Comparison of a new visual isothermal nucleic acid amplification test with PCR and skin snip analysis for diagnosis of onchocerciasis in humans

Alhassan, Andy; Osei-Atweneboana, Mike Y.; Kyeremeh, Kwadwo F.; Poole, Catherine B.; Li, Zhi‐Ru; Tettevi, Edward J.; Tanner, Nathan A.; Carlow, Clotilde K. S.

doi:10.1016/j.molbiopara.2016.07.006

Cited by 38 publications

(52 citation statements)

References 15 publications

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“…The ability of the RF4 LAMP assay to successfully detect as few as < 10 mf/ml and any developmental stage of the parasite that may be present in the insect hosts suggests the suitability of this method for identi cation of Chrysops with extremely low levels of infection which may be missed using conventional microscopy. A high level of sensitivity is particularly important in areas where prolonged administration of ivermectin has led to the drastic reduction of parasite in the host [49].…”

Section: Discussionmentioning

confidence: 99%

Validation of Loop Mediated Isothermal Amplification for the Detection of Loa loa Infection in Chrysops sp in Experimental and Natural Field Conditions

Amambo

Abong

Fombad

et al. 2020

Preprint

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Background/ObjectiveThe Mass Drug Administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that led to reduction of the levels of infection in Chrysops vectors. Accurate parasite detection is essential for the success of any filariasis control program. Although microscopy is the gold standard for detecting the infection, its sensitivity is compromised when the intensity of infection is low. The Loop-mediated isothermal amplification (LAMP) of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. This study aimed at validating the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions. MethodsTwo sets of 18 flies were fed on volunteers with either a low (<10mf/ml) or high (>30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed with LAMP for the detection of L. loa infection. In addition, a total of 9,270 flies were collected from the North West, East, and South West regions of Cameroon using sweep nets and subjected to microscopy (7,841 flies) and LAMP (1,291 flies plus 138 nulliparous flies) analyses. FindingsLAMP assay successfully detected parasites in Chrysops fed on both low and high microfilariaemic volunteers. Field validation and surveillance studies phase revealed LAMP-based infection rates ranging from 0,5 % to 31.6 %, with the lowest levels in the South West 2 and highest infection rates in South West 1. LAMP assay detected significantly higher infection rates than microscopy in 4 of 5 study sites. ConclusionThis study demonstrated that LAMP is a simple surveillance tool, and is more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

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Section: Discussionmentioning

confidence: 99%

Validation of Loop Mediated Isothermal Amplification for the Detection of Loa loa Infection in Chrysops sp in Experimental and Natural Field Conditions

Amambo

Abong

Fombad

et al. 2020

Preprint

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“…The applicability of LAMP to detect Onchocerca microfilariae in skin snips has also been tested using the O-150 tandem repeat as a target (Alhassan et al 2016). When applied to skin snips from 70 individuals living in endemic areas of Ghana, the LAMP assay showed a significantly higher sensitivity (67·5% prevalence) compared with both PCR using the external LAMP primers (37·1%) and microscopy (31·4%).…”

Section: Onchocerciasismentioning

confidence: 99%

“…When applied to skin snips from 70 individuals living in endemic areas of Ghana, the LAMP assay showed a significantly higher sensitivity (67·5% prevalence) compared with both PCR using the external LAMP primers (37·1%) and microscopy (31·4%). Additionally, the detection of amplified parasite DNA was accomplished visually using pH-sensitive dyes (Alhassan et al 2016). To date, the amplification of L3 larvae-specific transcripts by RT-PCR for the detection of infective black flies (as it has been for LF in mosquitoes) has yet to be reported.…”

Section: Onchocerciasismentioning

confidence: 99%

Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

et al. 2016

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The current mainstay for control of the four major helminth diseases in humans (lymphatic filariasis, onchocerciasis, soiltransmitted helminthiases and schistosomiasis) is with preventive chemotherapy by mass administration of key anthelminthics. Following the London Declaration on Neglected Tropical Diseases in 2012, a roadmap for the elimination and control of these helminthiases by 2020 has been devised. With expected declines in prevalence and intensity of these infections, there is urgent need for implementing more sensitive, high-throughput and cost-effective diagnostic tools. Currently available diagnostic approaches for surveying, monitoring and evaluating helminth control programmes are based on microscopical observation of eggs/larvae, and/or detection of antibodies or parasite antigens in stool, urine or blood; all relatively low-throughput and of limited sensitivity and specificity. Newly proposed approaches for helminthiases diagnosis include the nucleic acid-based methods of (multiplex) real-time polymerase chain reaction assays, loop-mediated isothermal amplification and recombinase polymerase amplification. However, as well as sensitivity/specificity evaluation, their comparison to current 'gold standard' diagnostics and future application in individual-/community-based diagnosis, or in xenomonitoring requires consideration of relative costs, agreement of standard methods and strategic interpretation of resulting data before control/elimination programmes might best utilize molecular diagnostics to inform decision making. We review current nucleic-acid-based molecular diagnostic methods and highlight the needs and future research required to refine these tools for monitoring and evaluation of control and elimination programmes for four major human helminthiases.

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“…Besides microscopy [10], immunological [17,18] and nucleic acid-based methods, speci cally PCR [19,20] and LAMP [21,22] have been used to diagnose O. volvulus in skin. Although there have been advances in the development of immunological methods involving detection of either antibody or antigen [17,18], cross-reactivity with other larial species has been reported [23].…”

Section: Introductionmentioning

confidence: 99%

Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

Abong

Amambo

Ndongmo

et al. 2020

Preprint

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Background. Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques.Method. Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, real-time PCR and LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of real-time PCR and LAMP.Results. In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) treated microfilaridemic participants where still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia.In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity.Conclusion. Patterns in skin mirofilarial clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA were less susceptible to ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

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Comparison of a new visual isothermal nucleic acid amplification test with PCR and skin snip analysis for diagnosis of onchocerciasis in humans

Cited by 38 publications

References 15 publications

Validation of Loop Mediated Isothermal Amplification for the Detection of Loa loa Infection in Chrysops sp in Experimental and Natural Field Conditions

Validation of Loop Mediated Isothermal Amplification for the Detection of Loa loa Infection in Chrysops sp in Experimental and Natural Field Conditions

Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

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