used in this study.

<p>Oligonucleotide primer sequences used in this study.</p

Novel <i>P</i>. <i>ovale</i> primers only amplify <i>P</i>. <i>ovale</i> and not the other human-infecting species.

<p><i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>malariae</i>, <i>P</i>. <i>knowlesi</i>, <i>P</i>. <i>ovale curtisi</i>, <i>P</i>. <i>ovale wallikeri</i> DNA samples were utilized for primer specificity testing. Only the positive control (a known <i>P</i>. <i>ovale</i> sample), <i>P</i>. <i>ovale curtisi</i> and <i>P</i>. <i>ovale wallikeri</i> were amplified using our primers (amplification plots with Ct values of 25.57, 33.38 and 35.2 respectively). No amplification (flat lines) was noted for the other species and the no template control (NTC).</p

<i>P</i>. <i>ovale</i> reticulocyte binding protein 2 (<i>rbp-2</i>) sequence alignment.

<p>Both <i>P</i>. <i>ovale curtisi</i> and <i>P</i>. <i>ovale wallikeri</i> sequences were aligned using Geneious software program in order to select a conserved region for the two <i>P</i>. <i>ovale</i> subspecies. Cytosine is labelled purple, adenine pink, guanine yellow and thymine green. The forward (PoRBP2FWD) and reverse (PoRBP2REV) primers are denoted in dark and light green boxes, respectively.</p

Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil

Abstract Background Microscopic detection of malaria parasites is the standard method for clinical diagnosis of malaria in Brazil. However, malaria epidemiological surveillance studies specifically aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and field-usable tools. The diagnostic accuracy of the colorimetric malachite green, loop-mediated, isothermal amplification (MG-LAMP) assay was evaluated in remote health posts in Roraima state, Brazil. Methods Study participants were prospectively enrolled from health posts (healthcare-seeking patients) and from nearby villages (healthy participants) in three different study sites. The MG-LAMP assay and microscopy were performed in the health posts. Two independent readers scored the MG-LAMP tests as positive (blue/green) or negative (clear). Sensitivity and specificity of local microscopy and MG-LAMP were calculated using results of PET-PCR as a reference. Results A total of 91 participants were enrolled. There was 100% agreement between the two MG-LAMP readers (Kappa = 1). The overall sensitivity and specificity of MG-LAMP were 90.0% (95% confidence interval (CI) 76.34–97.21%) and 94% (95% CI 83.76–98.77%), respectively. The sensitivity and specificity of local microscopy were 83% (95% CI 67.22–92.66%) and 100% (95% CI 93.02–100.00%), respectively. PET-PCR detected six mixed infections (infection with both Plasmodium falciparum and Plasmodium vivax); two of these were also detected by MG-LAMP and one by microscopy. Microscopy did not detect any Plasmodium infection in the 26 healthy participants; MG-LAMP detected Plasmodium in five of these and PET-PCR assay detected infection in three. Overall, performing the MG-LAMP in this setting did not present any particular challenges. Conclusion MG-LAMP is a sensitive and specific assay that may be useful for the detection of malaria parasites in remote healthcare settings. These findings suggest that it is possible to implement simple molecular tests in facilities with limited resources