Evaluation of the diagnostic accuracy of prototype rapid tests for human African trypanosomiasis

Peer reviewedPublisher PD

Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

<p><b>Background:</b> The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.</p> <p><b>Methodology/Principal Findings:</b> For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.</p> <p><b>Conclusions/Significance:</b> This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.</p&gt

Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination.

INTRODUCTION: Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority. METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia. RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105). CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings

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

Integrating innovations:a qualitative analysis of referral non-completion among rapid diagnostic test-positive patients in Uganda's human African trypanosomiasis elimination programme

BACKGROUND: The recent development of rapid diagnostic tests (RDTs) for human African trypanosomiasis (HAT) enables elimination programmes to decentralise serological screening services to frontline health facilities. However, patients must still undertake multiple onwards referral steps to either be confirmed or discounted as cases. Accurate surveillance thus relies not only on the performance of diagnostic technologies but also on referral support structures and patient decisions. This study explored why some RDT-positive suspects failed to complete the diagnostic referral process in West Nile, Uganda. METHODS: Between August 2013 and June 2015, 85% (295/346) people who screened RDT-positive were examined by microscopy at least once; 10 cases were detected. We interviewed 20 RDT-positive suspects who had not completed referral (16 who had not presented for their first microscopy examination, and 4 who had not returned for a second to dismiss them as cases after receiving discordant [RDT-positive, but microscopy-negative results]). Interviews were analysed thematically to examine experiences of each step of the referral process. RESULTS: Poor provider communication about HAT RDT results helped explain non-completion of referrals in our sample. Most patients were unaware they were tested for HAT until receiving results, and some did not know they had screened positive. While HAT testing and treatment is free, anticipated costs for transportation and ancillary health services fees deterred many. Most expected a positive RDT result would lead to HAT treatment. RDT results that failed to provide a definitive diagnosis without further testing led some to question the expertise of health workers. For the four individuals who missed their second examination, complying with repeat referral requests was less attractive when no alternative diagnostic advice or treatment was given. CONCLUSIONS: An RDT-based surveillance strategy that relies on referral through all levels of the health system is inevitably subject to its limitations. In Uganda, a key structural weakness was poor provider communication about the possibility of discordant HAT test results, which is the most common outcome for serological RDT suspects in a HAT elimination programme. Patient misunderstanding of referral rationale risks harming trust in the whole system and should be addressed in elimination programmes

Actigraphy in Human African Trypanosomiasis as a Tool for Objective Clinical Evaluation and Monitoring: A Pilot Study

The clinical picture of the parasitic disease human African trypanosomiasis (HAT, also called sleeping sickness) is dominated by sleep alterations. We here used actigraphy to evaluate patients affected by the Gambiense form of HAT. Actigraphy is based on the use of battery-run, wrist-worn devices similar to watches, widely used in middle-high income countries for ambulatory monitoring of sleep disturbances. This pilot study was motivated by the fact that the use of polysomnography, which is the gold standard technology for the evaluation of sleep disorders and has greatly contributed to the objective identification of signs of disease in HAT, faces tangible challenges in resource-limited countries where the disease is endemic. We here show that actigraphy provides objective data on the severity of sleep-wake disturbances that characterize HAT. This technique, which does not disturb the patient's routine activities and can be applied at home, could therefore represent an interesting, non-invasive tool for objective HAT clinical assessment and long-term monitoring under field conditions. The use of this method could provide an adjunct marker of HAT severity and for treatment follow-up, or be evaluated in combination with other disease biomarkers in body fluids that are currently under investigation in many laboratories

Late Stage Infection in Sleeping Sickness

At the turn of the 19th century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles

Estimating the economic and social consequences for patients diagnosed with human African trypanosomiasis in Muchinga, Lusaka and Eastern Provinces of Zambia (2004-2014)

Abstract Background Acute human African trypanosomiasis (rHAT) caused by Trypanosoma brucei rhodesiense is associated with high mortality and is fatal if left untreated. Only a few studies have examined the psychological, social and economic impacts of rHAT. In this study, mixed qualitative and quantitative research methods were used to evaluate the socio-economic impacts of rHAT in Mambwe, Rufunsa, Mpika and Chama Districts of Zambia. Methods Individuals diagnosed with rHAT from 2004 to 2014 were traced using hospital records and discussions with communities. Either they, or their families, were interviewed using a structured questionnaire and focus group discussions were conducted with affected communities. The burden of the disease was investigated using disability adjusted life years (DALYs), with and without discounting and age-weighting. The impact of long-term disabilities on the rHAT burden was also investigated. Results Sixty four cases were identified in the study. The majority were identified in second stage, and the mortality rate was high (12.5%). The total number of DALYs was 285 without discounting or age-weighting. When long-term disabilities were included this estimate increased by 50% to 462. The proportion of years lived with disability (YLD) increased from 6.4% to 37% of the undiscounted and un-age-weighted DALY total. When a more active surveillance method was applied in 2013–2014 the cases identified increased dramatically, suggesting a high level of under-reporting. Similarly, the proportion of females increased substantially, indicating that passive surveillance may be especially failing this group. An average of 4.9 months of productive time was lost per patient as a consequence of infection. The health consequences included pain, amnesia and physical disability. The social consequences included stigma, dropping out of education, loss of friends and self-esteem. Results obtained from focus group discussions revealed misconceptions among community members which could be attributed to lack of knowledge about rHAT. Conclusions The social and economic impact of rHAT on rural households and communities is substantial. Improved surveillance and strengthening of local medical services are needed for early and accurate diagnosis. Disease prevention should be prioritised in communities at risk of rHAT, and interventions put in place to prevent zoonotic disease spill over from domestic animals and wildlife. Supportive measures to mitigate the long-term effects of disability due to rHAT are needed