Eswab

Cervical, rectal, and amniotic fluid samples were collected from the childbearing women at the admission using cotton swabs (e-swab, Amies Medium, COPAN, Italy, cat.no.480CE) and were immediately transferred to the laboratory. Oral samples were collected from the newborn babies in the first 48 h after birth (e-swab, Amies Medium, COPAN, Italy, cat.no.480CE). Maternal and neonatal samples were first seeded on four different selective chromogenic agar plates—CHROMID ESBL agar (bioMérieux, Marcy l’Etoile, Lion, France), CHROMID MRSA agar (bioMérieux, Marcy l’Etoile, Lion, France), CHROMagar VRE (CHROMagar, Paris, France), and CHROMagar mSuperCARBA (Paris, France), which were incubated for 24–48 h at 37 °C under aerobic conditions.
Clinical and demographic data were gathered from medical records. The standard protocol is that the women gave birth and they and their newborn babies are hospitalized for three days. In the case of the women that presented positive bacterial cultures, the period of hospitalization was extended during their treatment, along with the treatment of their newborn babies, if there was needed, to seven days. After discharge, a follow-up was done for a period of 6 month by phone call once a month to find out if any infection occurred in the newborn babies that were initially infected with antibiotic-resistant bacteria at birth.

To increase the potential yield of the study, only samples from individuals who self-identified as men attending our sexual health clinic were included, as most reported cases to date in the current epidemic were men. Included sample types were anorectal swabs (Eswab, Copan Diagnostics), oropharyngeal swabs (Eswab, Copan Diagnostics) or a combination of a patient’s first-void urine, oropharyngeal swab and anorectal swab (pooled sample, as described in ref. ^{10 (link)}). All samples were collected for routine diagnostic testing or screening of gonorrhea/chlamydia in men with or without symptoms compatible with a gonorrhea/chlamydia infection. Samples were processed with the Abbott Real Time CT/NG assay, which includes the Abbott m2000sp for DNA extraction (Abbott Molecular). The original swab samples and their leftover DNA extracts were frozen (−20 °C) or refrigerated (2–8 °C), respectively, until processing in the current study. Repeat DNA extraction of the original samples was done with Maxwell Promega, using 300-µl sample input and 75-µl elution volume.

Both vaginal and skin maternal samples are taken with swabs and are collected in 1 ml liquid amies medium in a skirted tube (eSwab, Copan). These samples are obtained between 24 and 30 weeks of pregnancy. In the infant, a skin, mouth, and nose sample are taken using a swab (eSwab, Copan) at birth and at 12 and 24 months of age. All the samples are stored at − 80 °C until their use for microbiome determination.

Genital samples were collected from symptomatic male and female patients attending a large urban STI clinic in Durban, South Africa, as part of two clinical studies. Female patients (May 2016 –January 2017), aged 18–40 years, consented to vaginal swab collection (Eswab®, Copan, Brescia, Italy), as reported previously [37 (link)], and male patients (June–August 2015), aged 19–60 years, consented to urethral Eswab® collection. A total of 22 N. gonorrhoeae-positive specimens were included in this study. Ethics was approved for this study by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal, BREC97/2019.

The sample release efficiency of the microneedle swab was compared with that of the commercial Isohelix^® and Copan eSwab^® swabs. To prepare the DNA solution to be applied to the swab head, the oral mucosal tissue of the pig was extracted using the “DNA Purification from Tissues” protocol of the QIAamp DNA mini kit. Then, 40 μl of DNA solution was dropped on each swab head [MN swab, rayon swab (Isohelix^®), and nylon flocked swab (Copan eSwab^®)] with a pipette. Each swab head was cut off and placed in a 5 ml tube filled with 500 μl of buffer and vortexed for 1 min. Release efficiency (RE) was calculated as follows. $RE= \frac{V_{\mathit{buffer}}\left[sample\right]}{V_{\mathit{applied}}\left[origin\right]}100 \%$
The ratio of the amount of DNA released into the buffer compared to the amount of DNA applied to the swab head was expressed as a percentage. Vapplied in the denominator is the volume (40 μl) of the DNA solution applied to the swab head, and [origin] is the concentration of the DNA solution in the application solution. Vbuffer is the lysis buffer volume, 500 μl, and [sample] is the DNA concentration released into the buffer (Bruijns et al., 2018 (link)).

Aliquots of the respective viral strains containing ca. 10⁵ viral copies as determined by qRT-PCR were thawed and diluted ten-fold in DMEM (10⁵, 10⁴, 10³ and 10²). Five Copan eSwabs (Copan Italia S.p.A) were individually dipped for 10 seconds in these different dilutions to achieve a high, medium, low and very low viral load. For the Delta variant, this allowed for inoculation of ca. 10⁴ and 10³ pfu/ml on swabs. For the Omicron variant, this process allowed for inoculation of ca. 10⁴, 10³ and 10² pfu/ml on swabs. The inoculated swabs were placed in a 15 ml Falcon tube and stored at 4°C for 0 hrs, 24 hrs, 48 hrs, 72 hrs or 7 days. For the Omicron variants seeded at 10² pfu/ml, swabs were also stored at room temperature.