Multiple Swabs Research Articles

Investigating a simplified method for noninvasive genetic sampling in East African mammals using silica dried scat swabs.

Swabbing scat has proved to be an effective noninvasive method to collect DNA from mammals in the field. Previously, this method has relied on preservative liquids or freezing to preserve the DNA collected on swabs. In this study, we determine the effectiveness of using silica to simply dry the swab in field as an alternative way to prevent DNA degredation. Four species were included in the study; reticulated giraffe, impala, fringe‐eared oryx, and lion. Swabs were taken at multiple time points for giraffe and impala scat samples, with the lion and oryx sampled opportunistically. Mitochondrial DNA was successfully amplified and sequenced from scat swabs from all species; however, effectiveness varied between species, with 81.8% amplification success rate from swabs taken from impala scat compared to 25% amplification success rate in giraffe. This variation in success rate was overcome by taking multiple swabs, thus increasing the probability of a successful amplification. The true merit of this method is in its simplicity and cheapness; no preservative liquids were required to be brought into the field, at no stage in the 2 weeks of field sampling were samples frozen, and no commercial kits were used for DNA extraction.

Stroke in COVID-19: A Single-Center Initial Experience in a Hotspot of the Pandemic

Background: Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a global pandemic that has been an immense burden on the healthcare systems all over the world. Studies have shown that these patients may be at higher risk for acute ischemic stroke (AIS). We present our initial experience with AIS in patients with COVID-19. Methods: We reviewed all patients admitted to our hospital during a 6-week period with a positive nasopharyngeal swab test for SARS-CoV-2. Among these patients, we identified AIS by chart review. We reviewed the demographics, clinical, laboratory, imaging characteristics, treatments received, and outcomes of AIS in COVID-19 patients. Results: We identified 683 patients admitted with COVID-19 during the study period, of which 20 patients had AIS. Large vessel occlusion (LVO) was noted in 11 patients (55%). Intravenous alteplase (IV tPA) was administered in four patients (20%) and mechanical thrombectomy (MT) was performed in five patients (25%). More than half of the patients were out of window for IV tPA. Respiratory symptoms preceded the onset of stroke in most of the patients (70%), ranging between one to 21 days. The mortality in patients with AIS was 50% compared to 26% of all COVID-19 admissions. Most of these patients died due to non-neurological causes (70%). There were three patients with AIS, who had clinical, laboratory, and imaging findings consistent with COVID-19, but were negative for multiple nasopharyngeal swab tests. Interpretation: LVO was more common in patients with AIS and COVID-19. They had more severe disease and higher mortality rates. Most of the patients had respiratory symptoms preceding AIS by days to weeks. This could explain certain patients with clinical picture of COVID-19 but negative nasopharyngeal swab tests. We need larger studies to further understand the association and mechanisms of AIS in COVID-19.

The temporal RNA virome patterns of a lesser dawn bat (Eonycteris spelaea) colony revealed by deep sequencing.

The virosphere is largely unexplored and the majority of viruses are yet to be represented in public sequence databases. Bats are rich reservoirs of viruses, including several zoonoses. In this study, high throughput sequencing (HTS) of viral RNA extracted from swabs of four body sites per bat per timepoint is used to characterize the virome through a longitudinal study of a captive colony of fruit nectar bats, species Eonycteris spelaea in Singapore. Through unbiased shotgun and target enrichment sequencing, we identify both known and previously unknown viruses of zoonotic relevance and define the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. To our knowledge, this is the first study that combines probe-based viral enrichment with HTS to create a viral profile from multiple swab sites on individual bats and their cohort. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Given the known risk for bat-human zoonoses, a more complete understanding of the viral dynamics in South-eastern Asian bats has significant implications for disease prevention and control. The findings of this study will be of interest to U.S. Department of Defense personnel stationed in the Asia-Pacific region and regional public health laboratories engaged in emerging infectious disease surveillance efforts.

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

An unusual skin lesion caused by Lasiodiplodia theobromae

We describe a case of subcutaneous infection as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated in multiple swabs from the foot of an active healthy male. The fungus was identified by traditional mycology culture methods though this was slow with much time required for sporulation on only one of the agars used. Identification was confirmed by DNA sequencing. The patient was successfully treated with Voriconizole.

An unusual skin lesion caused by lasiodiplodia theobromae

Lasiodoplodia theobromae is a common plant pathogen of tea and fruiting plants from the Coelomycetes family. It is a rare plant pathogen in both immunocompetent and immunocompromised hosts with infections reported including sinusitis, keratitis, pneumonia and cutaneous lesions. We describe a case of subcutaneous infection as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated in multiple swabs from the foot of an active healthy male following a penetrating sea water injury. Following the trauma, the patient used wild aloe vera plant leaf to promote wound healing and this was thought to be a potential source of inoculation. Identification was performed by traditional mycology culture methods with sporulation occurring only with cornmeal agar at 28 days. Identification was confirmed by DNA sequencing. The patient was successfully treated with voriconizole. Lasiodoplodia theobromae is a common plant pathogen of tea and fruiting plants from the Coelomycetes family. It is a rare plant pathogen in both immunocompetent and immunocompromised hosts with infections reported including sinusitis, keratitis, pneumonia and cutaneous lesions. We describe a case of subcutaneous infection as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated in multiple swabs from the foot of an active healthy male following a penetrating sea water injury. Following the trauma, the patient used wild aloe vera plant leaf to promote wound healing and this was thought to be a potential source of inoculation. Identification was performed by traditional mycology culture methods with sporulation occurring only with cornmeal agar at 28 days. Identification was confirmed by DNA sequencing. The patient was successfully treated with voriconizole.

Patterns of detection of respiratory viruses in nasal swabs from calves in Ireland: a retrospective study

A retrospective analysis was conducted to investigate the prevalence and seasonality of bovine viral diarrhoea virus (BVDV), bovine coronavirus (BoCV), bovine herpesvirus-1 (BoHV-1), bovine respiratory syncytical virus (BRSV) and parainfluenza...

Characterizing the Temporal Dynamics of Human Papillomavirus DNA Detectability Using Short-Interval Sampling

Variable detection of human papillomavirus (HPV) DNA can result in misclassification of infection status, but the extent of misclassification has not been quantitatively evaluated. In 2005-2007, 33 women of ages 22 to 53 years self-collected vaginal swabs twice per week for 16 consecutive weeks. Each of the 955 swabs collected was tested for 37 HPV types/subtypes. Assuming that a woman's underlying infection status did not change over the short study period, biases in prevalence estimates obtained from single versus multiple swabs were calculated. Using event history analysis methods, time to recurrent gain and loss of at least one HPV type was determined, separately. Baseline any-type and high risk-type HPV prevalence was 60.6% and 24.2%, respectively. Cumulative any-HPV and high-risk HPV prevalence over the 16-week period was 84.8% and 60.6%, separately. Overall, there were 319 events of detection and 313 events of loss of detection. Median times to a recurrent detection and loss of detection were 11 and seven days, respectively. Neither vaginal sex nor condom use during follow-up was associated with recurrent viral detection or loss of detection. Assuming the cumulative 16-week prevalence reflects the true prevalence of infection, the baseline any-HPV prevalence underestimated infection status by 24.2%, with a bootstrapped mean of 20.2% [95% confidence interval (CI), 8.9%-29.6%]. These findings suggest that a substantial proportion of HPV-infected women are misclassified as being uninfected when using a single-time DNA measurement. Short-term variation in detectable HPV DNA needs to be considered while interpreting the natural history of infections using single samples collected at long intervals.

Inkjet-printed paper-based SERS dipsticks and swabs for trace chemical detection

We demonstrate a paper-based surface swab and lateral-flow dipstick that includes an inkjet-printed surface-enhanced Raman spectroscopy (SERS) substrate for analyte detection. Due to capillary-action wicking of cellulose, the paper dipstick enables extremely simple and pump-free loading of liquid samples into the detection device, and in addition provides inherent analyte concentration within the detection volume. Furthermore, the flexible nature of the paper-based SERS device also enables it to act as a swab to collect analyte molecules directly from a large-area surface; the collected analyte molecules can then be focused into a small-volume SERS-active region by lateral-flow concentration. These capabilities are unseen in today's SERS substrates and microfluidic SERS devices. Using these novel lateral-flow paper SERS devices, we achieved detection limits as low as 95 fg of Rhodamine 6G (R6G), 413 pg of the organophosphate malathion, 9 ng of heroin, and 15 ng of cocaine. Moreover, the measurements show that the technique is quantitative and is repeatable across multiple swabs and dipsticks. The results reported here may lead to ultra-low-cost portable applications in trace chemical detection.

The utility of Y-STR profiling in four-, six- and eight-day postcoital vaginal swabs

Y-STR profiles using the Promega PowerPlex(®) Y system were attempted on multiple vaginal swabs collected at four, six and eight days after intercourse from female partners of 11 couples. At four days postcoitus, full composite profiles (where swabs yielded confirmed alleles at all 11 loci) were obtained for five of the 11 couples and 78% of all possible alleles summed for all couples were confirmed (able to be duplicated in different swabs). Results for composite profiles for all couples taken at six days after intercourse showed that 53% of all alleles summed for all couples were confirmed. Only one couple yielded a full composite profile at six days after intercourse. Composite profiles from swabs taken at eight days after intercourse for all couples confirmed only 44% of all possible alleles summed for all couples. At eight days postcoitus, no couple yielded a full composite profile and the largest number of confirmed alleles for any couple was eight. However, one of 44 individual swabs taken from all couples combined at eight days postcoitus yielded a 10-locus profile. Composite partial profiles from the eight-day postcoital set with confirmed results at a minimum of five loci (8 of 11 couples) yielded haplotype frequencies from 0.000323 to 0.125862 using the Y chromosome haplotype reference database, suggesting that meaningful Y-STR information can still be obtained at much extended postcoital intervals.

English

Burn patients face an obviously high risk for infections due to the immunocompromising effects of their injury. This study aimed to detect and identify bacteria isolated from patients and hospital environment in the burn unit and determine their antibiogram pattern in response to commonly used antimicrobial agents; in order to give recommendations for management of bacterial infections and drug-resistance. Materials of this study were 100 samples of burn wounds and multiple swab samples of different hospital environments. One hundred and twelve isolates were analyzed, from which there was a single agent in the majority of cases (73.3%). Pseudomonas aeruginosawas the most common isolate (32.2%), followed by Enterobacter spp. (16.9%), coagulase-negative staphylococci (12.5%), Acinetobacter spp. (11.7%), Klebsiella spp. (8.9%), Staphylococcus aureus (7.2%), α-hemolytic streptococci (4.4%), and others (6.2%). The most commonly detected isolate from hospital environment was P. aeruginosa (35%) followed by Enterobacter spp., Klebsiella spp., coagulase-negative staphylococci. P. aeruginosa was the most resistant to third- and fourth-generation cephalosporins (100%), whereas other gram-negative bacteria were resistant to ciprofloxacin and cephalosporin (70 to 100%). Restriction in the abuse of antibiotics and establishment of an infection control unit will help lower the incidence of infection. Key words: Bacteriology, burn wounds, antibiogram, bacterial infections.

The new influenza A (H1N1/09): symptoms, diagnostics, and autopsy results

The first infections of the new H1N1 ("swine flu") virus were announced in April 2009. Soon after, case numbers were observed to be climbing virtually worldwide. In most cases, infections appeared rather mild with typical flu symptoms. In contrast to the seasonal flu, however, cases with vomiting and diarrhea were also reported, and the affected age group seemed to be younger. Sudden and fatal progression affected mostly those under 60years of age. The autopsy reports of two fatalities with the new influenza are discussed. Multiple postmortem swabs from the nose and throat area determined the H1N1-RNA.

The value of multiple surveillance cultures for methicillin-resistant Staphylococcus aureus

We evaluated our experience in a low prevalence setting to determine the extent to which multiple swabs increased detection rates and the incremental costs of doing so. Nasal and groin swabs submitted in pairs were cultured onto a single plate (Oxoid MRSA Denim Blue Agar; Oxoid Company, Napean, ON, Canada). We determined whether MRSA was detected when swabs submitted in the preceding 3 days were negative. We explored the costs associated with screening and of each additional colonized patient detected. In all, 60,049 paired nose and perineal swabs were submitted from 21,599 patients. In all, there were 12,750 duplicate, 1437 triplicate, and 112 instances when >4 swabs were processed within 3 days. The first culture was positive in 106 of 12,750 (0.83%%), 42 of 12,750 (0.33%) on the second when the first was negative, 7 of 1642 (0.43%) on the third or subsequent swab pair when the preceding 2 were negative. Overall, the sensitivity of the first of multiple cultures of a set was 74.3%. Had the 14,392 multiple samples not been submitted, 49 colonized patients would not have been identified. Additional laboratory costs associated with multiple samples equaled $2088 per patient identified.

14. Ascending listeria monocytogenes infection in a pregnant woman causing foetal death in utero

A 33-year-old pregnant woman, G1P0 at 30 weeks gestation, presented to her obstetrician with abdominal tightness associated with fever. On examination, she was febrile (388C) and her full blood count showed leukocytosis with neutrophi-lia. Abdominal ultrasound examination revealed a foetal death <i>in utero</i> (FDIU). She had a spontaneous vaginal delivery. The liquor was heavily meconium stained. The autopsy examination found a mildly macerated male foetus with features suggestive of oligohydramnios. The placental disc was small (241g) and showed multiple yellow cream lesions (3–18mm) on the maternal surface. There was absent coiling of the umbilical cord. Multiple microbiological swabs from the placenta, foetal abdomen and pleura, as well as culture of foetal blood, all showed a heavy growth of <i>Listeria mono-cytogenes</i>. Light microscopy showed vasculitis and funisitis of the umbilical cord as well as grade 3 (of 3) acute chorioamnionitis. Multiple microabscesses and necrotising granulomata were identified in the placental disc, liver and lung. Gram stains confirmed the presence of extracellular Gram positive bacilli within the placenta and liver. Thus there was microbiological and histological evidence of disseminated granulomatous septicaemia, a known and almost always fatal outcome for this infection. Listeriosis is an uncommon and perhaps under-diagnosed cause of FDIU. It frequently occurs in the third trimester secondary to a decline in cell-mediated immunity. Symptomatic pregnant women frequently present with a febrile episode, as in our case, following a history of ingestion of contaminated food, e.g., raw soft cheese, raw vegetables, meat or sausage. Treatment and follow up of this patient is crucial as persistent vaginal colonisation by <i>Listeria</i> may cause subsequent abortions or stillbirths in future pregnancies.

PCR Detection of Ranavirus in Adult Anurans from the Louisville Zoological Garden

Ranaviruses are known to cause mortality in a variety of anuran species and have the potential to significantly impact wild and captive frog populations. In this study, 16 captive frogs and toads from the Louisville Zoological Garden were examined for the presence of ranavirus; this group included 14 Cope's grey tree frogs (Hyla chrysoscelis), an American toad (Bufo americanus), and a southern toad (Bufo terrestris). All animals were wild caught and were evaluated via polymerase chain reaction (PCR), while animals that died were also assessed via histologic study to understand the role of ranaviral disease in these specimens. Of the animals that died, 82% were positive for ranavirus via PCR. Multiple swab samples collected over time from live tree frogs were positive for ranavirus via PCR. These findings reveal that ranaviral infection in captive adult anurans may occur without clinical signs or consistent histopathologic lesions.

Nucleic Acid Amplification Tests in the Diagnosis of Chlamydial and Gonococcal Infections of the Oropharynx and Rectum in Men Who Have Sex With Men

Several nucleic acid amplification tests (NAATs) are US Food and Drug Administration-cleared for detecting urogenital Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) infection, but they have not been adequately evaluated for the relatively common oropharyngeal or rectal CT and GC infections in men who have sex with men (MSM). Multiple swabs were collected from the oropharynx and rectum of MSM attending a city sexually transmitted disease clinic. The specimens were tested by standard culture and the following NAATs: Roche's Amplicor (PCR), Becton Dickinson's ProbeTec (SDA), and Gen-Probe's APTIMA Combo 2 (AC2) for the detection of CT and GC. Confirmatory testing of specimens with discrepant results was done by NAATs using alternate primers. A total of 1110 MSM were enrolled. Based on initial findings on 205 MSM, PCR had a 78.9% GC specificity with oropharyngeal swabs. Thus, we discontinued PCR testing for the rest of the study. For oropharyngeal GC (89 infections detected), sensitivities were 41% for culture, 72% for SDA, and 84% for AC2. For rectal GC (88 infections detected), sensitivities were 43% for culture, 78% for SDA and 93% for AC2. For oropharyngeal CT (9 infections detected), sensitivities were 44% for culture, 67% for SDA, and 100% for AC2. For rectal CT (68 infections detected), sensitivities were 27% for culture, 63% for SDA, and 93% for AC2. Specificities of SDA and AC2 were > or =99.4% for both organisms and anatomical sites. AC2 and SDA were far superior to culture for the detection of CT or GC from the oropharynx and rectum with AC2 detecting twice as many infections as culture. Further analyses with larger pharyngeal samples are needed, but clearly NAATs can improve our ability to diagnose rectal and oropharyngeal infection with CT or GC in MSM.

Successful Termination of a Furunculosis Outbreak Due to lukS-lukF-Positive, Methicillin-Susceptible Staphylococcus aureus in a German Village by Stringent Decolonization, 2002-2005

Skin infections due to Staphylococcus aureus have recently become a public concern, mainly because of emerging resistance against widely used antibiotics and specific virulence determinants. Strains harboring the lukS-lukF gene (which codes for Panton-Valentine leukocidin) are frequently associated with severe furunculosis. Generally applicable strategies for the control of community outbreaks of furunculosis have not been defined. We report the investigation and successful termination of an outbreak of furunculosis due to lukS-lukF-positive S. aureus in a German village (n=144). Nasal swab specimens were obtained from village residents. A retrospective cohort study was conducted. Nasally colonized persons, persons who had current furuncles or who had experienced relapsing furuncles since 2002, and their family members underwent stringent decolonization measures using mupirocin nasal ointment and disinfecting wash solution. Multiple nasal swab specimens were obtained to monitor the long-term outcome of decolonization measures. From January 1998 through December 2004, 42 cases and 59 relapses of furunculosis were identified by active case finding. Of 140 participants tested, 51 (36%) were found to be nasally colonized with S. aureus. In 9 participants, the strain was positive for lukS-lukF. No methicillin resistance was detected. Risk of furunculosis was associated with contact with case patients (relative risk, 6.8; 95% confidence interval, 3.2-14.3) and nasal colonization with a lukS-lukF-positive strain of S. aureus (relative risk, 3.6; 95% confidence interval, 2.3-5.9). Passive surveillance implemented in January 2005 did not detect any case of lukS-lukF-positive, S. aureus-associated furuncles in this village. This report describes a successful strategy for terminating the transmission of epidemic strains of S. aureus among a nonhospitalized population.

Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit.

The aim of this study was to determine if gastroenteric viruses were present on surfaces and equipment in a pediatric primary immunodeficiency unit (PPIU) by environmental sampling using swabs and subsequent nucleic acid extraction and reverse transcriptase PCR assays. A PPIU was chosen, and 11 swabs were taken at the same sites every 2 weeks for 6 months. Nested/heminested PCR assays were used to screen for astroviruses (AsV), noroviruses (NoV), and rotaviruses (RV). AsV, NoV, and RV were detected at multiple swab sites during the study period. NoV was the most frequently detected virus on environmental surfaces; however, RV was detected on 79% and NoV on 50% of swabbing dates during the study period. Toilet taps were the most contaminated sites. Fecal samples from selected patients in the unit were also screened during the study period, and patients excreted AsV, NoV, and RV at times during the study. New cleaning schedules and changes in some of the PPIU sanitary furniture have been suggested as a means of reducing environmental contamination.

Evaluation of separate vs pooled swab cultures, different media, broth enrichment and anatomical sites of screening for the detection of methicillin-resistant Staphylococcus aureus from clinical specimens

Early identification of methicillin-resistant Staphylococcus aureus (MRSA) carriers is a major component of an MRSA control programme. The cost and laboratory workload could be markedly reduced by processing multiple swabs from one person in one culture broth (specimen pooling). We evaluated the sensitivity for MRSA detection and the growth rate of pooled swabs compared with individual processing. In total, 1254 swabs from 423 subjects (two to five swabs per subject) were submitted for detection of MRSA. Swabs were suspended in 2-mL volumes of sterile Todd-Hewitt Broth and divided into two 1-mL aliquots. One aliquot of the suspension was processed as a single specimen, and the other aliquot was mixed (pooled) with other suspensions in which swabs from the same patient were suspended. Forty-four (10%) pooled samples were positive for MRSA. Specimens from seven additional patients that were negative when pooled were positive when processed separately. There was no case where the pooled specimen was positive but the separate specimens were negative. The diagnostic sensitivity of pooled surveillance cultures compared with single cultures, when only subjects colonized by MRSA were considered, was 86% and the false-negative rate was 14%. Eighty percent of the pooled positive cultures were detected by the third day and all were detected by the fourth day. Fifty-four percent of the specimens processed separately were detected by the second day and all were detected by the fourth day. Pooling of specimens decreases the sensitivity of MRSA detection compared with processing each swab separately, particularly in swabs with a low number of colony-forming units. In all subjects whose pooled samples were negative but whose swabs examined separately were positive, the swabs examined separately were negative on primary plates and positive only after culturing in enrichment broth.