Rotor q system

Equal amounts of cell cultures, corresponding to 1.5 × 10⁵ cells, were collected as appropriate at 2 or 48 h post-infection (hpi) and processed by using the Maxwell Viral Total Nucleic Acid kit on a Maxwell MDx platform (Promega), to obtain a total nucleic acid fraction in elution volumes of 150 μL. The quantitative evaluation of target nucleic acids was carried out by qPCR assays in a Rotor-Q system (Qiagen, Hilden, Germany). For the analysis of B19V DNA, aliquots of the eluted nucleic acids (corresponding to ~500 cells) were directly amplified in a qPCR assay (Maxima SYBR Green qPCR Master Mix, Thermo Scientific, Life Technologies, Monza, Italy). For the analysis of B19V RNA, parallel aliquots were first treated with the Turbo DNAfree reagent (Ambion, Life Technologies) before amplification in a qRT-PCR assay (Express One-step SYBR GreenER Kit, Invitrogen, Life Technologies). Standard cycling programs were used, followed by a melting curve analysis to define the $T_{\mathrm{m}}$ of amplified products. The primer pair R2210–R2355, located in the central exon of B19V genome, was used to amplify both viral DNA and total RNA, and a target sequence in the region of genomic DNA coding for 5.8S rRNA (rDNA) was amplified in parallel reactions for normalisation [5 (link),31 (link)].

Quantitative real-time PCR (qPCR) and RT-PCR (qRT-PCR) were performed using the QuantiTect SybrGreen PCR Kit and QuantiTect SybrGreen RT-PCR Kit on a RotorQ system (Qiagen, Hilden, Germany), including 0.5 µM of each specific primer pair, according to previously established protocols [10 (link)]. Quantitation of viral nucleic acids was obtained by the absolute quantitation algorithm, converting quantification cycle (Cq) values to geq number using external calibration curves obtained from respective standard targets. The primers used for the amplification of B19V DNA and the whole set of viral transcripts were the pair R2210–R2355, located in the central exon of the B19V genome, while determination of NS transcripts was obtained by using the pair R1882–R2033. For control and normalization with respect to the number of cells, a target sequence in the region of genomic DNA coding for 18S rRNA (rDNA) was amplified. All primers used are listed in Table 1.

Histological and molecular diagnosis were performed on skin or mucosal biopsies. For histological examination, biopsies were routinely formalin-fixed and paraffin-embedded (FFPE). Five- to six-µm sections were obtained from the block and stained with haematoxylin and eosin (HE) to detect leishmanial amastigotes. Slides were observed at 100×, 400×, and 600× magnification. If necessary, a Giemsa stain was evaluated in addition to HE. Molecular confirmation was performed on FFPE at RRL. DNA extraction was performed by the NucleoSpin DNA FFPE XS kit (Macherey-Nagel, Duren, Germany). Real-time PCR was carried out with two in-house assays, one targeting a segment of the small-subunit ribosomal RNA (rRNA) gene [25 (link)] and the other targeting a region of minicircle kinetoplast (k)DNA [26 (link)]. A real-time PCR assay targeting human β2-microglobulin was run simultaneously as a control of the amplification of the extracted DNA. PCR reactions were performed using a CFX real-time PCR detection system (Bio-Rad, Hercules, CA, USA) or Rotor-Q system (Qiagen, Hilden, Germany). Primers and probes employed for the real-time PCR assays are reported in Table S2.