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To determine the ZIKV viral load, we used the previously described primers known as ZIKV 1086 (5ʹ CCGCTGCCCAACACAAG 3ʹ) and ZIKV 1162c (5ʹ CCACTAACGTTCTTTTGCAGACAT 3ʹ) [ 12 ]. A 1-step reaction was performed using the SuperScript III Platinum SYBR Green One-Step quantitative reverse-transcription polymerase chain reaction kit (Life Technologies). For each sample, reactions were prepared with a final volume of 15 μL containing 10 μL 2X SYBR Green Reaction Mix, 0.4 μL SuperScript III RT/Platinum Taq Mix, 0.3 μL of each primer at 10 µM, ROX Dye to a final concentration of 50 nM, 5 µL of RNA, and water to complete the volume. Thermal cycling consisted of an initial step of 42°C for 3 minutes and 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and a final step of 40°C for 1 minute. Finally, a melting curve was applied to the amplicons. The standard curve was performed using ZIKV containing 10 9 –10 5 plaque-forming units (PFU)/mL.

To determine the DENV 2 viral load, a SYBR Green 2-step protocol was applied as previously described [ 14 ] and using Den F (5ʹ-TTAGAGGAGACCCCTCCC-3ʹ) and Den R2 (5ʹ-GAGACAGCAGGATCTCTGG-3ʹ). The standard curve was performed with DENV 2 PE 3808 containing 10 6 –10 1 PFU/mL.

All of the reactions were performed in the QuantStudio 3 Real-Time PCR System using a MicroAmp Fast Optical 96-well reaction plate (Applied Biosystems). The runs were analyzed in the QuantStudio Design and Analysis Software, version 1.4. The viral loads are presented as genomic equivalent per milliliter.

The patients’ sera were tested for the presence of DENV immunoglobulin G (IgG) using the human anti–DENV IgG enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, United Kingdom) according to the manufacturer’s instructions. The assay did not discriminate between the DENV serotypes. The plate was read at 450 nm using a Spectramax Plus ELISA reader (Molecular Devices).

Serum cytokine levels were determined through the use of a multiplex bead analysis in a Human Cytokine/Chemokine Magnetic Bead Panel (Millipore, Watford, United Kingdom). Levels of the following 10 molecules were measured according to the manufacturer’s instructions: interferon gamma (IFN-γ), interleukin (IL) 1β, IL-2, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, and IL-17. Finally, the beads were washed and analyzed in a Luminex IS-100 system (Luminex Corp, Texas). Standard curves of known concentrations of recombinant human cytokines were used to convert fluorescence units into cytokine concentration units (pg/mL). The data were stored and analyzed using GraphPad Prism software version 5.0.

All statistical analyses were carried out using JMP software version 10. Age was normally distributed and analyzed using parametric statistics, whereas number of days between symptoms and medical assessment, cytokine level, and viral load data were not normal and no transformation attempted rendered them normal, so these data were analyzed using nonparametric statistics.

B. Anti-Bb immunostaining.

I. Culture fluid – University of New Haven

Genital culture fluid from Patient 1 was fixed on a SuperFrost™ Plus microscope slide and was stained with FITC-labelled polyclonal anti-Bb antibody. Staining was strongly positive, revealing well-defined spirochetes morphologically consistent with Bb ( Free Shipping Manchester Sale Low Price Scarpa Mens Loafers Versace Jeans Couture Sale Footlocker Whole World Shipping Affordable For Sale 4gCRB
). The polyclonal antibody was not reactive to T. denticola (data not shown).

II. Culture pellets – McClain Laboratories

Anti-Bb immunostaining was positive for all genital cultures except for Patient 1, whose pellet was lost during processing ( Cupro Skirt Blue Diamond Cupro Skirt by VIDA VIDA Fashion Style For Sale Cheap Limited Edition Quality Free Shipping Low Price Shopping Online Original The Cheapest Online A4bxqR
). Immunostaining revealed both spiral and globular Bb forms ( Figure 2B ). Since control genital cultures had no visible spirochetes, the control samples were sent directly for PCR testing and were not subjected to immunostaining. See Silk Square Scarf Coneflower by VIDA VIDA Free Shipping Choice Discount New Q5OmLS
, data file 3.

Table 4. Results of immunostaining and FlaB molecular hybridization in genital culture concentrates.

See Dataset , data files 3 and 4. ND, not done.

*Positive Bb immunostaining of genital culture fluid. See Results section.

A : B. burgdorferi immunostaining of vaginal culture from Patient 1. Note intensely staining spiral and round forms in culture. 400× magnification. B : B. burgdorferi immunostaining of seminal culture from Patient 6. Note intensely staining spiral and round forms in culture. 400× magnification. See Dataset , data file 3.

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Hybridization with the Fla B probe was positive for genital culture pellets from Patients 2–9 ( Table 4 ). The culture pellet from Patient 1 was lost during processing. The molecular probe showed intense staining in vaginal secretions and less intense staining in semen samples ( Affordable Cheap Price Slipon Sneakers leather beige brown Hogan Buy Cheap Get To Buy Pictures Online rQLM4eq
). See Dataset , data file 4.

A : Molecular hybridization of B. burgdorferi -specific FlaB probe with seminal culture from Patient 6. Note intensely staining spiral and round forms in culture. 400× magnification. B : Molecular hybridization of B. burgdorferi -specific FlaB probe with vaginal culture from Patient 7. Note intensely staining spiral and round forms in culture. 400× magnification. See Dataset , data file 4.

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A. Australian Biologics. Borrelia 16S rRNA sequence was not detected by real-time PCR in any of the control genital culture pellets. In contrast, Borrelia 16S rRNA sequence was detected in genital culture pellets from 11 of 13 patients ( Modal Scarf NaTure by VIDA VIDA Sale Browse Best Place For Sale FTdHU7gH8
). Patient 2 had equivocal test results and Patient 3 had negative test results in seminal cultures. See Dataset , data file 5. Real-time PCR failed to detect treponemal gene sequences in any of the control or patient genital culture pellets. See Dataset , data file 5a. The 16S rRNA isolates from six patients were sequenced and subjected to BLAST analysis (see below).

last updated 06/26/2018