A loop-mediated isothermal amplification test for yaws: a multi-country diagnostic accuracy evaluation.

Background: To meet the WHO target of eradicating yaws by 2030, highly sensitive and specific diagnostic tools are needed. A multiplex Treponema pallidum-Haemophilus ducreyi loop-mediated isothermal amplification (TPHD-LAMP) test holds promise as a near-patient diagnostic tool for yaws and H ducreyi. We conducted a prospective evaluation in Cameroon, Côte d'Ivoire, Ghana, and the Republic of the Congo to determine the diagnostic accuracy of the TPHD-LAMP test, as well as to assess its acceptability, feasibility, and cost.

LAMP4yaws: , loop mediated isothermal amplification - protocol for a cross-sectional, observational, diagnostic accuracy study.

Introduction: Yaws, caused by the bacterium subsp. is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal.

Fully automated point-of-care differential diagnosis of acute febrile illness.

Background: In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk ("FeverDisk" for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device.

Methodology/principal Findings: A sample volume of 200 μL per FeverDisk was used.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies).

High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification.

We implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment.

Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Vaccine and Buffalo-Derived Strains.

Distinct pathogenic and epidemiological features underlie different strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin.

Development and Validation of Real-Time RT-LAMP Assays for the Specific Detection of Zika Virus.

Two one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of Zika virus (ZIKV) were developed, based on two different primer design approaches: (1) open source, based on a combination of sequence diversity clustering (phylogeny and principal component analysis) and LAVA algorithm, using 45 whole genome ZIKV sequences retrieved from the National Center for Biotechnology Information (NCBI) database; (2) standard software for LAMP primer design (Primer Explorer V4), using 59 sequences of the ZIKV 3' UTR. The assays were firstly evaluated with External Quality Assessment panels from INSTAND e.V.

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan.

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains.

Multiplex Mediator Displacement Loop-Mediated Isothermal Amplification for Detection of Treponema pallidum and Haemophilus ducreyi.

Yaws, a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue, manifests as ulcerative skin lesions. Nucleic acid amplification tests, like loop-mediated isothermal amplification (LAMP), are versatile tools to distinguish yaws from infections that cause similar skin lesions, primarily Haemophilus ducreyi. We developed a novel molecular test to simultaneously detect T.

A molecular prevalence survey on Anaplasma infection among domestic ruminants in Khartoum State, Sudan.

This study was conducted in Khartoum State, Sudan to determine the prevalence and the risk factors associated with Anaplasma and Ehrlichia species infections in domestic ruminants. Blood samples were collected from a total of 594 animals from 32 different farms distributed in the three provinces of Khartoum State. Among the 196 cattle, 200 sheep, and 198 goats examined using PCR, 13.

Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods.

Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered to be among the most important tick borne diseases in the Sudan. Information on the prevalence of the disease in different parts of the Sudan is limited. The purpose of this study was to estimate the prevalence of the disease in five states of the Sudan using molecular and serological assays.

Smear Microscopy for Diagnosis of Pulmonary Tuberculosis in Eastern Sudan.

Background: In Sudan, tuberculosis diagnosis largely relies on clinical symptoms and smear microscopy as in many other low- and middle-income countries. The aim of this study was to investigate the positive predictive value of a positive sputum smear in patients investigated for pulmonary tuberculosis in Eastern Sudan.

Methods: Two sputum samples from patients presenting with symptoms suggestive of tuberculosis were investigated using direct Ziehl-Neelsen (ZN) staining and light microscopy between June to October 2014 and January to July 2016.

Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome.

Background: A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015.

Methodology/principal Findings: A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used.

Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype.

Background: 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia.

Gene target selection for loop-mediated isothermal amplification for rapid discrimination of Treponema pallidum subspecies.

We show proof of concept for gene targets (polA, tprL, and TP_0619) that can be used in loop-mediated isothermal amplification (LAMP) assays to rapidly differentiate infection with any of the three Treponema pallidum subspecies (pallidum (TPA), pertenue (TPE), and endemicum (TEN)) and which are known to infect humans and nonhuman primates (NHPs). Four TPA, six human, and two NHP TPE strains, as well as two human TEN strains were used to establish and validate the LAMP assays. All three LAMP assays were highly specific for the target DNA.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization.

Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan.

A reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 1412 bp of the fusion protein gene (F gene) of four Newcastle disease virus (NDV) isolates; two velogenic (TY-1/90 and DIK-90) and two lentogenic isolates (Dongla 88/1 and GD.S.1).

Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium.

East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts.

Whole antigenic lysates of Ixodes ricinus, but not Der-p2 allergen-like protein, are potent inducers of basophil activation in previously tick-exposed human hosts.

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro.

Identification and characterization of Theileria annulata heat-shock protein 90 (HSP90) isoforms.

Heat-shock proteins (HSPs) refer to a group of proteins whose synthesis is enhanced upon sudden increase in temperature or exposure to a variety of other stressors. In this study, Theileria annulata (T. annulata) HSP90 was identified and characterized as a first step to understand the function of this molecule in T.

Coinfection of sheep with Anaplasma, Theileria and Babesia species in the Kurdistan Region, Iraq.

Infections of small ruminants with Anaplasma, Theileria and Babesia species are widely distributed in the old world and are of great economic impact. In Iraq, data on disease occurrence in sheep caused by above-mentioned infectious agents are scarce. This study provides information on various haemoparasitic agents infecting sheep in the Kurdistan Region, Iraq, using molecular diagnostic tools.