## 4 Discussion and Conclusions  
The low efficiency of germination on culture media of _P. larvae_ spores is known [50]. [PERSON] and [PERSON] (1983) [51] reported that the highest spore germination on MYPCP agar was about 6% of the direct microscopic spore counts. More recently, [PERSON] et al. (2008) [52] showed that different P. larvae genotypes had significant variability in the germination rate on solid media, and [PERSON] et al. [53] observed that not all the field strains were able to develop in vitro. For these reasons, the culture-based techniques systematically underestimated the number of spores present in the examined samples. The cultivation method detects only a limited percentage of the total spores, those capable to germinate in growing medium. Most of the others are still infectious, therefore potentially capable of causing disease, as not many spores are needed for the onset of the disease in young larvae [2,4,5]. In recent years, many molecular-based protocols have been published for the detection and quantification of _P. larvae_ in honey, bees, and debris. They have been used to evaluate the sanitary status of honeybee populations. The PCR-based methods are an alternative to the classic cultivation tests on agar and should also allow for the detection of _P. larvae_ spores that do not germinate in vitro, considerably improving the sensitivity. Conventional PCR has been used to detect _P. larvae_ in honey [2,31, 32, 33, 36, 54, 55] as well as in beehive debris [35]. Despite numerous advantages, such as higher sensitivity and specificity than the culture method, conventional PCR also has disadvantages and technical limits, because the true quantification of the pathogen is not achievable [45]. Quantitative real-time PCR (qPCR) assay overcomes this limitation of conventional PCR, and it is a time- and cost-effective choice with respect to the cultural assays. Cultural methods generally underestimate the _P. larvae_ spore load because of the weak and inconsistent germination [42, 44]. The qPCR protocol allows for a larger quantification of spores, mainly because spores that do not germinate in the culture substrate are also included. The number of spores found does not necessary translate to infection, as not all spores are viable and qPCR methods would not identify this aspect related to the spore viability. But qPCR is able to detect sub-clinical levels of _P. larvae_ spores. As noted by [PERSON] and [PERSON] [56], the PCR analysis of accumulated winter hive debris was the preferable method for the monitoring of the prevalence of the pathogen irrespective of disease symptoms; while cultural was more accurate for detecting the bacterium in clinically diseased colonies. This may be due to the presence of polymerase inhibitors in some hive materials and/or failures in DNA extraction protocols, among the main factors. [PERSON] et al. (2018) [42] proposed to adopt a qPCR protocol to get an early estimation of AFB prevalence, thanks to a reliable quantification of _P. larvae_ spores. This could help to prevent the diffusion of _P. larvae_ spores from hive to hive, without the need of clinical signs. Several qPCR methods, designed to detect the 16S rRNA gene of _P. larvae_, have been developed for the detection and/or quantification of _P. larvae_ spores in honey [39], in environmental honeybee larva or scales samples [37,38,40], and in honey and hive debris [42]. Unfortunately, all of these methods, although using specific primers targeting the 16S rRNA gene of _P. larvae_, are based on SyBr technology, which requires the analysis of the melting curve of the PCR products at the end of the amplification sessions. This is time consuming and yields vague interpretation. More recently, new probe-based real-time PCR protocols based on the use of specific fluorescence probes in addition to specific primers have been described. [PERSON] et al.