Molecular detection of 7SL‐derived small RNA is a promising alternative for trypanosomosis diagnosis

Verney, Mylène; Grey, Finn; Lemans, Charlène; Géraud, Tristan; Berthier, David; Thévenon, Sophie; Rincé, Alain; Hans, Aymeric; Morrison, Liam J.; Hébert, Laurent

doi:10.1111/tbed.13744

Cited by 6 publications

(6 citation statements)

References 27 publications

(51 reference statements)

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“…The SHERLOCK-7SL assay [15] appeared to be highly speci c in the present study but did not pick up the gHAT case. However, it also merits further evaluation as other studies, although using a different detection format, highlighted the promising diagnostic accuracy of 7SL small RNA detection for Trypanozoon detection in animals [45,46]. Speci city of conventional 18S-PCR is in line with a previous report [23] and although it did not detect the gHAT patient in the present study, its sensitivity has been previously estimated to be su cient [23,27].…”

Section: Discussionsupporting

confidence: 80%

Low specificity of rapid diagnostic tests and lack of coherence between laboratory tests to diagnose gambiense human African trypanosomiasis, as evidenced by a prospective clinical performance study in Côte d’Ivoire and in Guinea

N’Djetchi,

Camara,

Koffi

et al. 2024

Preprint

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Background:Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future “screen and treat” strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. Method: During active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (Bioline HAT 2.0, HAT Sero-K-SeT, CATT) and prototype (HAT Sero-K-SeT 2.0, DCN) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis, indirect ELISA) and molecular laboratory tests (conventional PCR, SHERLOCK, Trypanozoon S²-RT-qPCR, SNP RT-qPCR). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Results: One gHAT case was diagnosed. Overall test specificities (n=1094) were: CATT 98.9% (98.1-99.4%); HAT Sero-K-SeT 86.7% (84.5-88.5%); Bioline HAT 2.0 82.1% (79.7-84.2%); DCN HAT RDT 78.2% (75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Bioline HAT 2.0 and HAT Sero-K-Set 2.0. The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n=399) and 93.0-100% (n=302), respectively. Among 44 laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular laboratory test positivity. Conclusions:Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based “screen and treat” strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. Trial registration: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

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Section: Discussionsupporting

confidence: 80%

Low specificity of rapid diagnostic tests and lack of coherence between laboratory tests to diagnose gambiense human African trypanosomiasis, as evidenced by a prospective clinical performance study in Côte d’Ivoire and in Guinea

N’Djetchi,

Camara,

Koffi

et al. 2024

Preprint

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“…Interestingly, defects in quorum sensing pathways have recently been reported in T. evansi and T. equiperdum but also in artificially selected monomorphic lines of T. brucei (Cai et al, 2022). These parasites are normally almost indistinguishable from each other, using a wide range of molecular markers (Gibson et al, 1980;Lun et al, 1992a;Lun et al, 1992b;Lai et al, 2008;Verney et al, 2020), which makes it all the more interesting that these differences map onto the quorum sensing pathways. This implies a link between the acquisition of monomorphism and the loss of quorum sensing and supports the notion that T. evansi and T. equiperdum could have evolved from a malignant (or dedifferentiated) form of T. brucei (Lun et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

PAG3 promotes the differentiation of bloodstream forms in Trypanosoma brucei and reveals the evolutionary relationship among the Trypanozoon trypanosomes

Wen

Tang

Cai

et al. 2022

Front. Cell. Infect. Microbiol.

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IntroductionTrypanosoma brucei, T. evansi and T. equiperdum are members of the subgenus Trypanozoon and are highly similar morphologically and genetically. The main differences between these three species are their differentiation patterns in the hosts and the role of vectors in their life cycles. However, the mechanisms causing these differences are still controversial.MethodsPAG3 gene was accessed by PCR amplification in 26 strains of Trypanozoon and sequences were then analyzed by BLAST accompanied with T. evansitype B group. RNA interference and CRISPR/Cas9 were used for revealing possible role of PAG3 in slender to stumpy transformation.ResultsThe procyclin associated gene 3 (PAG3) can be found in the pleomorphicspecies, T.brucei, which undergoes differentiation of slender forms to the stumpy form. This differentiation process is crucial for transmission to the tsetse fly vector. However, a homologue of PAG3 was not detected in either T. evansi or in the majority of T. equiperdum strains which are allmonomorphic. Furthere xperiments in T. brucei demonstrated that, when PAG3 was down-regulated or absent, there was a significant reduction in the differentiation from slender to stumpy forms.ConclusionTherefore, we conclude that PAG3 is a key nuclear gene involved in the slender to stumpy differentiation pathway of T.brucei in the mammalian host. Loss of this gene might also offer a simple evolutionary mechanism explaining why T. evansi and some T. equiperdum have lost the ability to differentiate and have been driven to adapt to transmission cycles that by pass the tsetse vector or mechanical contact.

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“…The 7SL sRNA has also been found to be a sensitive diagnostic marker for equine infections with Trypanosoma brucei equiperdum , the causative agent of dourine; 7SL sRNA signal was detected in horses experimentally infected with T. b. equiperdum before parasite detection by microscopy, and earlier than seroconversion detection using a complement fixation test (CFT), which is the officially-recommended dourine test by OIE ( 30 ). The 7SL sRNA signal remained present in periods of sub-patent parasitaemia but decayed rapidly after trypanocidal treatment, indicating that presence of the marker correlated with active infection and that the test is specific (i.e., correctly identifies absence of infection as negative) ( 31 ). Verney et al also showed that 7SL sRNA is stable at 30°C for 7 days; a highly desirable characteristic for the application of the test in areas with limited or no cold chain capabilities.…”

Section: Introductionmentioning

confidence: 99%

Comparative Sensitivity and Specificity of the 7SL sRNA Diagnostic Test for Animal Trypanosomiasis

et al. 2022

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Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign Trypanosoma theileri, directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for T. brucei, T. vivax, and T. congolense performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with T. congolense, and in 3/5 cattle infected with T. vivax, with the signal being reduced 14,630-fold in the remaining two T. vivax cattle. Additionally, the assays did not cross-react with T. theileri. Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44–100%) and 100% specificity (95% CI, 96.53–100%), 96.73% (95% CI, 95.54–99.96%) and 99.19% specificity (95% CI, 92.58–99.60%), and 93.42% (95% CI, 85.51–97.16% %) and 82.43% specificity (95% CI, 72.23–89.44% %) for the T brucei, T. congolense and T. vivax assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with T. theileri, high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.

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Molecular detection of 7SL‐derived small RNA is a promising alternative for trypanosomosis diagnosis

Cited by 6 publications

References 27 publications

Low specificity of rapid diagnostic tests and lack of coherence between laboratory tests to diagnose gambiense human African trypanosomiasis, as evidenced by a prospective clinical performance study in Côte d’Ivoire and in Guinea

Low specificity of rapid diagnostic tests and lack of coherence between laboratory tests to diagnose gambiense human African trypanosomiasis, as evidenced by a prospective clinical performance study in Côte d’Ivoire and in Guinea

PAG3 promotes the differentiation of bloodstream forms in Trypanosoma brucei and reveals the evolutionary relationship among the Trypanozoon trypanosomes

Comparative Sensitivity and Specificity of the 7SL sRNA Diagnostic Test for Animal Trypanosomiasis

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