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. 2023 Jul 30;11(8):1950.
doi: 10.3390/microorganisms11081950.

Relationship between the Cycle Threshold Value (Ct) of a Salmonella spp. qPCR Performed on Feces and Clinical Signs and Outcome in Horses

Hélène Amory  1   2 , Carla Cesarini  1   2 , Lorie De Maré  1   2 , Clémence Loublier  1   2 , Nassim Moula  2   3 , Johann Detilleux  1   2 , Marc Saulmont  4 , Mutien-Marie Garigliany  2   5 , Laureline Lecoq  1   2
Affiliations

Relationship between the Cycle Threshold Value (Ct) of a Salmonella spp. qPCR Performed on Feces and Clinical Signs and Outcome in Horses

Hélène Amory et al. Microorganisms. .

Abstract

The objective of this retrospective study was to evaluate the clinical significance of fecal quantitative real-time polymerase chain reaction (qPCR) Salmonella results when taking the cycle threshold values (Ct) into account. The study included 120 Salmonella qPCR-positive fecal samples obtained from 88 hospitalized horses over a 2-year period. The mean Ct of the qPCR test was evaluated in regard to (1) clinical outcome and (2) systemic inflammatory response syndrome (SIRS) status (no SIRS, moderate SIRS, or severe SIRS) of the sampled horses. An ROC analysis was performed to establish the optimal cut-off Ct values associated with severe SIRS. The mean ± SD Ct value was significantly lower in samples (1) from horses with a fatal issue (27.87 ± 5.15 cycles) than in surviving horses (31.75 ± 3.60 cycles), and (2) from horses with severe SIRS (27.87 ± 2.78 cycles) than from horses with no (32.51 ± 3.59 cycles) or moderate (31.54 ± 3.02 cycles) SIRS. In the ROC analysis, the optimal cut-off value of Ct associated with a severe SIRS was 30.40 cycles, with an AUC value of 0.84 [95% confidence interval 0.76-0.91] and an OR of 0.64 [0.51-0.79]. Results suggest that including the Ct value in the interpretation of fecal qPCR results could improve the diagnostic value of this test for clinical salmonellosis in horses.

Keywords: SIRS; bacterial load; equine; fecal shedding; qPCR; salmonellosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Boxplots of the cycle threshold value (Ct) of positive Salmonella spp. qPCR tests (a) and of the corresponding log CFU/mL estimated from standard calibration curves (b) obtained on fecal samples from horses hospitalized for gastrointestinal problems and not presenting SIRS (n = 72), presenting a moderate SIRS (n = 26), or presenting a severe SIRS (n = 22) at the time of sampling. a and b = significantly different from samples obtained on cases with no SIRS or on cases with a moderate SIRS, respectively, using the one-way ANOVA test (p < 0.05).
Figure 2
Figure 2
Receiver-operating characteristic curves showing the performance of the cycle threshold value (Ct) of a Salmonella spp. qPCR performed on fecal samples in predicting the association with a severe systemic inflammatory response syndrome (SIRS) in 82 horses hospitalized for gastro-intestinal problems.
Figure 3
Figure 3
Boxplots of the cycle threshold value (Ct) of positive Salmonella spp. qPCR tests (a) and of the corresponding log CFU/mL estimated from standard calibration curves (b) obtained on fecal samples from horses hospitalized for gastrointestinal problems that were discharged from the hospital (survivors, n = 73) or that died or were euthanized during hospitalization (non-survivors, n = 9). a = significantly different from samples obtained on surviving cases, using the unpaired T-test (p < 0.05).
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