A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings.

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45-98.42) and 88.85% (95% CI: 85.08-91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

Differentiation of Capripox Viruses by Nanopore Sequencing.

The genus capripoxvirus (CaPV), family Poxviridae, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Flesh ID: Nanopore Sequencing Combined with Offline BLAST Search for the Identification of Meat Source.

Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing, in addition to avoid allergy and religious consequences. Gold standard is the real-time PCR assays, which has a limited target capability. In this study, we have established a rapid sequencing protocol to identify animal species within hours. Sequencing was achieved by nanopore sequencing and data analysis via offline BLAST search. The whole procedure was conducted in a mobile suitcase lab. As per national and international regulations, the developed assay detected adulteration of pork meat with 0.1% of horse, chicken, turkey, cattle, sheep, duck, rabbit, goat, and donkey. The developed test could be used on-site as a rapid and mobile detection system to determine contamination of meat products.

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

Molecular and Serological Footprints of Mycobacterium avium Subspecies Infections in Zoo Animals.

BACKGROUND: Mycobacteria of the Mycobacterium avium complex (MAC) pose a significant risk to zoological collections. Mycobacterium avium subspecies paratuberculosis (MAP) is a member of MAC and the causative agent of Johne's disease. Despite many reports in animals kept in zoological gardens, systemic surveillance has rarely been reported. METHODS: In this study, archived serum samples collected from animal species at the Wilhelma Zoological and Botanical Gardens in Stuttgart, Germany, were screened for the presence of antibodies against MAC and MAP. In addition, molecular investigations were performed on necropsy, fecal, and environmental samples. RESULTS: In total, 30/381 serum samples of various mammalian species were positive for MAC antibodies in ELISA, while one sample of a reticulated giraffe (Giraffa camelopardalis reticulata) was positive in MAP-specific ELISA. Samples from many species were positive in pan-Mycobacterium real-time PCR (40/43 fecal samples, 27/43 environmental samples, and 31/90 necropsy samples). Surprisingly, no sample was positive in the MAP-specific molecular assays. However, two environmental samples from primate enclosures were positive in Mycobacterium avium subspecies hominissuis (MAH)-specific real-time PCR. CONCLUSIONS: The results reveal serological indications of MAC infections in the zoological collection. However, the presence of a MAP-contaminated environment by a high-shedding individual animal or MAP-infected population is unlikely.

Zika Virus Amplification Using Strand Displacement Isothermal Method and Sequencing Using Nanopore Technology.

Development of novel point of care diagnostic methods in order to help in implementing disease control program and identifying the causative agent of an outbreak is crucial. Classical diagnostic techniques, e.g., real-time polymerase chain reaction (PCR), rely on the presence of the nucleic acid sequence of the target in GenBank. In the case of an emerging new strain of a known or novel pathogen, false-negative results will be recorded by PCR. On the other hand, next-generation sequencing technologies allow rapid whole genome sequencing without previous knowledge of the target. One of these methods is the Oxford Nanopore sequencing technique, which utilizes a portable device named MinION and has a short run time. In this protocol, we describe the development of a novel nanopore sequencing protocol by combining random isothermal amplification technology and nanopore sequencing. The established protocol is rapid (<7 h) and sensitive as less than 4% of the sequenced RNA belonged to the target virus, Zika. Interestingly, we have established an offline BLAST search for the data analysis that facilitates the use of the whole protocol at remote settings without the need of an Internet connection.

Recombinase polymerase amplification assays for the identification of pork and horsemeat.

Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing. Gold standard is the real-time PCR assays, which can be conducted at highly equipped laboratories. Toward the development of point-of-need test, two rapid molecular assays based on recombinase polymerase amplification (RPA) for the detection of pork and horse DNA were established. Target genes are the porcine mitochondrial ND2 and equine ATP 6-8 genes. The pork and horse_RPA assays detected 16 and one DNA molecules/µl in eleven to six minutes, respectively. The myoglobin in the meat did not influence the assays performances, while the presence of high background-DNA induced a one log decrease in the sensitivity. Both assays are highly specific and identify down to 0.1% of their target DNA in meat mixtures. Both RPA assays could be used on-site as a rapid and mobile detection system to determine contamination of meat products.

Recombinase polymerase amplification assay for rapid detection of Monkeypox virus.

In this study, a rapid method for the detection of Central and West Africa clades of Monkeypox virus (MPXV) using recombinase polymerase amplification (RPA) assay targeting the G2R gene was developed. MPXV, an Orthopoxvirus, is a zoonotic dsDNA virus, which is listed as a biothreat agent. RPA was operated at a single constant temperature of 42°C and produced results within 3 to 10 minutes. The MPXV-RPA-assay was highly sensitive with a limit of detection of 16 DNA molecules/µl. The clinical performance of the MPXV-RPA-assay was tested using 47 sera and whole blood samples from humans collected during the recent MPXV outbreak in Nigeria as well as 48 plasma samples from monkeys some of which were experimentally infected with MPXV. The specificity of the MPXV-RPA-assay was 100% (50/50), while the sensitivity was 95% (43/45). This new MPXV-RPA-assay is fast and can be easily utilised at low resource settings using a solar powered mobile suitcase laboratory.

Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples.

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from "sample in" to "result out" was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Serotyping of foot-and-mouth disease virus using oxford nanopore sequencing.

Foot-and-mouth disease virus (FMDV), belonging to the family of Picornaviridae, infects mostly cloven-hoofed animals and leads to huge economic losses. Since there is no cross-protection between the seven serotypes of FMDV, effective vaccination relies on the knowledge of the serotype causing the outbreak. The most common methods of serotyping are antigen ELISAs and amplification-based sequencing. Serotype-specific PCR methods exist but have limitations due to emerging mutants within serotypes. Sequencing is a promising technology, but currently suffers from cumbersome procedures and long turnaround times. In this study, we have established a novel sequencing protocol relying on nanopore sequencing and offline BLAST search. The procedure was completed in 5 h including RNA extraction, reverse transcription, second-strand synthesis, barcoding, sequencing and data analysis, which did not require a bioinformatician. In total, 12,193 sequence files were obtained. The offline BLAST search to the P1 region revealed the most successful categorization of the seven FMDV serotypes (specificity: 98.3%) over whole genome (24.8%), P2 (23.6%) and P3 (21.4%). In conclusion, our protocol enables rapid and reliable FMDV serotyping. The whole procedure can be conducted with a mobile suitcase laboratory, which is easy to use at the point of need in endemic countries.

Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka.

BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.

Combination random isothermal amplification and nanopore sequencing for rapid identification of the causative agent of an outbreak.

BACKGROUND: Outbreaks of fever of unknown origin start with nonspecific symptoms and case definition is only slowly developed and adapted, therefore, identifying the causative agent is crucial to ensure suitable treatment and control measures. As an alternative method for Polymerase Chain Reaction in molecular diagnostics diagnostic, metagenomics can be applied to identify the pathogen responsible for the outbreak through sequencing all nucleic acids present in a sample extract. Sequencing data obtained can identify new or variants of known agents. OBJECTIVES: To develop a rapid and field applicable protocol to allow the identification of the causative agent of an outbreak. STUDY DESIGN: We explored a sequencing protocol relying on multiple displacement isothermal amplification and nanopore sequencing in order to allow the identification of the causative agent in a sample. To develop the procedure, a mock sample consisting of supernatant from Zika virus tissue culture was used. RESULTS: The procedure took under seven hours including sample preparation and data analysis using an offline BLAST search. In total, 63,678 sequence files covering around 10,000 bases were extracted. BLAST search revealed the presence of Zika virus. CONCLUSION: In conclusion, the protocol has potential for point of need sequencing to identify RNA viruses. The whole procedure was operated in a suitcase laboratory. However, the procedure is cooling chain dependent and the cost per sequencing run is still high.

Development of a pan-rickettsial molecular diagnostic test based on recombinase polymerase amplification assay.

Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections.

Rapid Molecular Detection of Zika Virus in Acute-Phase Urine Samples Using the Recombinase Polymerase Amplification Assay.

BACKGROUND: Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%. CONCLUSIONS/SIGNIFICANCE: The developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering).

Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.

BACKGROUND: Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD. METHODS: A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh. RESULTS: The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent. CONCLUSIONS: The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes.