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

Abstract

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.

Keywords: animal trypanosomiasis; diagnostic; sensitivity; small RNA; specificity.

Figure 1

Comparison of detection by buffy coat microscopy, TBR PCR and 7SL sRNA RT-qPCR in T. brucei infected calves. Four calves (Holstein-Friesian, approximately 4–6 months old) were infected with T. brucei Antat 1.1 (day 0) and monitored for 32 days. On day 28, calves were treated with diminazene aceturate, indicated by a red diamond. Parasitaemia (right y axis) was determined on average every 2 days by the BCT; days on which this was measured are indicated by gray bars. Approximate parasitaemia equivalences (trypanosomes/mL) of the score used: 1 = 1 × 10²; 2 = 1 × 10³; 3 = 5 × 10³-5 × 10⁴; 4 = 1 × 10⁴-5 × 10⁵; 5 = 5 × 10⁵-5 × 10⁶; 6 = >5 × 10⁶. Gray bars measuring 0 indicate time points in which parasitaemia was measured but no parasites were detected (0 on y axis is shown by red dotted line). Relative expression (dCt) of 7SL sRNA by RT-qPCR (left y axis) is shown with a black line and was calculated by normalizing to an uninfected control animal. TBR PCR results are shown by plus (+) or minus (–) signs indicating the presence or absence of the expected PCR band on an agarose gel, respectively.

Figure 2

Comparison of detection by buffy coat microscopy, TCS PCR and 7SL sRNA RT-qPCR in T. congolense infected calves. Four calves (Holstein-Friesian, approximately 4–6 months old) were infected with T. congolense IL3000 (day 0) and monitored for 32 days. On day 28, calves were treated with diminazene aceturate, indicated by a red diamond. Parasitaemia (right y axis) was determined on average every 2 days by the BCT and is indicated by gray bars. Approximate parasitaemia equivalences (trypanosomes/mL) of the score used: 1 = 1 × 10²; 2 = 1 × 10³; 3 = 5 × 10³-5 × 10⁴; 4 = 1 × 10⁴-5 × 10⁵; 5 = 5 × 10⁵-5 × 10⁶; 6 = >5 × 10⁶. Gray bars measuring 0 indicate time points in which parasitaemia was measured but no parasites were detected (0 on y axis is shown by red dotted line). Relative expression (dCt) of 7SL sRNA by RT-qPCR (left y axis) is shown with a black line and was calculated by normalizing to an uninfected control animal. TCS PCR results are shown by plus (+) or minus (–) signs indicating the presence or absence of the expected band on an agarose gel, respectively.

Figure 3

Decay of 7SL sRNA signal in T. congolense infected cattle after trypanocide treatment. Cattle (Holstein-Friesian, n = 5 per treatment group) were infected with T. congolense Kont2/133 (day 0) at peak parasitaemia (1 × 10⁴-5 × 10⁵ trypanosomes/ml), cattle were treated with a low (equivalent to 0.5 × minimal effective dose), medium (1×) or high dose (2×) of an experimental trypanocide. Relative expression of 7SL sRNA by RT-qPCR is shown with a black line and was calculated by normalizing to an uninfected control animal.

Figure 4

Decay of 7SL sRNA signal in T. vivax infected cattle after trypanocide treatment. Cattle (Holstein-Friesian, n = 5 per treatment group) were infected with T. vivax STIB 719 (day 0) at peak parasitaemia (1 × 10⁴-5 × 10⁵ trypanosomes/ml), cattle were treated with a low (equivalent to 0.5 × minimal effective dose), medium (1×) or high dose (2×) of an experimental trypanocide. Relative expression of 7SL sRNA by RT-qPCR is shown with a black line and was calculated by normalizing to an uninfected control.

Figure 5

(A) alignment of 7SL sRNA sequences from African trypanosomes and the commensal Trypanosoma theileri. Consistent with the African trypanosomes previously analyzed, the T. theileri 7SL sRNA sequence exhibits highly conserved flanking regions with a variable internal sequence, which importantly does not match that of the other trypanosome species. (B) 7SL sRNA sequences alignment in representative T. brucei subspecies isolates (from a total of 88 analyzed). Genome data were obtained from a previous study (34) (ENA accession: PRJEB2486) or from TriTrypDB. Strain information: GYBO: T. b. rhodesiense, isolated in 1984 in Tanzania; N4ANNEE_13_KIVI: T. b. gambiense group I, 2002, Guinea; TB386: T. b. gambiense group II, 1978, Côte d'Ivoire; BIM_1: T. b. gambiense group I, 1975, Cameroon; STIB247: T. b. brucei, 1971, Tanzania; S14_5_1: T. b. gambiense group I, 2002, Côte d'Ivoire; TB927: T. b. brucei, 1969 Kenya; STIB805: T. b. evansi, 1985, China. (C) 7SL sRNA sequence alignments in T. congolense isolates. Data from 55 genomes were obtained from a previous study (35) (ENA accession: PRJEB15251), and a representative subset is shown. Strain information (Savannah subtype unless stated): DIND: Forest subtype, 1986, Burkina Faso; ALME: 2005, Cameroon; GUTR28: 1975, The Gambia; MALI/1312/95: 1995, Mali; TOGO222: 2014, Togo; DJUMA: 1988, Congo; TRT1: 1996, Zambia. (D) 7SL sRNA sequence alignments in T. vivax isolates. Data from 27 genomes were obtained from a previous study (36) (ENA accession number: PRJNA486085) and a representative subset is shown. Strain information: TviBrMi: 1999, Brazil; TvBobo-2009: 2009, Burkina Faso; IL3171: year unknown, The Gambia; IL3638: 1990, Côte d'Ivoire; IL1392: 1981, Nigeria; TvMagna: 2011, Togo; IL2714: 1969, Uganda.

Figure 6

ROC curve of RT-qPCR detection of trypanosome specific 7SL sRNA. ROC analysis was performed using the Wilson/Brown method. The ROC curve of the 7SL sRNA test is represented by the series of black dots. The red diagonal line works as a reference and represents random chance, the characteristics of a test that is not useful for in detecting infection (AUC = 0.5). (A) 107 T. brucei Antat 1.1 infected samples and 104 uninfected samples were included in the analysis (AUC=1). (B) 153 T. congolense (IL3000 or Kont2/133) infected samples and 123 uninfected samples were included in the analysis (AUC = 0.99). (C) 76 T. vivax STIB 719 infected samples and 74 uninfected samples were included in the analysis (AUC = 0.96).