## 3 Results  
The presence/absence of _P. larvae_ spores by the two tests is reported in Table 1. All of the samples tested by real-time PCR produced a reliable IC value; therefore, they showed no inhibition factors and did not have to be tested diluted. The proportions of positive colonies were 54% (CI 95% 44.3-64.0, 57/105) with the culture method and 66% (CI 95% 55.8-74.7, 69/105) with real-time PCR. A significant difference was found with [PERSON]'s test (_p_ = 0.02). Discordance was observed in 24/105 (23%) samples: 18 samples resulted in being negative by the culture method but positive \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Culture** & **Culture** & \\  & **(+)** & **(\(-\))** & \\ \hline RT-PCR (\(+\)) & **51** & 18 & 69 \\ \hline RT-PCR (\(-\)) & 6 & **30** & 36 \\ \hline  & 57 & 48 & Tot. **105** \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Presence/absence of _P. larvae_ according to molecular and microbiological analyses of 105 powdered sugar samples. Figure 1: Amplification plot of the _P. larvae_ genome standard curve. Relation between serial dilutions of DNA from _P. larvae_ ATCC 9545 and the threshold cycle (Ct). The Ct values increased in proportion to initial DNA template quantities at a range from 0.3 to \(3\times 10^{6}\) genome copies. Standard curves showed values of efficiency and linearity of the reaction (slope = \(-\)3.365; R\({}^{2}\) = 0.999). by real-time PCR, and six samples resulted in being positive by the culture method but negative by real-time PCR. Coincidence was observed in 81 samples (77%): 51 resulted in being positive and 30 in being negative by both methods. The spore counts obtained by the culture method and qPCR on sugar samples are reported in Figure 2. In 45 samples out of the 51 positives determined by both methods, qPCR found a greater number of spores than the culture method did. In Table 2, spore counts grouped by contamination level are reported. All of the sugar samples of _Sub-group A1_ had a spore load greater than 1 \(\times\) 10\({}^{4}\) by the microbiological method, and greater than 1 \(\times\) 10\({}^{5}\) by qPCR. In _Sub-group A2_, the spore load by the microbiological method was less than 100 for 60% (27/45) of the samples, while for 71% (32/45) of the samples by qPCR the spore load was between 1 \(\times\) 10\({}^{2}\) and 1 \(\times\) 10\({}^{5}\). In _Group B_, 82% of the samples had a spore load > LOD by the microbiological method, while 38% of the samples by qPCR had a spore count between 20 and 1000. Detailed results of all of the samples obtained from both methods are reported in Table S4. Detailed results of all of the samples obtained from both methods are reported in Table S4. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{4}{c}{_Group A_} & _Group B (u = 45)_ \\ \cline{2-6}  & \multicolumn{2}{c}{_Sub-Group A1 (u = 15)_} & _Sub-Group A2 (u = 45)_ & _Group B (u = 45)_ \\ \hline
**Classes of Contamination** & **Culture** & **qPCR** & **Culture** & **qPCR** & **Culture** & **qPCR** \\ \hline
**(CFU/g–Spore/g)** & **Method** & **qPCR** & **Culture** & **qPCR** & **Culture** & **qPCR** \\ \hline \(<\)20 & 0 & 0 & 11 (24\%) & 8 (18\%) & 37 (82\%) & 28 (62\%) \\ \hline
20–100 & 0 & 0 & 16 (36\%) & 5 (11\%) & 6 (13\%) & 8 (18\%) \\ \hline
101–1000 & 0 & 0 & 13 (29\%) & 18 (40\%) & 2 (4\%) & 9 (20\%) \\ \hline
1001–10,000 & 0 & 0 & 4 (9\%) & 8 (18\%) & 0 & 0 \\ \hline
10,001–100,000 & 3 (20\%) & 0 & 1 (2\%) & 6 (13\%) & 0 & 0 \\ \hline
100,001–1,000,000 & 8 (53\%) & 5 (33\%) & 0 & 0 & 0 & 0 \\ \hline \(>\)1,000,001 & 4 (27\%) & 10 (67\%) & 0 & 0 & 0 & 0 \\ \hline
**Total** & **15** & **15** & **45** & **45** & **45** & **45** \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Distribution of results obtained by microbiological and qPCR assays according to the different classes of contamination level expressed in terms of the CFU/g or spore/g of _P. larvae_, respectively.